InteractiveFly: GeneBrief

Protein C kinase 53E and Protein C kinase 98E : Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Activation of kinases is central to signal transduction during neural activation. Familiarity with two cellular kinases is essential for understanding this process: Protein kinase A (PKA), is activated by elevated levels of cyclic AMP generated by adenylyl cyclase (Rutabaga in Drosophila); a second, Protein kinase C (PKC), is activated by a lipid dependent mechanism. Activation of both kinases takes place in the two main routes of signal transduction during neural activation, and both kinases have been implicated in the cellular processes involved in learning.

Protein kinase C operates at one of the most unusual crossroads of cellular activation, one dependent on hydrolysis of membrane lipids. The membrane lipid component phosphatidylinosityl 4,5-bisphosphate (PIP2), generated by the addition of phosphate residues to the sugar (inositol) moiety of the membrane lipid phosphatidylinositol, is hydrolyzed by the enzyme phospholipase C-beta to give the lipid diacylglycerol (DAG) and the sugar phosphate known as inositol 1,4,5-trisphospate (IP3). While diacylglycerol activates protein kinase C, IP3 releases Ca2+ from the endoplasmic reticulum. Each of these enzymatic processes are sensitive to the level of Ca2+ in the cell. Initiating the cellular activation cascade is the activation of phospholipase C-beta by heterotrimeric G proteins linked to G protein coupled receptors. The activation of PKC by the membrane lipid DAG is mimicked by phorbol esters whose structure resembles the structure of DAG.

Drosophila has three PKC enzymes, one eye specific [inactivation no afterpotential C or dPKC53E(ey)] and two others whose expressions are confined to the head. The isoform called dPKC53E(brain) is closely linked to dPKC53E(eye). Among the Drosophila genes, dPKC53E(brain) (Rosenthal, 1987) is most similar to the classical genes (PKCalpha, beta and gamma), while dPKC98F has greater amino acid identity with, and a domain arrangement characteristic of, PKCdelta (also termed nPKC) (Schaeffer, 1989).

The concept of memory as a unified process involving a linear series of biochemical and cellular activation steps and a single anatomical localization is no longer tenable. There are multiple kinds of memory, residing in multiple locations and generated by multiple biochemical and cellular processes (Tully, 1994). Studies in insects, in particular the honeybee (Hammer, 1995) and Drosophila, have been one of the main stimuli for reaching this conclusion. For a discussion of the multiple memory systems in mammals, see CaM Kinase II: Evolutionary homologs: CaM kinase II and learning and behavior. Studies in humans reveal that selective effects on a single component of memory have been correlated with damage to particular brain regions, such that short- and long-term memory can function independently of one another. In some cases, an affected individual cannot repeat a string of three items immediately after presentation but may nonetheless perform normally in tests for 24 hour memory. In this situation, short-term memory is not a prerequisite for long-term memory (Squire, 1992 and references).

Mutants for genes involved in memory in Drosophila (linotte, latheo, dunce, rutabaga, amnesiac, Protein kinase A, dCREB2, and radish) produce specific memory defects that are classified by mutant phenotype. linotte and latheo produce a defect in the acquisition phase (learning) of memory formation while subsequent memory formation is normal. Dunce and Rutabaga, both involved in the cyclic AMP cascade, are involved in short term memory that decays within the course of hours. Mutants for amnesiac and PKA are defective in medium term memory, which declines over the course of a day or two. dCREB2 and radish are involved in memory of longer duration (long term memory and anesthesia resistent memory). These are memory components that last for at least a day (Greenspan, 1995 and references).

Since mutants in PKC are not available in Drosophila, a biochemical genetic trick is performed to study the role of PKC in neural growth and fly memory. The inactive form of PKC results from the presence of an autoinhibitory domain that occupies the catalytic site. Flies were made homozygous for a transgene that expresses a protein fragment consisting of a specific pseudosubstrate inhibitor of PKC. The transgene pseudosubstrate inhibitor is derived from PKC from the fly eye. Controlling the PKC inhibitor by means of heat shock circumvents any possibility of developmental effects such as those that may occur in knockout mutants. This strategy also has the advantage of avoiding the non-specific effects of drugs. Following heat shock, flies express the pseudosubstrate inhibitor at high levels. This system was used to study the role of PKC in the growth of cultured, differentiating neuroblasts. Cell cultures expressing a low concentration of the pseudosubstrate inhibitor show an alteration in neurite growth. Such cultures exhibit a significant decrease in the number of neural processes as compared to control cultures.

Drosophila larvae form two types of neuronal processes. Type I processes have large buttons with few branches, and type II processes are thinner and beaded, with more branches. Both type I and type II processes are diminished in vitro in cells from flies expressing the pseudosubstrate inhibitor. In in vitro neuronal cell clusters the proportional amounts of neuroblasts, ganglion mother cells (GMCs), and mature neurons are little affected. It is concluded that endogenously inhibited protein kinase C in transgenic Drosophila embryonic neuroblasts down regulates the outgrowth of type I and II processes of cultured mature neurons (Broughton, 1996a). The effect of PKC on development of neural processes in cultured cells is likely to be due to modification of cytoskeletal targets by PKC. Another enzyme, CaM kinase II, evokes an inhibition of neural arborization and outgrowth in Drosophila cultured neurons. CaM kinase II is transported along processes after neuronal contact has been made, at a time when functional synapses might be forming (Broughton, 1996b)

A cytoskeletal target for PKC has been identified in mammals. Dynamin is a microtubule-binding protein with a microtubule activating GTPase activity. The gene encoding Dynamin is mutated in shibire, a Drosophila mutant defective in endocytosis in nerve terminals and other cells. Mammalian dynamin is phosphorylated by Protein kinase C and is dephosphorylated upon neural excitation. The GTPase activity of dynamin is enhanced 12-fold when phosphorylated by PKC. The dynamins are therefore a group of nerve terminal phosphorproteins whose GTPase is regulated by phosphorylation in parallel with synaptic vesicle recycling. The regulation of dynamin GTPase could serve as a trigger for the rapid endocytosis of synaptic vesicles after exocytosis (Robinson, 1993).

As PKC modifies neural process formation in Drosophila, it might also act to modify components of learning and memory. A test for such a role has been made in flies expressing the PKC pseudosubstrate. The effects of heat-inducible PKC inhibitor on courtship conditioning (an associative conditioning paradigm) were examined. Normally, a male's courtship activity is suppressed after exposure to a mated female. (For a more detailed description of this study see Calcium/calmodulin dependent protein kinase II, Biological overview). Flies mutant for PKC show an alteration in performance, that is, they fail to show immediate suppression of courtship, a trait exhibited by normal flies. Mutant flies nevertheless develop a normal memory of training episodes. In other words, they remember and forget no differently from controls (Kane, 1997).

The behavior of transgenic flies shows that disrupting PKC function dissociates immediate performance from ultimate retention and decay rate of conditioned memory. In so doing, it demonstrates that immediate performance, at least for this form of learning, occurs in parallel with memory formation, not as a precursor. These results suggest a mechanism in which a common stimulus, the mated female, triggers separate events: a PKC-dependent process that feeds back immediately on the animal's behavior and one or more separate processes that lead to memory formation. The existence of such a mechanism supports the idea that distinct biochemical mechanisms are needed, each with its own characteristic time course, to fill all of the gaps from the organism's first experience of an effective conditioning stimulus until the consolidation of its most durable memory. No single, unitary mechanism appears to be able to be able to span all of these intervals (Kane, 1997).

A similar result has been obtained in the nudibranch mollusk Hermissenda (Crow, 1993). Blockade of PKC in Hermissenda selectively affects the in vitro expression of short-term conductance changes associated with learning, but has no effect on those that are long-term. Similarly, in a physiological preparation of Aplysia, long-term synaptic facilitation can be set up in the absence of short-term facilitation after pharmacological blockade of one type of serotonin (Emptage, 1993) thought to activate PKC (Sugita, 1992). Moreover, the time course for the development of long-term facilitation, which is diagnostic of the normal duration of short-term facilitation, resembles the time course seen in the Drosophila study for the PKC-mediated effect on performance (Kane, 1997).

Given the pleiotropy of PKC (the fact that it affects many pathways) it is not obvious how to account for the apparent specificity of the behavioral defect in flies. One would expect that interference with PKC, implicated in neural process formation, would interfere with long term memory, thought to involve alterations in neural connectivity. However PKC is involved in early learning processes and not long term memory. The failure of PKC-inhibited flies to manifest expression of immediate conditioning can be interpreted as a transient failure of retrieval in the earliest stages of memory (Kane, 1997). These flies are truely LD, not "learning disabled" but those that may be said to "learn differently."

Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca²⁺ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn⁹⁷ mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn⁹⁷ mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn⁹⁷ mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut¹, rut²⁰⁸⁰ and dnc¹ mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

REGULATION

RNAi screening for kinases and phosphatases identifies PKC53E as a FoxO activator

FoxO transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. This study reports a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays several enzymes were identified that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of the approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3beta, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. These results contribute to a comprehensive understanding of FoxO regulatory processes (Mattila, 2008).

By using a combination of transcriptional reporter and localization assays, twenty one dFoxO regulators were discovered. Some positive hits from the screen had an effect in dFoxO activity, localization, and protein stability, whereas other hits affected only transcriptional activity, suggesting that more mechanisms beyond subcellular localization and degradation are used by cells to regulate dFoxO activity. In addition to the 18 proteins that affected dFoxO transcriptional activity, the screening produced three more hits. Two of them seem to affect only dFoxO localization (dgkd and ptp69d), and one, neurospecific receptor kinase (nrk), affected exclusively dFoxO protein stability. It is possible that these proteins regulate dFoxO transcription on specific promoters in conjunction with other activators and that such factors are missing in Drosophila S2 cells. This would explain their lack of effect on the dInR promoter. Alternatively, they could affect dFoxO stability resulting in a net effect of dFoxO protein accumulation in the nucleus (Mattila, 2008).

Initially, the screening strategy was designed to identify both positive and negative regulators of dFoxO activity; however, no dFoxO repressors were found. Putative dFoxO repressors were present in the primary hit list of 31 targets, but those were later excluded in the secondary screen. This surprising observation suggests that the screen may be biased against dFoxO repressors. dFoxO is a well known inhibitor of protein biosynthesis in vivo, so under conditions of increased dFoxO activity, a reduction of general translation is expected that could affect GFP and luciferase translation too. Therefore, it is hypothesized that in the case of enhanced dFoxO activity it is possible that the concomitant inhibition of protein biosynthesis overruled a slight increase in reporter accumulation. This would explain the lack of dFoxO repressors among the targets of the screen. Moreover, the design of the screening based on S2 cells excludes the identification of regulatory mechanisms specific for other cell types, and instances where dFoxO is acting as a cofactor thereby regulating transcription indirectly (Mattila, 2008).

The results demonstrate that Drosophila PKC53E isoform is a dFoxO activator. Similar results were obtained in mammalian cells pointing out that the interaction is conserved. PKC isoforms have very important roles in insulin signaling, and each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Importantly, PKC isoforms can both activate or inhibit insulin signaling: Atypical PKC isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. In contrast, certain conventional and novel PKC isoforms are known to antagonize insulin signaling in vertebrates. This interaction is implicated in the pathogenesis of free fatty acid mediated insulin resistance. Drosophila possesses six PKC isoforms whose role in this context has not yet been addressed. PKC53E homolog is closest to human conventional PKCα. Interestingly, it has been shown that PKCα inhibits insulin signaling through binding and phosphorylation of IRS1. Thus, PKCα would serve as a constitutively active inhibitory regulator of the insulin cascade through its association with IRS1. On stimulation with insulin, PKCα would dissociate from IRS1, thus releasing this protein from its down-regulated state. This would open the 'gate' for transmission of the insulin signal. It has been found that dFoxO/FOXO1 increases insulin sensitivity by up-regulating insulin receptor transcription. The observation that Drosophila PKCα activates dFoxO adds an additional twist in the complex regulatory network that dFoxO has on insulin signaling. Interestingly, in the experimental system used in this study AKT dependent dFoxO bandshift and AKT Ser-505 phosphorylation was not affected by PKC53E, indicating that PKC53E regulation of dFoxO is independent of AKT signaling (Mattila, 2008).

Another well known enzyme implicated in the control of metabolism identified as a regulator of dFoxO transcriptional activity is the Drosophila ortholog of Glycogen synthase kinase 3β (GSK-3β, Shaggy). GSK-3β is a regulator of glucose metabolism through the phosphorylation and inhibition of glycogen synthase, the rate limiting enzyme of glycogen deposition. GSK-3β is inhibited by AKT, so it was not surprising to see that GSK-3β activates dFoxO. GSK-3β protein level and activity is elevated in type II diabetic skeletal muscle cells reflecting the impairment of whole body glucose uptake characteristic to this disease. In addition, selective inhibition of GSK-3β by lithium chloride represses the expression of g6pase and pepck in rat hepatoma cells, both known targets of FoxO. Taken together, these observations suggest that some of the metabolic effects of GSK-3β are achieved by directly modulating dFoxO activity (Mattila, 2008).

An interesting dFoxO regulator is Polo-like kinase. Polo-like kinases (Plks) are known regulators of cell cycle progression. In addition, Plks have a role in the protection against cellular stress through the transcription factor HSF1. Recently it was proposed that an intricate tradeoff between lifespan and cancer results from opposing effects of enzymes regulating FoxO and p53 activity. Plks are known to inhibit p53 transcriptional activity, so the results raise the possibility that Plks mediate the common but opposing regulators of p53 and FoxO. Interestingly, FoxOs are necessary in the completion of the cell cycle, which is partly mediated by cell cycle dependent activation of Plk expression by FOXO3a. The results show that Drosophila dFoxO is regulated by Polo, suggesting the existence of a positive feedback mechanism that has a role in achieving periodic M-phase gene expression and proper cell cycle exit (Mattila, 2008).

dFoxO localization was affected by eight modulators; however, band shifts demonstrated that none of these proteins phosphorylated dFoxO in the three conserved Ser/Thr amino acids known to regulate nuclear/cytoplasmic status through AKT. This observation raises the possibility that some of the newly identified dFoxO regulators could affect dFoxO nuclear/cytoplasmic localization by phosphorylating dFoxO in additional residues that do not alter its electrophoretic mobility, or that dFoxO regulation by these proteins is indirect. Further studies will be needed to clarify this point (Mattila, 2008).

In summary, this study has identified 21 dFoxO modulators. The results underscore the complexity underlying dFoxO regulation and establish dFoxO as a transcription factor controlled exquisitely by an intricate network of kinases and phosphatases achieving a perfect balance of activity. This balance ensures the correct execution of key cellular processes in metabolism, response to stress, and life span (Mattila, 2008).

Protein Interactions

The Drosophila mutant turnip was initially isolated based on poor learning performance. turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. These results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila (Choi, 1991).

Activation of the Drosophila visual cascade is extremely rapid and results in opening of the cation influx channels transient receptor potential (TRP) and transient receptor potential-like (TRPL) within ~10-20 msec of photostimulation of rhodopsin. The G-protein-signaling cascade that leads to opening of the ion channels has been extensively characterized and is known to involve the inositol phospholipid-signaling system. Termination of the photoresponse, after cessation of the light stimulus, is also rapid and is a Ca2+-regulated process; however, understanding of the mechanism by which Ca2+ contributes to termination of the photoresponse is quite incomplete (Li, 1998 and references).

Several proteins have been identified that seem to mediate Ca2+-dependent termination of phototransduction. These include the Ca2+-binding regulatory protein Calmodulin, which functions in both light adaptation and termination of the light response. The NinaC (neither inactivation nor afterpotential C) locus, which encodes two isoforms, p132 and p174, each of which consists of a protein kinase domain fused to a myosin head domain, also functions in negative feedback regulation of the photoresponse. The two NINAC proteins differ because of unique C-terminal ends. p174 is localized exclusively to the microvillar portion of the photoreceptors, the rhabdomeres, and p132 is restricted to the cell bodies. Null mutations in ninaC cause defects in adaptation and response termination. These functions are caused by p174 because elimination of p174, but not p132, causes each of these phenotypes. Because negative feedback regulation seems to be mediated by Ca2+, it is plausible that p174 is regulated by Ca2+. However, p174 does not contain a known Ca2+-binding motif, such as an EF hand or C2 domain, and there is no evidence that it binds Ca2+ directly. Thus, p174 seems to respond to the light-dependent Ca2+ flux indirectly. One NINAC Ca2+ sensor is Calmodulin because NINAC binds to Calmodulin and the NINAC-Calmodulin interaction is required for both adaptation and termination. NINAC might also be regulated by Ca2+-dependent phosphorylation because p174 contains multiple protein kinase C (PKC) consensus sites including several in its unique C-terminal tail. Moreover, mutation of an eye-specific PKC (ePKC) causes perturbations in adaptation and termination. The role of PKC in negative feedback regulation may be more significant than that indicated by mutation of ePKC because a second PKC, brain PKC (brPKC), is known to be enriched in the Drosophila retina and a third PKC, PKC98F, is highly expressed in adult heads. Two retinal substrates for PKC have been identified. These are the TRP cation influx channel and the PSD95, DLG, and ZO-1 (PDZ)-containing protein inactivation, no afterpotential D (INAD), which binds to most of the proteins that function in phototransduction and organizes a supramolecular signaling complex. However, the consequences of disrupting PKC phosphorylation of any retinal substrate that functions in Drosophila vision have not been determined (Li, 1998 and references).

The current work shows that NINAC p174, which consists of a protein kinase domain joined to the head region of myosin heavy chain, is a phosphoprotein and is phosphorylated in vitro by PKC. Mutation of either of two PKC sites in the p174 tail results in an unusual defect in deactivation that has not been detected previously for other ninaC alleles or other loci. After cessation of the light stimulus, there appeared to be a transient reactivation of the visual cascade. This phenotype suggests that a mechanism exists to prevent reactivation of the visual cascade and that p174 participates in this process. The termination mechanisms controlling Drosophila phototransduction seem to be more complicated than previously envisioned. In addition to a requirement for NINAC in facilitating rapid deactivation after cessation of the light stimulus, there is an additional requirement for this unconventional myosin in preventing transient reactivation of the plasma membrane conductances. Because p174 also functions in adaptation, it seems that NINAC has a central role in many aspects of negative feedback regulation of the visual cascade. Recently, a homolog of NINAC has been identified in the mammalian retina (D. Hillman, A. Dose, and B. Burnside, personal communication to Li, 1998). Thus, it is intriguing to speculate that vertebrate NINAC also functions in negative feedback regulation and that an active mechanism may also exist in mammalian photoreceptor cells to ensure stable termination of phototransduction (Li, 1998).

Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, the Drosophila myosin-II tail expressed in Escherichia coli was purified and it was shown to be phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, it has been shown that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, transgenic flies were generated that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way (Su, 2001).

β-Adducin is required for stable assembly of new synapses and improved memory upon environmental enrichment

Learning is correlated with the assembly of new synapses, but the roles of synaptogenesis processes in memory are poorly understood. This study shows that mice lacking β-Adducin fail to assemble new synapses upon enhanced plasticity and exhibit diminished long-term hippocampal memory upon environmental enrichment. Enrichment enhanced the disassembly and assembly of dynamic subpopulations of synapses. Upon enrichment, stable assembly of new synapses depends on the presence of β-Adducin, disassembly involves β-Adducin phosphorylation through PKC, and both are required for augmented learning. In the absence of β-Adducin, enrichment still leads to an increase in spine structures, but the assembly of synapses at those spines is compromised. Virus-mediated re-expression of β-Adducin in hippocampal granule cells of β-Adducin^-/- mice rescues new synapse assembly and learning upon enrichment. These results provide evidence that synapse disassembly and the establishment of new synapses are both critically important for augmented long-term learning and memory upon environmental enrichment (Bednarek, 2011).

Phosphorylation via PKC regulates the function of the Drosophila odorant co-receptor

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the G^q protein signaling cascade has been shown to reduce the odor response. This study investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo, single sensillum electrophysiological recordings were combined with microinjection of agents affecting PLC and PKC function, and an altered response of olfactory sensory neurons (OSNs) to odorant stimulation was observed. Injection of the PLC inhibitor U73122 or the PKC inhibitor Go6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. It is concluded that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response (Sargsyan, 2011).

Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction

Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by Protein kinase C (PKC). The Drosophila adducin, Hu-li tai shao (Hts), has been shown to play a role in larval neuromuscular junction (NMJ) growth. This study finds that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), this study shows that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. Evidence is provided that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. It was also shown that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014: PubMed ID).

The Drosophila neuromuscular junction (NMJ) is the site of contact between motor neuron and muscle, and is stably maintained but remodelled during the growth and development of the fly. To permit these differing functions, the NMJ uses an actin- and spectrin-based cytoskeleton both pre- and post-synaptically, where a number of synaptic proteins modify the cytoskeleton dynamically. One such protein involved in the dynamic responses of the synapse to stimuli in vertebrates is the actin- and spectrin-binding protein adducin, a heteromeric protein composed of α, β and γ subunits that is widely expressed in many cell types including neurons and myocytes. The adducins are composed of a globular N-terminal head domain, a neck domain and a C-terminal myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain containing an RTPS-serine residue which is a major phosphorylation site for protein kinase C (PKC), as well as cAMP-dependent protein kinase (PKA). Phosphorylation of adducin in the MARCKS-homology domain inhibits adducin-mediated promotion of actin-spectrin interactions, resulting in cytoskeletal reorganization (Wang, 2014).

Multiple studies have demonstrated that the mammalian MARCKS protein, or more specifically its MARCKS effector domain, can bind to and sequester the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP₂), in artificial lipid vesicles. This interaction has been linked to the regulation of the actin cytoskeleton during the growth and branching of dendrites in rat brains, as well as the directed migration of bovine aortic endothelial cells in wound healing assays. Notably, it has been proposed that aberrant MARCKS regulation of PIP₂ signaling may be implicated in the formation of amyloid plaques in Alzheimer's disease. A recent study has also provided evidence that reduced hippocampal levels of MARCKS, and thus PIP₂, in mice contributes to age-related cognitive loss (Wang, 2014).

MARCKS binds to PIP₂ as the MARCKS effector domain carries basic residue clusters that can interact with acidic lipids in the inner leaflet of the cell membrane. By analogy to other MARCKS-homology domain-containing proteins, it is hypothesized that phosphorylation of adducin at the RTPS-serine may alter the electrostatic interaction between adducin and phosphoinositides, thus reversing the binding between them and causing translocation of adducin from the membrane to the cytosol. In this way, adducin might act as a molecular switch in its regulation of synaptic plasticity, with its localization at the synapse controlled by phosphorylation (Wang, 2014).

In Drosophila, orthologs of adducin are encoded by the hu-li tai shao (hts) locus, and the Hts protein is present at both the pre- and post-synaptic sides of the larval NMJ where it regulates synaptic development. Previous studies have shown that Hts interacts with the scaffolding protein Discs large (Dlg), and regulates Dlg localization at the postsynaptic membrane by promoting its phosphorylation through Partitioning-defective 1 (PAR-1) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), two known regulators of Dlg postsynaptic targeting. Dlg is an important regulator of synaptic plasticity, and likely constitutes a major route by which Hts controls NMJ development. This study found that the main isoforms of Hts at the NMJ are the MARCKS-homology domain-containing isoforms, Add1 and/or Add2. There, the adducin-like isoforms form complexes with Dlg and PIP₂, interactions that were identified through Proximity Ligation Assay (PLA). Evidence is provided that Hts promotes the phosphorylation, and thus delocalization, of Dlg at the postsynaptic membrane by regulating the re-distribution of par-1 and camkII transcripts from the muscle nucleus to the cytoplasm. It was also shown that these Hts interactions with Dlg and PIP₂ are impeded through phosphorylation of the MARCKS-homology domain, further establishing that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014).

Through the use of PLA, this study has shown that Hts forms complexes with Dlg and PIP₂ at the postsynaptic region of the larval NMJ, with its ability to associate with these proteins being negatively regulated through phosphorylation of the MARCKS-homology domain. Studies on mammalian adducin have demonstrated that phosphorylation of the MARCKS-homology domain impedes its actin-binding and spectrin-recruiting functions, reduces its affinity for these cytoskeletal components and the membrane, and targets it for proteolysis. It is proposed that phosphorylation of the MARCKS-homology domain in the Add1/Add2 isoforms of Hts in response to upstream signaling events at the synapse reduces their affinity for spectrin-actin junctions and Dlg at the NMJ, but may also hinder their interactions with PIP₂ and other phosphoinositides in line with the electrostatic switch model for phosphoinositide binding by the MARCKS-homology domain (Wang, 2014).

It was proposed previously that Hts regulates Dlg localization at the NMJ by controlling the protein levels of PAR-1 and CaMKII, which phosphorylate Dlg and disrupt its postsynaptic targeting. This study now shows that regulation of these kinases appears to occur at the level of transcript processing, with Hts promoting the accumulation of par-1 and camkII transcripts in the muscle cytoplasm. Cytoplasmic accumulation of the transcripts would then presumably lead to higher levels of PAR-1 and CaMKII protein. How is Hts achieving this mode of regulation when it is residing with Dlg at the postsynaptic membrane? One possibility is that Hts at the NMJ is activating a signaling pathway that promotes the transcription and/or stability of par-1 and camkII transcripts, as well as their transport out of the nucleus. Another possibility is that Hts itself, which contains predicted NLS and NES sequences, translocates to the nucleus in response to events at the NMJ, similar to the way that mammalian α-adducin translocates to the nucleus upon loss of cell-cell adhesion in epithelia. This study was unable to detect endogenous Hts in muscle nuclei, however, nuclear Hts levels might be tightly restricted and undetectable under wild-type conditions. Over-expressed wild-type Hts, on the other hand, is readily observable in the nucleus, though not its phosphorylated form - a result also seen with α-adducin. Whatever the mechanism may be, the presence of Hts in a complex with Dlg may allow it to evaluate the status of Dlg and the synapse, and execute a response in the form of regulating Dlg localization through PAR-1 and CaMKII mediated phosphorylation (Wang, 2014).

A recent study has uncovered a novel nuclear envelope budding mechanism that can export select transcripts from muscle nuclei during larval NMJ development, and involves Lamin C (LamC) and the Wnt receptor, DFrizzled2 (DFzz2) (Speese, 2012). Interestingly, camkII, but not dlg, transcripts are regulated by this process, which is consistent with the findings that CaMKII, but not Dlg, expression is regulated by Hts. Future work will determine whether Hts is involved in this LamC/DFzz2-dependent mechanism (Wang, 2014).

Two papers have underscored the importance of phosphoinositides in synaptic development at the Drosophila NMJ (Forrest, 2013; Khuong, 2010). Binding of Hts to PIP₂ and probably other phosphoinositides at the NMJ, as seen with other MARCKS-homology domain-containing proteins, may affect the availability of these lipids for processes such as signal transduction, thus affecting synaptic development. Conversely, the localization of Hts at the NMJ may be regulated by the distribution of phosphoinositides. In line with this, postsynaptic knockdown of the phosphoinositide phosphatase Sac1 via transgenic RNAi expression disrupts Hts localization at the NMJ (Wang, 2014).

The observations reported in this study may have important implications for understanding diseases that affect synaptic function in humans and other mammals. Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a disorder characterized by the progressive loss of motor neurons, have been assumed until recently to be a consequence of neuronal death within the central nervous system. However, there is substantial recent evidence indicating that neuron pathology in ALS and other neurodegenerative diseases is due to a degenerative process that begins in the presynaptic terminal, NMJ or distal axon. This may also be the case in normal aging (Wang, 2014).

Initial interested in adducin arose when elevated levels of phospho-adducin protein was found in the spinal cord tissue of patients who died with ALS, compared to individuals who died without neurological disease. Similar observations were also made in mSOD-expressing mice, a transgenic animal model of ALS. Multiple studies have shown that adducin plays important roles in synaptic plasticity, and that mice mutant for β-adducin display defects in memory, learning and motor coordination. It is clear that modulation of Hts expression and phosphorylation can affect synaptic development. This study provides evidence here that phosphorylation of Hts impedes its function at the larval NMJ, a result that is consistent with the mammalian adducins. In addition, overexpression of phospho-mimetic Hts has dominant negative effects over endogenous Hts. Thus, loss of adducin function through aberrant phosphorylation of the MARCKS-homology domain may be a contributing factor for human neurodegenerative diseases (Wang, 2014).

DEVELOPMENTAL BIOLOGY

Each of the three PKC genes is expressed in the Drosophila adult head but transcripts are not detected in the body. The dPKC98F gene is expressed throughout development, with a lower level of expression in embryonic flies. The dPKC98F gene 5.5 kb transcript is greatly reduced in embryos and correlates with the increased expression of two additional transcripts of 4.3 and 4.5 kb. The dPKC98F gene is the only Drosophila PKC that is transcribed during embryonic stages. dPKC53E(brain) is transcribed in all head neural tissue. In contrast inactivation no afterpotential C or dPKC53E(ey) is specifically expressed in photoreceptor cells, both in the compound eyes and ocelli (Schaeffer, 1989).

A role for Drosophila Wnt-4 in heart development - Is PKC involved?

In vertebrates, different Wnt signaling pathways are required in a temporally coordinated manner to promote cardiogenesis. In Drosophila, wingless holds an essential role in heart development. Among the known Drosophila Wnts is DWnt4, the function of which has been studied in various developmental processes except for heart development. This study re-evaluated the expression pattern of DWnt4 during embryogenesis and showed that transcripts are not restricted to the dorsal ectoderm but are also present in the cardiogenic mesoderm. Moreover, DWnt4 mRNA transcripts were detected in myocardial cells by stage 16. The heart phenotype in DWnt4 mutant embryos is characterized by various degrees of disrupted expression of different cardiac markers. Overexpression of Dwnt4 also affects heart marker expression, which can be partially rescued by simultaneous inhibition of PKC. These data reveal a role for DWnt4 in cardiogenesis, however integration of DWnt4 with other known signaling pathways that function in heart development still awaits further investigation (Tauc, 2012).

Previously published data indicate that DWnt4 expression in the visceral mesoderm is regulated by Hox genes, in particular by Ultrabithorax (Ubx) and abdominal A (abd-A). Four Hox genes, Antennapedia (Antp), Ubx, abd-A and abd-B are expressed in the Drosophila heart where they specify different regions along the anterior-posterior axis of the heart tube. Therefore, it may be that members of the Hox gene family regulate DWnt4 expression also in the heart tube. Ubx is expressed in the aorta where low levels of DWnt4 mRNA expression and the higher expression levels of Dwnt4 in the heart proper correlate with the expression of Abd-A. The strong accumulation of DWnt4 transcripts in the heart proper is detected at what appears to be the border between high-level Abd-A and Abd-B expression. Ubx and abd-A were also shown to be involved in the establishment and patterning of alary muscles that project from the dorsal vessel. There are seven pairs of alary muscles that attach the heart to the dorsal epidermis in larvae and in adult flies. The extracellular matrix marker Pericardin (Prc) is not only expressed around pericardial cells and in the basal membrane of myocardial cells but also accumulates along the alary muscles. It was noticed that in DWnt4^EMS23 mutants the number of Prc positive projections is affected. Although the phenotype was not quantified, it was observed that the number of projections varied. For example additional Prc positive projections were seen at positions different from where the seven pairs of alary muscles normally attach. Not much is known about the embryonic origin of the alary muscles and the molecules required for their development. The current data may spur investigations on the role of DWnt4 in the development of these muscles. It is intriguing to hypothesize that DWnt4 acts as a guidance cue (attractive or repulsive) for the alary muscle attachment site. A guidance function for DWnt4 has been previously described in the context of dorsoventral projections of retinal axons, of motor neuron target specificity and in salivary gland migration (Tauc, 2012).

The early expression pattern of DWnt4 in the cardiac mesoderm and in the overlying ectoderm suggested that DWnt4 could be involved in early steps of cardiogenesis such as cardiac specification and differentiation of cardiac cell types. All cardiac marker genes that were analyzed in DWnt4^EMS23 mutant embryos exhibited a range of degrees of disruption, none of which had serious detrimental effects though. Hence, in contrast to wg, DWnt4 does not appear to be essential for Drosophila cardiogenesis. Nevertheless, DWnt4 does play a role to ensure normal cardiac marker gene expression. Whereas in DWnt4 mutants a mild loss of Svp positive cells (or only Svp expression) was observed, DWnt4 overexpression resulted in the loss and increase of Svp expressing cells. It has been suggested that DWnt4 modulates cell fate specification within the Hedgehog-dependent domain and hedgehog was shown to regulate Svp expression. Hence, it is intriguing to speculate that the Svp phenotype is caused by defective Hh signaling that results from inappropriate amounts of DWnt4. Next attempts were made to investigate which components may mediate the DWnt4 signal. Immunostainings for Prc revealed two phenotypes in DWnt4 mutants. Embryos were characterized by gaps in Prc expression along the heart tube and/or by a detachment of Prc expressing cells from the Prc positive basal membrane, which indicates the detachment of pericardial cells from myocardial cells. These phenotypes are reminiscent of the phenotypes described for embryos that are mutant for the α-subunit of the heterotrimeric Go protein bkh. Gαo was shown to couple to the seven transmembrane Fz receptors and mediate Wnt signaling as well as planar polarity signaling. fz mutants, like bkh mutants, exhibit both phenotypes: gaps in Prc and a detachment of pericardial cells from myocardial cells. Of note, DWnt4 was shown to be able to bind to three Fz receptors Fz, Fz2 and Fz4. Unlike Fz2, which was shown to solely activate the arm-dependent Wg signaling pathway, Fz can also mediate a non-canonical, planar polarity signal (Tauc, 2012).

Since similar phenotypes were observed for Prc in DWnt4, fz and bkh mutants tests were performed to see whether fz and bkh may be components of the DWnt4 signaling pathway. The rationale was that if these molecules act in the same pathway, an increase of severity and/or penetrance of the phenotype in would be expected double heterozygous embryos. The results do not support such a simple linear relationship with respect to Prc expression along the heart tube. Nevertheless, changes were observed in the number of embryos showing a particular phenotype, which suggests that these factors could be genetically interacting. Due to the complexity of the data, a straight-forward interpretation is somewhat difficult at this point. Reasons for such phenotypic changes could be due to the involvement of different molecular mechanisms underlying either the gap or detachment phenotype. For example, gaps in Prc expression could result from a defective mesenchymal-epithelial transition required for proper heart morphogenesis as was shown for bkh mutants. One cause for the detachment phenotype is a misregulation of septate junction proteins present in myocardial and pericardial cells where bkh is also involved. It is feasible that DWnt4 may function as a guidance cue for the proper migration of mesodermal cells. Irregularities in mesoderm migration can also lead to heart defects similar to the ones seen in DWnt4 mutants, which was shown for example in embryos mutant for the FGF receptor heartless or for the proteoglycan syndecan (Tauc, 2012).

In addition to Prc possible genetic interactions between DWnt4 and fz or DWnt4 and bkh were examined with respect to the reduction in Odd expressing pericardial cells that was detected in all single mutants. This analysis indeed revealed that the phenotype increased in embryos that were double heterozygous for DWnt4 and bkh compared to embryos that were single heterozygous for each factor (Tauc, 2012).

As to a possible genetic interaction between DWnt4 and fz, the data is less convincing since the phenotype was already highly penetrant in fz/+ heterozygous embryos. Multiple publications have indicated that DWnt4 acts through a non-canonical, arm-independent Wnt pathway. Therefore whether JNK, a core component of the planar polarity pathway, may be part of the DWnt4 signal transduction machinery was tested. Since ectodermal JNK signaling is essential for the morphogenetic process of dorsal closure, which impinges on normal heart development as a secondary effect, the pathway was interrupted in a tissue-specific manner. Mesodermal inhibition of JNK signaling using the dominant-negative bsk construct did not elicit a heart phenotype. Mesodermal inhibition of the canonical Wg signaling pathway using a dominant-negative construct of TCF was detrimental to heart development as expected. These results exclude a primary function for JNK in cardiogenesis and due to the lack of resemblance to the phenotypes in DWnt4 mutants, it is unlikely that JNK is a component of the DWnt4 signaling pathway in this context. Although inhibition of the canonical Wg pathway has a much more dramatic effect on cardiogenesis than lack of DWnt4, the data still leaves the option that DWnt4 may also act through the canonical pathway. Despite several publications that indicate that DWnt4 activates a non-canonical pathway, Harris and Beckendorf (2007) concluded from their data that DWnt4 acts through a canonical Wnt pathway during salivary gland migration. PKCs were shown to mediate non-canonical Wnt signaling in vertebrates and were implicated in mediating the DWnt4 signal during ovarian morphogenesis. This study performed experiments to determine whether PKCs could function in the DWnt4 signaling pathway in heart development expressing a characterized PKC pseudosubstrate that inhibits all PKCs present in Drosophila. The results show that by inhibiting PKC signaling, the amount of embryos exhibiting a reduction in Svp and Odd expressing cells was decreased after the overexpression of DWnt4. Admittedly this is an indirect indication that PKC may mediate the DWnt4 signal but this piece of data together with previously published results encourages further investigation of this possibility (Tauc, 2012).

In summary, although the definite function of DWnt4 in cardiogenesis still awaits further investigation, the data provides a good platform for subsequent analyses of DWnt4 in heart development. In particular with respect to the newly described cardiac expression pattern of DWnt4, future results can be anticipated that demonstrate a function for DWnt4 in heart tube formation and heart function (Tauc, 2012).

PKC in the Adult: Function in Neurons

The calcium-dependent PKC purified from honeybee (Apis mellifera) brain shows remarkable similarities to the corresponding vertebrate PKC. Interestingly, the staining of honeybee brain with a polyclonal PKC antiserum reveals a strong, distinct labeling of the antennal lobes (ALs) and the mushroom bodies (MBs). ALs and MBs are both involved in olfactory learning in the honeybee. Local cooling experiments and local injections of octopamine into the ALs indicate that the AL may establish a memory trace independent of the MB. However, the ALs primarily process chemosensory information, and PKC immunoreactivity is located predominantly in the interneurons of the ALs. Sensory neurons projecting via the antennal nerve and innervating the rind areas of the glomeruli are stained very weakly. In contrast to the AL, the MB is involved in higher integrative functions and receives input from different sensory modalities. The MB shows high levels of PKC expression. Thus, because the ALs predominantly process olfactory information, the study concentrated on this structure of the honeybee brain to examine the role of PKC in associative olfactory learning (Grunbaum, 1998 and references).

To detect changes in PKC activity induced by in vivo stimulation, a fast phosphorylation assay was applied, using MARCKS as a PKC-specific substrate. MARCKS is phosphorylated specifically by PKC, but not by PKA or CaMII kinase from honeybee brain. MARCKS also is phosphorylated by the calcium-independent constitutively active PKC activity of PKM (generated by partial proteolysis of purified PKC). In brain homogenate that is depleted of PKC, MARCKS phosphorylation is below 10% of the phosphorylation in the original homogenate containing PKC. To confirm further the specific phosphorylation of MARCKS by PKC, the PKC inhibitor peptide was included in the phosphorylation assay. This PKC inhibitor peptide selectively inhibits MARCKS phosphorylation by PKC purified from honeybee brain without affecting PKA and CaMII kinase activity. In brain homogenates, it reduces MARCKS phosphorylation to below 10% of the phosphorylation in the absence of the inhibitor peptide. These data strongly confirm that MARCKS phosphorylation of honeybee brain homogenates is mediated exclusively by PKC (Grunbaum, 1998).

To discriminate between two different forms of PKC activation, (1) transient calcium-dependent activation and (2) constitutive calcium-independent activation, PKC activity was measured in either the presence or the absence of activators for classical PKC [calcium and diacylglycerol (DAG)]. All of the stimuli used for olfactory conditioning induce comparable transient PKC activation in the AL regardless of the stimuli and the sequence of application. A similar, although prolonged, transient activation also is induced by three forward or backward pairings with intertrial intervals (ITI) of 2 min. In the absence of PKC activators (calcium and DAG) in the in vitro assay, the PKC activity is reduced to <5% of the activity measured in the presence of the activators. This suggests that in vivo stimulation during conditioning causes a transient calcium-dependent activation of PKC in the AL (Grunbaum, 1998).

Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes. Multiple conditioning trials inducing a memory different from that induced by a single conditioning trial specifically cause an increase in PKC activity. This increase begins 1 hr after conditioning, lasts up to 3 d, and is attributable to an increased level of constitutive PKC. The increased level of constitutive PKC consists of an early proteolysis-dependent phase and a late phase that requires RNA and protein synthesis. Whereas constitutive activity is marginal in untrained animals (<5% of basal calcium-dependent PKC activity), three-trial conditioning produces an at least 600% increase in constitutive activity, as compared with that in untrained animals. This increase is similar at 3 and 18 hr after conditioning and corresponds to the learning-induced elevation of PKC activity measured in the presence of calcium and DAG. Hence, the constitutive PKC activity may be fully responsible for the long-lasting PKC activation induced by conditioning. Whereas the transient activation of PKC induced by single or paired stimuli is blocked in the presence of the PKC inhibitor Gö 7874, long-lasting activation 3 and 18 hr after conditioning is not susceptible to this inhibition. Thus, the calcium independence and the lack of inhibition by Gö 7874 support the hypothesis that long-lasting and transient PKC activation are mediated by different mechanisms (Grunbaum, 1998).

Because the direct detection of PKM is not possible, the in vivo effect of protease inhibitors on conditioning-induced PKC activation was investigated. The formation of PKM probably is mediated by the thiol protease calpain. Calpain homologs have been purified from honeybee brains and can be inhibited by the thiol protease inhibitor E 64. E 64 was injected before conditioning, because the induction of a proteolytic mechanism in LTP was described as taking place during or shortly after training. Elevation of PKC activity 3 hr after conditioning was blocked in E 64-treated bees, as compared with that in PBS-injected control animals. However, 18 hr after conditioning, PKC activation in E 64-treated bees was not distinguishable from that in PBS-injected controls. PKC activity in untrained animals was not changed by E 64. Hence, a proteolytic mechanism is required for the induction of PKC activation during the first hour after conditioning. To investigate the contribution of gene expression to PKC activation after conditioning, transcription and translation inhibitors were tested for their ability to interfere with PKC activation in vivo. It was assumed that RNA and protein synthesis are not required during, but after, training. Therefore, inhibitors of RNA (actinomycin D) and protein synthesis (a mixture of cycloheximide and anisomycin) were injected 1 hr after training. Actinomycin D does not interfere with PKC activation 3 hr after conditioning. However, 18 hr after conditioning, no activation of PKC was measured in bees treated with transcription or translation inhibitors. Taken together, these data suggest that the early phase of conditioning-induced PKC activation is dependent on proteolysis and is not required for the induction of the late phase of conditioning-induced PKC activation, which requires the synthesis of RNA and proteins. Thus Inhibition of the pathways resulting in constitutive PKC selectively impairs distinct phases of multiple-trial induced memory. The inhibition of the proteolytic mechanism has an instant effect on an early phase of multiple-trial induced memory but does not affect acquisition and the late phase of memory. Blocking of the transient PKC activation during conditioning does not affect the induction of memory formation. Thus, the constitutive PKC in the antennal lobe seems to contribute to the early phase of memory that is induced by multiple-trial conditioning (Grunbaum, 1998).

EVOLUTIONARY HOMOLOGS

Drosophila PKCs

Genomic and cDNA clones encoding a Drosophila protein kinase C (PKC) homolog were identified using a bovine PKC cDNA probe. The cDNA clones contain a single open reading frame that encodes a 639 amino acid, 75-kd protein having extensive homology with bovine, human and rat PKC and homology with the kinase domains of other serine, threonine and tyrosine kinases. The Drosophila PKC, Pkc53E, is localized to region 53E of chromosome 2. The gene spans approximately 20 kb and contains at least 14 exons. Messenger RNA for PKC could not be detected in 0-3 h Drosophila embryos. Adult flies contain three PKC transcripts of 4.3, 4.0 and 2.4 kb (Rosenthal, 1987).

Of two protein kinase C genes from D. melanogaster, one ( dPkc98F), maps to chromosome region 98F and displays over 60% amino acid sequence identity with members of a recently described "PKC-related" subfamily in mammals. The other, dPKC53E(ey), now known as inactivation no afterpotential C, maps to region 53E4-7 on the second chromosome and lies within 50 kb of the PKC gene previously characterized by A. Rosenthal (1987). While Pkc98E transcripts are expressed throughout development, expression of the two genes mapping at cytogenetic location 53E is primarily in adults. Pkc98E and the previously reported 53E gene are transcribed predominantly in brain tissue. In contrast, inactivation no afterpotential C [PKC53E(ey)] is transcribed only in photoreceptor cells (Schaeffer, 1989).

Drosophila eye specific PKC

Invertebrate photoreceptors use the inositol-lipid signaling cascade for phototransduction that involves the eye specific PKC. An excellent opportunity to investigate the function of PKC has been provided by the identification of an eye-specific PKC in Drosophila and a null PKC mutant, inaCP209. Bright conditioning lights delivered to inaC mutant photoreceptors lead to an abnormal loss of sensitivity in whole cell recordings from dissociated ommatidia; this has been interpreted as 'hyper-adaptation' and PKC's role has been suggested to be distinct from light adaptation. A presumably related finding is that during intense light, the response of inaC declines to baseline. Invertebrate photoreceptors use the phosphoinositide signaling cascade, responding to single photons with so-called quantum bumps that sum to form the macroscopic response to light. Light adaptation allows photoreceptors to adjust their sensitivity over the enormous range of ambient intensities. Although the molecular mechanism of light adaptation remains obscure, it is a negative-feedback process mediated by a rise in cytosolic calcium and a decrease in bump size. Under physiological conditions light adaptation is severely reduced in inaC, suggesting that eye-specific PKC, itself activated by a rise in cytosolic calcium and diacylglycerol, is required for adaptation. In the absence of PKC individual bumps fail to terminate normally, an effect that can account for the pleiotropic manifestations of the inaC phenotype (Hardie, 1993).

A useful approach to dissecting the inositol-lipid signaling cascade for phototransduction and its regulation has been provided by the isolation of Drosophila visual mutants. Extracellular changes of Ca2+ were measured in Drosophila retina using Ca(2+)-selective microelectrodes in both the transient receptor potential (trp) mutant, in which the calcium permeability of the light-sensitive channels is greatly diminished and in the inactivation-but-no-afterpotential C (inaC) mutant, which lacks photoreceptor-specific protein kinase C (PKC). Illumination induces a decrease in extracellular Ca2+ with kinetics and magnitude that changes with light intensity. Compared to wild-type, the light-induced decrease in the Ca2+ signal is diminished in the transient receptor potential (trp) channel mutant but significantly enhanced in inaC. The enhanced Ca2+ signal is diminished in the double mutant inaC;trp indicating that the effect of the trp mutation overrides the enhancement observed in the absence of eye-PKC. The decrease in Ca2+ signal may reflect light-induced Ca2+ influx into the photoreceptors; the trp mutation is thought to block a large fraction of this Ca2+ influx, while the absence of eye specific PKC leads to enhancement of light-induced Ca2+ influx. This suggestion was supported by Ca2+ measurements in isolated ommatidia loaded with the fluorescent Ca2+ indicator, Ca Green-5N, which indicated an approximately threefold larger light-induced increase in cellular Ca2+ in inaC relative to WT. These observations are consistent with the hypothesis that TRP is a light activated Ca2+ channel and that the increased Ca2+ influx observed in the absence of PKC is mediated mainly via the TRP channel (Perez, 1996).

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds calmodulin. Two calmodulin-binding sites are present in the C-terminal domain of the Trpl protein, CBS-1 and CBS-2. CBS-1 binds calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3-0.5 microM for calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 microM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+ (Warr, 1996).

Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light-sensitive channels activate spontaneously, generating a "rundown current" (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents that inactivate with increasing rapidity as Ca2+levels are raised. On depolarization inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild-type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition (12 ms with 1.5 mM external Ca2+ at -60 mV) is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induce transient currents that appear similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivate more rapidly, with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached (Hardie, 1994).

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains (see Drosophila Discs large 1) known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C (INAC) and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis the essential elements of the NORPA-INAD association have been defined and three critical residues in the C-terminal tail of NORPA, required for the interaction, have been identified. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD-NORPA association in vivo, transgenic flies were derived expressing a modified NORPA: NORPAC1094S, which lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation. Similarly, delayed activation may result from NORPAC1094S being unable to localize in close proximity to the TRP channel. It is concluded that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells (Shieh, 1997).

Yeast PKC

Protein kinase C, encoded by PKC1, regulates construction of the cell surface in vegetatively growing yeast cells. Pkc1 in part acts by regulating Mpk1, a MAP kinase. Mutants lacking Bck1, a component of the MAP kinase branch of the pathway, fail to respond normally to nitrogen starvation, which causes entry into quiescence. Given that the Tor1 and Tor2 proteins are key inhibitors of entry into quiescence, the Pkc1 pathway may regulate these proteins. pkc1Delta and mpk1Delta mutants rapidly die by cell lysis upon carbon or nitrogen starvation. The Pkc1 pathway does not regulate the TOR proteins: transcriptional changes dependent on inhibition of the TORs occur normally in pkc1Delta and mpk1Delta mutants when starved for nitrogen; pkc1Delta and mpk1Delta mutants die rapidly upon treatment with rapamycin, an inhibitor of the TORs. Mpk1 is transiently activated by rapamycin treatment via a novel mechanism. Finally, rapamycin treatment or nitrogen starvation induces resistance to the cell wall-digesting enzyme zymolyase by a Pkc1-dependent mechanism. Thus, the Pkc1 pathway is not a nutrient sensor but acts downstream of TOR inhibition to maintain cell integrity in quiescence (Krause, 2002).

Hydra PKC

Two different cDNA clones from Hydra (HvPKC1a and HvPKC1b) have been characterized that encode members of the cPKC family of protein kinase Cs (PKCs). The two predicted proteins differ only in their amino-terminal sequences and thus probably represent the products of alternatively spliced mRNAs from a single gene. In situ hybridization with a probe recognizing sequences in common between the two mRNAs detects HvPKC1 RNA in all parts of the adult polyp except the foot. The mRNA is contained in ecto- and endodermal epithelial cells as well as a certain subset of gland cells and pairs of interstitial cells. During head and foot formation, induced by either regeneration, budding, lithium treatment or repeated application of a diacylglycerol, HvPKC1 expression is upregulated immediately prior to the evagination of tentacles and downregulated by foot formation. Although PKC activity is clearly inducible in vitro by diacylglycerol and a tumour promoting phorbol ester, structural features detected in the regulatory domains of HvPKC1a and 1b indicate that endogenous activators for Hydra PKC might differ from those of other organisms. The results corroborate the hypothesis that signal transduction systems using protein kinase C are key elements controlling the formation of head structures in Hydra (Hassel, 1998a).

Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. A gene (HvPKC2) from Hydra vulgaris has been cloned that encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations: the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of membrane-bound PKC activity is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms (Hassel, 1998b).

PKC isoforms

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions. Not only may some PKC isoforms be active while others may not, for a given response, but the actions of different isoforms may even be antagonistic. In NIH 3T3 cells, for example, PKCdelta arrests cell growth whereas PKCepsilon stimulates it. To probe the contribution of the regulatory and the catalytic domains of PKC isozymes to isozyme-specific responses, chimeras were prepared between the regulatory and the catalytic domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in mouse fibroblasts. A major objective was to characterize the growth properties of the cells that overexpress the various PKC constructs. Both the regulatory and the catalytic domains play roles in cell proliferation. The regulatory domain of PKCepsilon enhances cell growth in the absence or presence of phorbol 12-myristate 13-acetate (PMA); in the presence of PMA, all chimeras with the PKCepsilon regulatory domain also give rise to colonies in soft agar. The role of the catalytic domain of PKCepsilon is evident in the PMA-treated cells that overexpress the PKC chimera containing the delta regulatory and the epsilon catalytic domains (PKCdelta/epsilon). The important contribution of the PKCepsilon catalytic domain to the growth of PKCdelta/epsilon-expressing cells is also evident in terms of a significantly increased saturation density in the presence of PMA, their formation of foci upon PMA treatment, and the induction of anchorage-independent growth. Aside from the growth-promoting effect of PKCepsilon, most chimeras with PKCalpha and -delta regulatory domains inhibit cell growth. These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC (Ács, 1997).

The implication of protein kinase C (PKC) isoforms cPKC-alpha, nPKC-epsilon, aPKC-lambda and aPKC-zeta in the transcriptional activation of a c-fos promoter-driven CAT-reporter construct by transforming Ha-Ras has been investigated. This was achieved by employing antisense constructs encoding RNA directed against isoform-specific 5' sequences of the corresponding mRNA, and expression of PKC mutants representing either kinase-defective, dominant negative, or constitutively active forms of the PKC isoforms. The data indicate that for the transcriptional activation of c-fos in HC11 mouse mammary epithelial cells, transforming Ha-Ras requires the activities of the three PKC isozymes: aPKC-lambda, nPKC-epsilon and aPKC-zeta, but not cPKC-alpha. Co-expression of oncogenic Ha-Ras with combinations of kinase-defective, dominant negative and constitutively active mutants of the various PKC isozymes are in agreement with a tentative model suggesting that in the signaling pathway from Ha-Ras to the c-fos promoter, aPKC-lambda acts upstream of nPKC-epsilon, whereas aPKC-zeta functions downstream of this isozyme (Kampfer, 1998).

Initial results reveal that the effects of the various PKC isotypes are, at least in part, mediated through the Raf-1/ERK pathway. As outlined above, aPKC-zeta acts downstream of the nPKC-epsilon and aPKC-lambda isotypes. Expression of the kinase-defective, dominant negative aPKC-zeta K/W mutant abrogates the transcriptional activation of c-fos by Raf-BxB or MEK-1, indicating that aPKC-zeta is implicated in signal transmission through ERK1,2 and acts somewhere downstream of MEK-1. Induction of c-fos by Raf-BxB or MEK-1 is not affected by expression of the dominant negative aPKC-lambda mutant, suggesting that aPKC-lambda acts upstream of Raf-1. However, the data available so far do not exclude an alternative scenario in which aPKC-lambda transmits signals independent of Raf-1 and MEK-1, perhaps via PI3 kinase or phospholipase D (PLD). As depletion or inhibition of aPKC-zeta blocks c-fos induction by a constitutively active aPKC-lambda mutant, this alternative pathway, if it exists, requires the cooperation of the zeta isotype. aPKC-lambda is thought to be required for the activation of nuclear ERKs. In any case, the exact function of aPKC-lambda still remains to be elucidated. Expression of the kinase-defective, DN nPKC-epsilon K436R mutant, which depresses c-fos induction by oncogenic Ras or (CA)aPKC-lambda A119E, inhibits transcriptional activation of the c-fos-CAT reporter by Raf-BxB or MEK-1, indicating that nPKC-epsilon is implicated in the ERK pathway. However, inhibition by (DN)nPKC-epsilon K436R of Raf-BxB or MEK-1 induced transcriptional activation of c-fos was incomplete and definitely weaker than the interference with the Ras- or (CA)aPKC-lambda A119E-stimulated c-fos induction. These results suggest that nPKC-epsilon may in addition to its effect downstream of MEK, also be involved in Raf-1 activation, or that nPKC-epsilon signals through Raf-1-/MEK-1-dependent and independent pathways. At this point it is satisfying to note that all three PKC isoforms that have been identified in these studies as required for the transmission of signals from Ha-Ras to the c-fos promoter have been previously described as downstream targets of Ha-Ras and regulators of the Ha-Ras-MAP kinase pathway. The novelty of this paper is the demonstration that, at least in the system studied here, a combination of all three isozymes is required; they appear to cooperate in a hierarchically ordered sequence (Kampfer, 1998 and references).

A novel effector of Rac and Cdc42, hPar-6, has been identified that is the human homolog of a cell-polarity determinant in C. elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKC, and forms a stable ternary complex with either Rac1 or Cdc42 and PKC. This association results in stimulation of PKC kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKC-dependent pathway distinct from the pathway mediated by Raf (Qui, 2000).

Many direct targets of Rac1 and Cdc42 have been identified, but none has been shown to have a direct role in cell transformation by Rac1 and Cdc42. hPar-6 is a novel effector of Rac1 and Cdc42 that promotes PKCzeta-dependent transformation by both GTPases. Although it has been suggested that PAK1 may also contribute to transformation by Rac1 in Rat1 fibroblasts, PAK1 does not enhance transformation by activated Raf or activated Rac1 in NIH-3T3 cells, and studies using effector domain mutants indicate that interaction of PAK1 with Rac1 does not correlate with cell-cycle progression or transformation. Thus, hPar-6 appears to be the first effector shown to directly mediate transformation by Rac1 and Cdc42. The identification of PKCzeta as a downstream effector of hPar-6 represents the first elucidation of a signaling pathway linking Rac1/Cdc42 to cell transformation. A model is presented depicting two separate pathways downstream of Ras that lead to cell polarity and growth control: these pathways can contribute to cell transformation. One pathway is comprised of Rac/Cdc42, hPar-6 and PKCzeta, and the other is mediated by Raf, MEK and MAP kinase (Qui, 2000).

The mechanism by which hPar-6 regulates the kinase activity of PKCzeta is currently under investigation. Subcellular targeting by interaction with specific proteins provides an attractive mechanism for PKC isozyme-specific regulation. It is possible that hPar-6 and PKCzeta are translocated by Rac1 or Cdc42 to the membrane, where PKCzeta could interact with an activator. One candidate activator is the phosphatidylinositol 3-kinase (PI3-kinase) target PDK1, since PDK1 and PKCzeta associate in vivo via their catalytic domains, and both PI3-kinase and PDK1 stimulate PKCzeta activity. Consistent with this model, it has been demonstrated that PI3-kinase can act as a link between Ras and Rac in transformation and that membrane-targeted PKCzeta is constitutively active. The observation that hPar-6 alone exhibits little, if any, transforming activity is also consistent with the membrane-targeting model. It should also be noted that although overexpression of hPar-6 alone (i.e., in the absence of Rac1[G12V]) is sufficient to activate PKCzeta kinase activity, overexpression of hPar-6 and PKCzeta only marginally promotes focus formation, suggesting that activated Rac1 is necessary to target PKCzeta to substrates involved in transformation. However, the possibility that Rac1 activates some other pathway that is also necessary for transformation cannot be ruled out. In addition to being activated by hPar-6, PKCzeta might in turn phosphorylate hPar-6. In this regard, it should be noted that there is a putative PKCzeta-phosphorylation site in mammalian Par-6 (Qui, 2000).

The mechanism underlying transformation by hPar-6 and PKCzeta is not yet clear. Stimulation of cell proliferation and inhibition of apoptosis are, however, important characteristics of cell transformation. In this regard, it has been shown that Rac1 and Cdc42 induce cyclin D1 transcription and accumulation, phosphorylation and inactivation of the tumor suppressor protein Rb, and activation of the transcription factor E2F. Inactivation of Rb may be necessary for Rac1/Cdc42 stimulation of cell proliferation, and it is possible that hPar-6 and PKCzeta have a role in this pathway. In addition, Ras, Rac1, Cdc42 and PKCzeta are all able to activate the transcription factor NF-kappaB. NF-kappaB activation is associated with mitogenesis, anti-apoptotic activity and cell transformation. Thus, the hPar-6-PKCzeta pathway might mediate NF-kappaB activation, and thereby contribute to cell transformation by Rac1 and Ras. Another possibility is that the hPar-6-PKCzeta pathway may mediate growth control by Rac1/Cdc42 by inducing downregulation of the pro-apoptotic protein Par-4 (prostate apoptosis response-4; unrelated to the C. elegans Par gene product). Par-4 interacts with PKCzeta and overexpression of PKCzeta downregulates Par-4, an event that appears important for Ras transformation and tumor progression. Thus, cyclin D1, Rb, E2F, NF-kappaB and Par-4 all warrant further investigation as possible downstream targets of the hPar-6-PKCzeta pathway (Qui, 2000).

Polarity is a fundamental feature of all eukaryotic cells. Rac, Cdc42, Par-6 and atypical PKCs appear to be conserved in diverse metazoans, including Drosophila, C. elegans, Xenopus, mouse and humans. The CRIB motif of Par-6 is also conserved, suggesting that it interacts with Rac and/or Cdc42 in these different species. In C. elegans, inhibition of Cdc42 function by RNA-mediated gene interference (RNAi) produces defects in cell polarity similar to those observed in par and pkc-3 mutants, while in mammalian cells, Par-6 is localized to tight junctions, together with atypical PKC and ASIP, the mammalian homolog of Par-3. Moreover in C. elegans, Par-6 interacts with Par-3, and in Drosophila the Par-3 homolog has an important role in the asymmetric cleavage of epithelial cells and neuroblasts. Taken together, these observations suggest that Rac or Cdc42, Par-6, atypical PKC, and perhaps Par-3, constitute a conserved pathway that regulates cell polarity. As hPar-6 and PKCzeta mediate cell transformation by Rac1 and Cdc42, there may be a link between cell-polarity signaling and growth control: aberrant cell-polarity signaling could lead to oncogenic transformation. In the light of the important roles of Rac1/Cdc42 in Ras-induced transformation, hPar-6 and PKCzeta could represent potential targets for anti-cancer therapeutics (Qui, 2000).

Selective erasure of distinct forms of long-term synaptic plasticity underlying different forms of memory in the same postsynaptic neuron

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, this study found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC; see Drosophila PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA (see Drosophila Kibra), which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories (Hu, 2017).

PKC targets

The c-kit-encoded tyrosine kinase receptor for stem cell factor (Kit/SCFR) is crucial for the development of hematopoietic cells, melanoblasts, and germ cells. Ligand stimulation of Kit/SCFR leads to receptor dimerization and autophosphorylation on tyrosine residues. Protein kinase C (PKC) acts in an SCF-stimulated negative feedback loop, which controls Kit/SCFR tyrosine kinase activity and modulates the cellular responses to SCF. Two serine residues in the kinase insert, Ser-741 and Ser-746, are PKC-dependent phosphorylation sites in vivo and account for all phosphorylation by PKC in vitro. Together they comprise more than 60% of the total SCF-stimulated receptor phosphorylation in living cells and 85-90% of its phosphorylation in resting cells. Two additional serine residues, Ser-821 (close to the major tyrosine autophosphorylation site in the kinase domain) and Ser-959 (in the carboxyl terminus) are SCF-stimulated PKC-dependent phosphorylation sites. However, they are not phosphorylated directly by PKC-alpha in vitro. Both the specific receptor tyrosine autophosphorylation and specific receptor-associated phosphatidylinositide 3'-kinase activity is approximately doubled in response to SCF in PAE cells stably expressing Kit/SCFR(S741A/S746A). The kinase activity of Kit/SCFR(S741A/S746A) toward an exogenous substrate is increased, which is reflected as a decreased Km and an increased Vmax, in accordance with the negative regulatory role of PKC on Kit/SCFR signaling (Blume-Jensen, 1995).

A cDNA clone isolated from a Xenopus embryo (stage 8 blastula) library, was predicted to encode a variant form of the type 1 fibroblast growth factor receptor (FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located within the juxtamembrane region. Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region demonstrates that this variant form arises from use of an alternative 5' splice donor site. RNase protection analysis revealed that both VT- and VT+ forms of the FGFR1 are expressed throughout embryonic development, VT+ being the major form. Amino acid position 424 is located within a consensus sequence for phosphorylation by a number of Ser/Thr kinases. A VT+ peptide is specifically phosphorylated by protein kinase C (PKC) in vitro, but not by protein kinase A (PKA). In contrast, the VT- peptide is not a substrate for either enzyme. Phosphorylation levels of in vitro synthesized FGFR-VT+ protein by PKC are twice that of FGFR-VT- protein. In a functional assay, Xenopus oocytes expressing FGFR-VT- or FGFR-VT+ protein are equally able to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However, pretreatment with phorbol ester significantly reduces this mobilization in oocytes expressing FGFR-VT+ while having little effect on oocytes expressing FGFR-VT-. These findings demonstrate that alternative splicing of Val423-Thr424 generates isoforms that differ in their ability to be regulated by phosphorylation; thus, alternative splicing represents an important mechanism for regulating FGFR activity (Gillespie, 1995).

Protein kinase C is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibit development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, is paralleled by a delay in the sorting of the tight-junction protein, Zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction is not detectably affected. ZO-1 is phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevents approximately 85% of the phosphorylation increase. In vitro measurements indicate that ZO-1 may be a direct target of PKC. Membrane-associated PKC activity more than doubles during junction assembly, and immunocytochemical analysis reveals a pool of PKC zeta that appears to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins is detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may well turn out to be occludin. Neither the mass of this complex nor the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were PKC dependent (Stuart, 1995).

Three desmoglein (Dsg) isoforms are expressed in a differentiation-specific fashion in the epidermis, with Dsg2 being basal, Dsg3 (pemphigus vulgaris antigen) basal and spinous, and Dsg1 (pemphigus foliaceus antigen) predominately granular. To better understand the mechanism(s) regulating Dsg isoform expression, the expression pattern of Dsg1, Dsg2, and Dsg3 were examined in normal human epidermal keratinocytes (NHEKs), the immortalized, nontumorigenic HaCaT cell line, and several squamous cell carcinoma cell lines (SCC-9, SCC-12F, SCC-13, and SCC-25). In all cells, the accumulation of high Dsg protein levels requires calcium and is not observed in low calcium (0.05-0.07 mM) media. NHEKs express Dsg1 in all media tested, consistent with their normal differentiation capacity. HaCaT and SCC-25 also express Dsg1; however, the presence of serum in the media dramatically decreases Dsg1 protein levels. Serum also inhibits Dsg1 mRNA levels in HaCaT cells. Dsg1 is not detected in extracts from SCC-9, SCC-12F, and SCC-13 under any conditions. Since activation of protein kinase C (PKC) is involved in keratinocyte differentiation, the effects of PKC down-regulation were evaluated on Dsg isoform expression. Long-term treatment with either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or bryostatin 1 inhibits levels of Dsg1 and Dsg3, but not Dsg2 in NHEKs and HaCaT cells. Chronic TPA also decreases Dsg1 and Dsg3 mRNA levels in NHEKs, further supporting a role for PKC activation in the expression of the suprabasal Dsg1 and Dsg3. These results identify several regulatory mechanisms by which the differentiation-specific pattern of desmosomal cadherins is established in the epidermis (Denning, 1998).

The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. Several invasive human colon carcinoma cell lines were studied that lack alpha-catenin. They showed a solitary and rounded morphotype that correlates with increased invasiveness. These round cell variants acquire a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, and also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations show that the cells start to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect is not associated with alpha-catenin induction. There are only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins are dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. TPA induces the appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion is E-cadherin dependent, as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibits the TPA effect. These studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin (See Drosophila Shotgun) remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin (van Hengel, 1997).

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin. Adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibits actin capping (as measured by using pyrene-actin polymerization) and it abolishes activity of adducin in recruiting spectrin to the ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increases two-fold in rat hippocampal slices, eight- to nine-fold in human embryonal kidney (HEK 293) cells, three-fold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin is no longer concentrated at the cell membrane at sites of cell-cell contact, and instead is distributed in a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibit increased levels of cytoplasmic spectrin, which colocalizes with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody reveals the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin are detected in the dendritic spines of cultured hippocampal neurons. Spectrin also is a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures (Matsuoka, 1998).

Activation of the protein kinase C (PKC) family with phorbol esters induces endothelial proliferation and angiogenesis, but which of the events that constitute angiogenesis are affected by individual members of the PKC family is unknown. In rat capillary endothelial (RCE) cells, serum stimulation increases expression of a single PKC isoenzyme, PKCtheta, and its translocation to the periphery. Conditional overexpression of a dominant-negative mutant of PKCtheta markedly inhibits RCE proliferation, as well as closure of a "wound" by RCE migration and formation of capillary rings and tubules in vitro. PKCtheta inhibition delays the endothelial cell cycle at the G2/M phase and prevents formation of actin stress fibers and filopodia but not lamellipodia. The defect in cell morphology and wound closure in PKCtheta-kn cells is reversed by overexpressing kinase-active PKCtheta, indicating that these RCE functions depend upon PKCtheta substrates. Thus, PKCtheta is required for multiple processes essential for angiogenesis and wound repair, including endothelial mitosis, maintenance of a normal actin cytoskeleton, and formation of an enclosed tube (Tang, 1997).

This study investigated the phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca2+ pump isoforms 2 and 3. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms are phosphorylated easily with PKC, isoform 2b is phosphorylated only a little, and isoform 3b is not phosphorylated at all. Phosphorylation of isoforms 2a and 3a does not affect their basal activity, but does prevent the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicates that phosphorylation prevents activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed (Enyedi, 1997).

In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium fails to stimulate the extracellular signal-regulated protein kinase pathway but is well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (See Drosophila JNK). In contrast, Ang II, which increases both protein kinase C (PKC) activity and intracellular calcium, stimulates extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. These findings suggest either that CADTK is not involved in JNK activation or that PKC activation inhibits the CADTK to JNK pathway. A 1-min phorbol pretreatment of GN4 cells inhibits thapsigargin-dependent JNK activation by 80-90%. The consequence of PKC-dependent JNK inhibition is reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induces c-Fos, produces a much greater and more prolonged c-Jun response than Ang II. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK and PKC; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal (Li, 1997).

A growing family of proteins is found to be regulated by protein kinase C and calmodulin through IQ domains. The IQ domain, a regulatory motif originally identified in neuromodulin EWS, a nuclear RNA-binding prooncoprotein that contains an IQ domain, is phosphorylated by protein kinase C, and interacts with calmodulin. Interestingly, PKC phosphorylation of EWS inhibits its binding to RNA homopolymers; conversely, RNA binding to EWS interferes with PKC phosphorylation. Several other RNA-binding proteins, including TLS/FUS and PSF, co-purify with EWS. PKC phosphorylation of these proteins also inhibits their binding to RNA in vitro. These data suggest that PKC may regulate the interactions of EWS and other RNA-binding proteins with their RNA targets and that IQ domains may provide a regulatory link between Ca2+ signal transduction pathways and RNA processing (Deloulme 1997).

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 has been thought to be Ras independent. The extracellular signal-regulated kinases (ERKs) are mitogen-activated protein kinases (MAPKs) that are activated by PKC. ERKs appear to mediate the effects of PKC on differentiation, secretion, proliferation, and hypertrophy. A study was undertaken to explore the role of Ras in transducing signals from PKC to the ERKs. Stimulation of PKC in COS cells leads to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras block activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC is not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases (Marais, 1998).

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions, such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) that phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study the possibility that different PKCs may control NF-kappaB through the activation of the IKKs is addressed. AlphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha. In contrast, the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. Cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation (Lallena, 1999).

Protein kinase C (PKC) alpha has been implicated in ß1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and ß1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha has been shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy shows an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. This is the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo (Ng, 2001).

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. Phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity (Firulli, 2003).

Activation of Protein kinase C

In Saccharomyces cerevisiae, the phosphatidylinositol kinase homolog Tor2 controls the cell-cycle-dependent organization of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2. Four Rho1 effectors are known: protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signaling protein Skn7. It has been suggested that Rho1 signals to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organization, however. The role of Rho1 in controlling actin organisation has been investigated and analysed as well as which of the Rho1 effectors mediates Tor2 signaling to the actin cytoskeleton was anayzed. Some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganized actin cytoskeleton. Both the growth defect and the actin organization defect of the rho1-2ts mutant are suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescues a tor2ts mutant, and deletion of BNI1 or SKN7 does not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppresses the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade (Helliwell, 1998).

To study protein kinase C (PKC) activation during sea urchin egg fertilization three different fluorescent probes specific for PKC were used: fim-1 (which recognizes the catalytic site of the enzyme), and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. PKC activation was followed during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increases and clusters in the cortical zone of the cell. The process is Ca2+ dependent and is inhibited in the presence of staurosporine, a PKC inhibitor. This initial phase of activation is followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization (de Barry, 1997).

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues. In protein kinase C betaII, this residue is Ser-660. Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position has no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor onthe conformational change accompanying membrane binding. In contrast, phosphate causes a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increases the protein's thermal stability and decreases its Km for ATP and peptide substrate. These data indicate that at the extreme carboxyl terminus of protein kinase C, the process of phosphorylation structures the active site so that it binds ATP and substrate with higher affinity and structural determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure (Edwards, 1997).

Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer is regulated by a single Ca(2+)-binding site. The dependence of PKC association with phosphatidylserine-containing membranes on the concentration of Ca2+ is linear in the submicro- to submillimolar range. The Ca(2+)-regulated association of PKC with the membrane is sensitive to the factors that alter the diffuse double-layer potential produced by anionic lipids such as phosphatidylserine (PS). This indicates that the Ca(2+)-binding site on the membrane-bound enzyme senses a higher concentration of Ca2+ than is present in bulk solution. This is a consequence of the accumulation of Ca2+ in the layer adjacent to the plane of the membrane by the double-layer potential. Calculations yield a unique value of the Ca2+ dissociation constant for the Ca(2+)-PKC-bilayer complex equal to approximately 700 nM. The soluble form of the enzyme has a 3.5 order of magnitude lower affinity for Ca2+. The free energy of interaction between the Ca(2+)- and PS-binding sites is large (approximately 5 kcal/mol). In contrast, the interaction between the diacylglycerol-binding site and either the Ca(2+)- or PS-binding site appears to be weak (Mosior, 1994).

The activation of protein kinase C involves sequestering the reaction components into membrane domains. The lateral membrane organization of phosphatidylserine, diacylglycerol, substrate, and Ca(2+)-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging microscopy. The formation of phosphatidylserine domains can be induced by either Ca2+, the MARCKS (myristoylated alanine-rich C kinase substrate) peptide, or protein kinase C. However, only Ca2+ can induce diacylglycerol to partition into the phosphatidylserine domains. In the complete protein kinase C assay mixture, two separate triple-labeling experiments demonstrate the colocalization of phosphatidylserine, protein kinase C, diacylglycerol, and the MARCKS peptide in domains. The amounts of all the labeled components in whole vesicles and in domains were measured at various concentrations of either phosphatidylserine, Ca2+, diacylglycerol, or the MARCKS peptide or with the addition of polylysine. The role of each component in forming membrane domains and in mediating the enzyme activity was analyzed. The results indicate that the inclusion of the MARCKS peptide in the domains, not just the binding of the substrate to vesicles, is especially important for PKC activity. The formation of PKC domains required the presence of DAG and Ca2+ at physiological ionic strength. The PKC activity is proportional to the amounts of PKC and substrate in the domains. The results also show that the MARCKS peptide leaves the domains after being phosphorylated. The efficiency of the reaction is greatly increased by concentrating the activators, the enzyme, and the substrate into domains (Yang, 1996).

The gamma-subspecies of protein kinase C (gamma-PKC) fused with green fluorescent protein (GFP) was expressed in various cell lines and the movement of this fusion protein was observed in living cells under a confocal laser scanning fluorescent microscope. gamma-PKC-GFP fusion protein had enzymological properties very similar to that of native gamma-PKC. The fluorescence of gamma-PKC- GFP is observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester but not by an inactive phorbol ester induces a significant translocation of gamma-PKC-GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induces a more rapid translocation of gamma-PKC-GFP than TPA. The A23187-induced translocation is abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of gamma-PKC-GFP is unidirected, while Ca2+ ionophore-induced translocation is reversible; that is, gamma-PKC-GFP translocates to the membrane returns to the cytosol and finally accumulates as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of gamma-PKC in translocation, mutant gamma-PKC-GFP fusion protein was expressed in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant is translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant is translocated by TPA but not by Ca2+ ionophore. To examine the translocation of gamma-PKC-GFP under physiological conditions, it was expressed either in NG-108 cells, N-methyl-D-aspartate (NMDA) receptor-transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induces rapid translocation of gamma-PKC-GFP. In NMDA receptor-transfected COS-7 cells, application of NMDA plus glycine also translocates gamma-PKC-GFP. Rapid translocation and sequential retranslocation of gamma-PKC-GFP are observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affects the translocation of gamma-PKC-GFP, indicating that translocation of gamma-PKC is independent of actin and microtubule. gamma-PKC-GFP fusion protein is a useful tool for investigating the molecular mechanism of gamma-PKC translocation and the role of gamma-PKC in the central nervous system (Sakai, 1997).

Delta subspecies of protein kinase C (delta-PKC) fused with green fluorescent protein (GFP) was expressed in CHO-K1 cells and the movement of this fusion protein was observed in living cells after three different stimulations. The delta-PKC-GFP fusion protein has enzymological characteristics very similar to those of the native delta-PKC and is present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM causes a transient translocation of delta-PKC-GFP to the plasma membrane approximately 30 s after the stimulation and a subsequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induces a slower translocation of delta-PKC-GFP, and the translocation is unidirectional. Concomitantly, the kinase activity of delta-PKC-GFP is increased by these two stimulations, when the kinase activity of the immunoprecipitated delta-PKC-GFP is measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide fails to translocate delta-PKC-GFP but increases the protein's kinase activity more than threefold. delta-PKC-GFP is strongly tyrosine phosphorylated when treated with H2O2 but is tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induce the translocation of delta-PKC-GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activates delta-PKC-GFP up to more than fivefold. TPA treatment of cells overexpressing delta-PKC-GFP leads to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increases the number of cells in S phase and induces no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of delta-PKC (Ohmori, 1998).

Protein kinase C zeta (PKC zeta) is a member of the PKC family of enzymes and is involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival and transcriptional regulation. PKC zeta has received considerable attention recently as a target of phosphoinositide 3-kinase (PI 3-kinase), although the mechanism of PKC zeta activation is, as yet, unknown. Recent reports have also shown that the phosphoinositide-dependent protein kinase-1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate (Ptdlns-3,4,5-P3), phosphorylates and potently activates two other PI 3-kinase targets: the protein kinases Akt/PKB and p70S6K. PDK-1 has therefore been investigated to determine if it is the kinase that activates PKC zeta. In vivo, PI 3-kinase is both necessary and sufficient to activate PKC zeta. PDK-1 phosphorylates and activates PKC zeta in vivo, and this is due to phosphorylation of threonine 410 in the PKC zeta activation loop. In vitro, PDK-1 phosphorylates and activates PKC zeta in a Ptdlns-3,4,5-P3-enhanced manner. PKC zeta and PDK-1 are associated in vivo, and membrane targeting of PKC zeta renders it constitutively active in cells. Thus, these results have identified PDK-1 as the kinase that phosphorylates and activates PKC zeta in the PI 3-kinase signaling pathway. This phosphorylation and activation of PKC zeta by PDK-1 is enhanced in the presence of Ptdlns-3,4-5-P3. Consistent with the notion that PKCs are enzymes that are regulated at the plasma membrane, a membrane-targeted PKC zeta is constitutively active in the absence of agonist stimulation. The association between PKC zeta and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase signaling pathway (Chou, 1998).

Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. Studies in vivo reveal that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKCbetaII with a catalytically inactive form of PDK-1 in COS-7 cells results in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Studies in vitro using purified proteins establish that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme does not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggers carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. It is concluded that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation (Dutil, 1998).

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. The second messenger functions of diacylglycerol were examined in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induce a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation is mimicked by addition of the diacylglycerol analog DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies show that PMA nearly immobilizes Cys1-GFP in the membrane, whereas DiC8 leaves Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localizes Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization is lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocate from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP is only observed in response to PMA addition. These studies introduced GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes (Oancea, 1998).

The heterotrimeric G proteins mediate a variety of cellular processes by coupling transmembrane receptors to different effector molecules, such as adenylyl cyclases and inositol-phospholipid-specific phospholipase C (PLC). Activation of adenylyl cyclases results in the production of cyclic AMP and activation of cAMP-dependent protein kinase (PKA). Phospholipase C catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to generate diacylglycerol and inositol-1,4,5-triphosphate (InsP2), leading to the activation of protein kinase C (PKC) and the mobilization of intracellular calcium. The various PLC isoforms appear to be activated by different receptors, and in some cases by different G-protein components. There are four well-characterized forms of PLC-beta and all of them are activated to various extents by the G alpha q family of G proteins. Specific activation of PLC isoforms beta 2 and beta 3 by G-protein beta gamma subunits has also been reported. Although it has been suggested that PLC activity might be modulated by the adenylyl cyclase pathway, no clear link has been established between the two pathways. cAMP-dependent protein kinase specifically inhibits G beta gamma-activated PLC-beta 2 activity but not that of the G alpha-activated PLC isoforms, and that the effect of PKA is not mimicked by PKC isozymes. PKA directly phosphorylates serine residues of the PLC-beta 2 protein both in vivo and in vitro. These results provide an insight into the specificity and nature of the crosstalk between the two G-protein-coupled signal transduction pathways (Liu, 1996).

TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increase the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induce an autophosphorylation of TrkB receptors, subsequently resulting in a phosphorylation and binding of phospholipase C-gamma (PLC-gamma) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-gamma, BDNF increases the phosphatidylinositol (PI) turnover and elevates intracellular calcium levels. Cerebellar PKC is activated after BDNF or TPA treatment. Survival-promoting effects of BDNF and TPA are blocked with calphostin C, a specific PKC inhibitor. BDNF activates c-ras (See Drosophila Ras) in a concentration-dependent manner. These results suggest that two different pathways, the c-ras and the PLC-gamma pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF (Zirrgiebel, 1995).

A series of PDGFR "add-back" mutants were used to evaluate the signaling pathways involved in the activation of PKC epsilon when stimulated by platelet-derived growth factor (PDGF) receptor (PDGFR). Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) causes translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also causes the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) is inhibited by wortmannin, an inhibitor of PI 3-kinase. Purified PKC epsilon is activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase, whose downstream targets remain to be identified (Moriya, 1996).

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 (Drosophila homolog Rolled) by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because treatment with phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocks Raf-Ras association in VSMC but does not inhibit activation of MEK and ERK1/2 by angII. It is thought that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediates ERK1/2 activation by angII. All isoforms except PKC-zeta are down-regulated by PDBU for 24 h suggesting that PKC-zeta is responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associates with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreases PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2 (Liao, 1997).

14-3-3 proteins are ubiquitous in eukaryotes associated with many fundamental functions in signal transduction pathways and cell cycle regulation. Different protein kinase C isozymes have distinct roles in signal transduction pathways; protein kinase C epsilon is of particular interest because its overexpression leads to oncogenic transformation. The 14-3-3 protein has been reported to regulate the activity of protein kinase C, albeit with equivocal results. In this study, when activated by 14-3-3 zeta protein, various protein kinase C isoforms respond differently: the classical isozymes show an approximately twofold increase in activation; protein kinase C delta shows no significant increase in activity, whereas protein kinase C epsilon, another novel isozyme, is highly activated (Acs, 1995).

Using immunoprecipitation and phosphotyrosine detection by Western blotting, intracellular signaling intermediates were analyzed in human primary dermal fibroblasts, either seeded as monolayers on collagen I coats (2D) or seeded within three-dimensional collagen I lattices (3D). Integrin activation in these systems results in a cascade of protein tyrosine phosphorylation, including focal adhesion kinase. Further downstream signaling events have now been shown to include coordinate activation of ERK1 and ERK2 at 2 h after cell-collagen contact, irrespective of 2D or 3D culture conditions. Application of U-73122, an inhibitor of PLC, inhibits collagen lattice contraction in a dose-dependent fashion. Immunoprecipitation identified the isoform PLCgamma-1 as a signaling intermediate in fibroblast-collagen interactions. PLCgamma-1 becomes phosphorylated within 10 min after culture initiation and declines after 2 h. So far, no qualitative differences in signaling intermediates between 2D and 3D cultures have been identified (Langholz 1997).

The protein kinase C (PKC) isoforms (alpha, betaI, and gamma of cPKC subgroup; delta and epsilon of nPKC subgroup, and zeta of aPKC subgroup) are tyrosine phosphorylated in COS-7 cells in response to H2O2. These isoforms isolated from the H2O2-treated cells show enhanced enzyme activity to various extents. The enzymes (PKC alpha and delta) recovered from the cells are independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC delta shows that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H2O2. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids (Konishi, 1997).

The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. Overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-kappaB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, because a dominant-negative MEK and a MEK inhibitor (PD98059) do not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Calpha (PKCalpha) in vivo; however, deletion of the CAAX site on V14Rho severely diminishes this association. Evidence for a role for PKCalpha as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCalpha blocks the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation (Chang, 1998).

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. Signal transduction pathways in the chicken limb bud have been explored. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induces RACK1 and such induction of RACK1 expression is accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region results in buds that do not express sonic hedgehog (Shh) and develop into wings that are severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcomes the effects of blocking PKC with chelerythrine and results in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs (Lu, 2001).

Inactivation of PKC

Treatment of cells with tumor-promoting phorbol esters first results in the activation of phorbol ester-responsive protein kinase C (PKC) isoforms, and then in their depletion. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. In 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms alpha, delta, and epsilon in response to tumor-promoting phorbol ester TPA. Proteasome inhibitors also block the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which result from the depletion of PKC delta. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC alpha, delta, and epsilon are detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulates ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevent both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. A kinase-dead ATP-binding mutant of PKC alpha cannot be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway (Lu, 1998).

PKC in C. elegans and Aplysia

The par genes are required to establish polarity in the Caenorhabditis elegans embryo. Three of the PAR proteins themselves exhibit asymmetric distributions. PAR-1, a putative serine/threonine kinase, and PAR-2, a protein containing a putative ATP-binding site and a zinc-binding motif of the RING finger class, are localized to the posterior periphery, whereas PAR-3, a novel protein containing three PDZ domains, is localized to the anterior periphery of a 1-cell embryo. Mutations in two par genes, par-3 and par-6, exhibit similar phenotypes. A third gene, pkc-3, gives a similar phenotype when the protein is depleted by RNA interference. PAR-3 and PKC-3 protein are colocalized to the anterior periphery of asymmetrically dividing cells of the germline lineage. The peripheral localizations of both proteins depends upon the activity of par-6. The molecular cloning of par-6 and the immunolocalization of PAR-6 protein are reported. par-6 encodes a PDZ-domain-containing protein and has homologs in mammals and flies. The PDZ domain of PAR-6 shares most similarity to the PDZ domain of Tax clone 40, a human protein that interacts with the Tax protein of human T-cell leukemia virus (TLV). The alignment of PAR-6 PDZ with PSD-95 PDZ3 shows that amino acids forming the beta-sheets and alpha-helix structures in PSD-95 PDZ3 are well conserved in PAR-6 PDZ. The conservation is greatest among these homologs over a 115 amino acid region containing the PDZ domain; worm PAR-6 is 88% and 80% identical to the fly (Fly EST #LD08317) and mouse proteins (#440139), respectively. PAR-6 colocalizes with PAR-3; par-3 and pkc-3 activity are both required for the peripheral localization of PAR-6. The localization of both PAR-3 and PAR-6 proteins is affected identically by mutations in the par-2, par-4 and par-5 genes. The co-dependence of PAR-3, PAR-6 and PKC-3 for peripheral localization and the overlap in their distributions lead to a proposal that they act in a protein complex (Hung, 1999).

The C. elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. [3H]Phorbol 12,13-dibutyrate [3H]PDBu binds with high affinity to the cysteine-rich region of Unc-13. This affinity is similar to that found in other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 binds diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis reveals significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 requires slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, is also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. Although these results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13 (Kazanietz, 1995b).

Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning. Serotonin (5-HT) is a key modulatory transmitter and it exerts its short-term actions via cAMP-dependent activation of protein kinase A. Subsequently, it has become clear that other kinase systems such as protein kinase C (PKC) also may be involved in the actions of 5-HT. Application of phorbol esters, which activate PKC, produced a slowly developing spike broadening but have little effect on excitability (a process known to be primarily cAMP dependent). Moreover, the effects of phorbol esters and 5-HT on spike duration are not additive, suggesting that they may share some common mechanisms. The protein kinase inhibitor staurosporine suppresses both 5-HT-induced slowly developing spike broadening and, under certain conditions, facilitation of transmitter release. Staurosporine does not inhibit 5-HT-induced enhancement of excitability. The effectiveness of staurosporine on spike broadening is dependent on the time at which spike broadening was examined after application of 5-HT. Staurosporine appears to have little effect on spike broadening 3 min after application of 5-HT, whereas it inhibits significantly 5-HT-induced spike broadening at later times. The staurosporine-insensitive component of 5-HT-induced spike broadening may be mediated by cAMP. The results suggest that the activation of PKC plays a key role in components of both 5-HT-induced spike broadening and facilitation of synaptic transmission (Sugita 1992).

There are three isoforms of protein kinase C (PKC) in Aplysia, two of which, Apl I and Apl II, are expressed abundantly in the nervous system. Two major kinase activities were resolved from nervous tissue by column chromatography on DEAE-cellulose and hydroxylapatite (HAP), one Ca(2+)-activated and the other Ca(2+)-independent. These two activities correspond to the previously cloned Apl I and II. These two isoforms appear to be the only major PKCs present in nervous tissue. The Apl I kinase is strongly activated by cis-fatty acids but only in the presence of Ca2+. Functionally, Apl I is more like the alpha and beta isoforms of vertebrate PKC, than the vertebrate neural gamma isoform. The Apl II kinase is Ca(2+)-independent and resembles vertebrate PKC epsilon. The simplicity of the PKC isoform distribution in Aplysia makes this mollusc an attractive animal for understanding the differential regulation and physiological activities of Ca(2+)-activated and Ca(2+)-independent PKCs (Sossin, 1993).

Translocation of PKC in intact Aplysia ganglia requires higher concentrations of phorbol esters than would be expected based on: (1) their affinity for Aplysia PKCs measured in vitro; (2) their physiological effects on cultured Aplysia neurons; and (3) their actions on PKC in synaptosomes. Although phorbol esters enter intact ganglia slowly, delayed access to neurons is insufficient to account for the high concentrations needed for translocation. Increasing accessibility to the neural components of ganglia increases the rate at which translocation occurs, but does not affect the concentration of phorbol ester required. It is suggested that this might best be explained by the presence of a competitive inhibitor at the binding site for phorbol esters in PKC. An indication for an inhibitor is that the concentration of phorbol esters needed for translocation in homogenates of nervous tissue is markedly decreased by diluting the extract. Preliminary characterization shows that the inhibitory activity is unusual: in addition to being competitive with lipid activators, it is soluble and tissue-specific. This type of inhibitor may be an important regulator of protein phosphorylation by PKC in neurons (Sossin, 1994).

The Aplysia nervous system contains two phorbol ester-activated protein kinase C isoforms, the Ca(2+)-activated Apl I and the Ca(2+)-independent Apl II. Short-term applications of the facilitatory transmitter serotonin (5-HT) activates Apl I, but not Apl II. In contrast, Apl II, but not Apl I, can form an autonomous kinase. To investigate the biochemical characteristics of the Aplysia kinases that might underlie their differential activation, Apl I, Apl II, and two derivatives of Apl II with deletions in the amino-terminal 150 amino acid E region were expressed in insect cells using the baculovirus system. Similar to nervous system extracts, expressed Apl II has more autonomous activity than Apl I. Removal of the E region lowers the amount of phosphatidylserine required for activation of Apl II, but does not remove the autonomous kinase activity. In addition, phosphatidylserine vesicles can sediment fusion proteins containing the E region, consistent with a role for the E region in lipid interactions. A partial deletion of the E region modifies activation of Apl II by phorbol esters and oleic acid, suggesting that in the intact enzyme the E region interacts with the phorbol ester-binding domain of the kinase. These results introduce a model whereby the E region acts as a negative regulator of Apl II activation and suggest that this inhibition may explain the inability of short-term applications of 5-HT to activate Apl II (Sossin, 1996).

In the sensory neurons of Aplysia, 5-HT acts through cAMP to reduce current flow through two classes of K+ channels: the S-K + channel and a transient K+ channel (Ikv). In addition, 5-HT increases a voltage-dependent, nifedipine-sensitive Ca2+ current. While the effect on the S-K+ channel is mediated exclusively by cAMP, the effect on the Ca2+ current can be mimicked by phorbol as well as by intracellular injection of cAMP. Specific blockers of protein kinase C (PKC) and the cAMP-dependent protein kinase A (PKA) were used to examine the roles of PKC and PKA in mediating the effect of 5-HT on the nifedipine-sensitive Ca2+ current. H-7, a kinase inhibitor that appears to inhibit PKC more effectively than PKA in intact Aplysia neurons, reverses the increase in the Ca2+ current produced by PDBu. H-7 partially blocks the effect of 5-HT on the Ca2+ current without affecting the decrease in the S-K+ current. A more specific PKC inhibitor (the 19-31 pseudosubstrate of PKC) also partially blocks the increase in the Ca2+ current produced by 5-HT, suggesting that this increase is mediated by PKC. Rp-cAMPS, a specific blocker of PKA, does not block the increase in the Ca2+ current produced by 5-HT, suggesting that PKA mediates the effect of 5-HT on this current to only a small extent. The effect of 5-HT on the S-K+ current and the Ca2+ current can also be separated on the basis of the time course of their appearance. The fact that the decrease in the S-K+ current precedes the increase in Ca2+ current suggests that there may be a temporal difference in the activation of the two kinase systems (Braha, 1993).

Serotonin (5-HT) plays important roles in various behavioral and physiological processes in Aplysia californica. These include feeding, locomotion, circadian rhythm, learning and memory, synaptic plasticity, and synaptic growth. Serotonin modulates these various functions by interacting with different 5-HT receptor subtypes that are coupled to various second-messenger systems. Two serotonergic receptors have been isolated from Aplysia californica: Ap5-HTB1 and Ap5-HTB2, using a strategy based on the amino acid sequence homology among G-protein-coupled biogenic amine receptors. Ap5-HTB1 and Ap5-HTB2 are both intronless and highly homologous to one another, sharing 79.5% sequence identity at the amino acid level. Sequence comparison reveals that these receptors are 33.1 to 23.3% identical to various mammalian 5-HT receptors. Both Ap5-HTB1 and Ap5-HTB2 encode functional 5-HT receptors. When expressed in cultured cells, these receptors stimulate phospholipase C in response to 5-HT in a dose-dependent manner. This stimulation can be blocked by specific 5-HT receptor antagonists. These receptors are present in the CNS (Ap5-HTB2) and in the reproductive system (Ap5-HTB1) (Li, 1995).

Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. This study shows that one olfactory neuron in C. elegans, AWC(ON), has the potential to direct both attraction and repulsion. Attraction, the typical AWC(ON) behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWC(ON) axons. gcy-28 mutants avoid AWC(ON)-sensed odors; they have normal odor-evoked calcium responses in AWC(ON) but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWC(ON) odor preferences. A behavioral switch in AWC(ON) may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWC(ON). These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs (Tsunozaki, 2008).

Protein kinase C and asymmetric division

Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. An atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. The predicted amino acid sequence of PKC-3 shows extensive similarity to mammalian atypical PKC subfamily members PKCzeta and PKClambda. The amino terminal half contains one cysteine-zinc finger motif and lacks a potential Ca 2+ -binding domain conserved in the conventional PKC family members. These structural features characterize atypical PKCs, which are dependent on neither Ca 2+ nor diacylglycerol for their activation. The carboxy-terminal half of the predicted PKC-3 protein exhibits about 70% similarity to the kinase domain of atypical PKCs. A separate study (Wu, 1998) shows that purified PKC-3 protein requires phosphatidylserine but is independent of Ca 2+ and diacylglycerol for its activation, characteristics of aPKCs (Tabuse, 1998).

Embryos depleted of PKC-3 by RNA interference die showing Par-like phenotypes, including defects in early asymmetric divisions and mislocalized germline-specific granules (P granules). The defective phenotypes of PKC-3-depleted embryos are similar to those exhibited by mutants for par-3 and another par gene, par-6. Direct interaction of PKC-3 with PAR-3 is shown by in vitro binding analysis. This result is reinforced by the observation that PKC-3 and PAR-3 co-localize in vivo. Furthermore, PKC-3 and PAR-3 show mutual dependence on one another and on three of the other par genes for their localization. It is concluded that PKC-3 plays an indispensable role in establishing embryonic polarity through interaction with PAR-3 (Tabuse, 1998).

The exact functional relationship between PKC-3 and PAR-3 is not clear. Three possibilities are suggested. (1) PAR-3 could be acting to recruit PKC-3 to the cell periphery where it acts as a signaling molecule. This idea is supported by the observation that PKC-3 does not become peripheral in the absence of PAR-3. It seems unlikely, however, that PAR-3 is functioning exclusively through PKC-3 because the gut differentiation phenotype of pkc-3(RNAi) is not as severe as that of par-3 mutants. Although this difference in severity could be due to incomplete depletion of PKC-3, peripheral PKC-3 is undetectable in pkc-3(RNAi) embryos. Thus, it appears that there is residual PAR-3 activity in the absence of detectable PKC-3. Furthermore, an exclusively downstream role for PKC-3 is not consistent with the cell-cycle- dependent mislocalization of PAR-3 in pkc-3(RNAi) embryos. This phenotype suggests a second possibility: (2) that PKC-3, like the par-6 product, acts to recruit PAR-3 to the cell periphery or maintain it there throughout the cell cycle or both. It could do this by phosphorylating PAR-3 directly or by modifying the cortical cytoskeleton. The two possibilities discussed above are not mutually exclusive; PAR-3 could recruit PKC-3 to the cell periphery where its kinase activity has the dual effect of providing a signal leading to anterior/posterior differences as well as maintaining PAR-3 at the periphery. (3) PKC-3 and PAR-3, perhaps along with the product of the par-6 gene, may act together to form a functional complex whose stability or localization requires the presence of both (Tabuse, 1998).

Cell polarity is fundamental to the differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends on cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. The present study reports the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and shows that ASIP colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy reveals that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, these results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry (Izumi, 1998a).

Protein kinase C subcellular distribution

Protein kinase C (PCK) epsilon has been found to have unique properties among the PCK isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. PKC epsilon localizes to the Golgi network via its zinc finger domain; both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKC epsilon, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKC epsilon. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding. Several regions of PKC epsilon contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins results in association with the plasma membrane and cytoskeletal components. The catalytic domain is found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation is dependent on the protein conformation. Based on these observations, a model has been proposed, depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites (Lehel, 1995).

The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79-PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity in a Ca2+-dependent manner. Both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reverses the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduces inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide is shown to be partially dependent on Ca2+. Ca2+/calmodulin reduces PKC coimmunoprecipitated with AKAP79 and results in a 2.6 increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein (Faux, 1997).

The message encoding the nPKC theta isoform, a member of the novel calcium-independent class of PKCs, has recently been shown to be abundant in mouse skeletal muscle. The message for cPKC alpha, a calcium-dependent isoform, was also found to be highly expressed in this tissue. In an effort to distinguish between the physiological roles of these two isoforms of PKC in rat skeletal muscle, their subcellular distribution, developmental expression and intracellular localization were examined. An isotype-specific antiserum directed against a peptide sequence unique to nPKC theta recognizes a 79 kDa protein highly enriched in rat skeletal muscle, which is likely to be nPKC theta. cPKC alpha is also readily detectable in skeletal muscle, using another isotype-specific antibody, but it appeared to be ubiquitously expressed in all of the tissues examined. Together these results suggest that nPKC theta, rather than cPKC alpha, is involved in physiological functions that are specific for skeletal muscle. The immunoreactivity for nPKC theta is highest in the membrane subcellular fraction compared to the cytosolic fraction of skeletal muscle. In contrast, cPKC alpha is found to be predominantly distributed in the cytosolic rather than the membrane fraction. nPKC theta appears to be developmentally regulated postnatally in rat skeletal muscle, with a 4-fold increase in expression occurring exclusively in the membrane fraction during postnatal days 3 through 21. This time course coincides with the period in rat development associated with maturation of neuromuscular junctions. Expression of nPKC theta in rat spleen, another tissue expressing detectable levels of this isoform, is not found to be developmentally regulated during this time. nPKC theta is detected in association with the sarcolemma of skeletal muscle and was found to be localized in the neuromuscular junction. Enhanced staining for nPKC theta in the neuromuscular junction appears as early as 4 days after birth. Staining for nPKC theta in the neuromuscular junction persists after prolonged denervation, suggesting that the enzyme is distributed postsynaptically. Taken together, these data suggest that nPKC theta may play a specific role in skeletal muscle signal transduction in both the developing and the mature neuromuscular synapse (Hilgenberg, 1995).

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples. The study reveals the tonic and stimulated juxta-membrane vesicular location of activated PKCalpha (Ng, 1999).

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane (Perez, 2001).

PKC and the cell cycle

The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1-S transition of the cell cycle and by perturbations to the cell surface, for example, those induced by heat shock. The activity of the Pkc1 pathway is partially dependent on Cdc28 activity. Swi4 activates transcription of many genes at the G1-S transition, including CLN1 and CLN2. swi4 mutants are defective specifically in bud emergence. The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1. This suppression requires CLN1 and CLN2. Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants. Another dose-dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein. Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity. It is proposed that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28-Cln1/Cln2 at the G1-S transition and by Hcs77 upon heat shock. Hcs77 may monitor the state of the cell surface (Gray, 1997).

Protein kinase C (PKC) is activated at the nucleus during the G2 phase of cell cycle, where it is required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear PKC are not known. Nuclear levels of the major physiologic PKC activator diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G2/M phase cells are 2.5 to 3 times higher than in G1 phase cells. In synchronized cells, nuclear DAG levels rise to a peak coincident with the G2/M phase transition and return to basal levels in G1 phase cells. This increase in DAG level is sufficient to stimulate betaII PKC-mediated phosphorylation of its mitotic nuclear envelope substrate lamin B (see Drosophila Lamin) in vitro. Isolated nuclei from G2 phase cells contain an active phospholipase activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG generation. Treatment of synchronized cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and cell cycle blockade in the G2 phase, suggesting a role for nuclear PI-PLC in the G2/M phase transition. These data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear betaII PKC, whose activity is required for mitosis (Sun, 1997).

The resumption of meiosis in the developing starfish oocyte is the result of intracellular signaling events initiated by 1-methyladenine (known as maturation inducing hormone) stimulation. 1MA interacts with an oocyte surface receptor that initiates intracellular signaling through the activation of the betagamma subunit of heterotrimeric G proteins and, results in the activation of maturation-promoting factor (MPF, a complex of p34cdc2 and cyclin B). One of the earliest detectable kinase activities during meiotic maturation of starfish oocytes is a protein kinase C or PKC-like activity. In this study, several isoforms of protein kinase C were cloned from the oocyte; however, the most abundant PKC-like maternal transcript corresponds to protein kinase C-related kinase 2 (PRK2). PRK2 is an atypical PKC since it is unresponsive to phorbol esters. PRK2 is expressed in the immature oocyte, at least until germinal vesicle breakdown. Subcellular localization of PRK2 reveals a cytoplasmic distribution in the immature oocyte; during meiotic maturation, this remains in the cytoplasm but it is also localized to the disintegrating germinal vesicle. Significantly, PRK2 is phosphorylated in vivo in response to 1-methyladenine, which precedes MPF activation, making PRK2 a candidate regulator of early signaling events of meiotic maturation (Stapelton, 1998).

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-beta I and -beta II isozymes. PKC-beta I and -beta II are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-beta I and PKC-beta II (beta I-A10 and beta II-A10). PKC levels are increased three- to four-fold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells is 36 h, the cell doubling time in beta I-A10 cells decreases by 12 h, and, in contrast, the doubling time of beta II-A10 cells increases by 12 h, as compared to A10 cells. The increase of [3H]thymidine (TdR) incorporation is accelerated and increases in beta I-A10 cells, but slows and diminishes in beta II-A10 cells, compared to A10 and control cells transfected with empty vector. Cell cycle analysis of beta I-A10 cells shows an acceleration of S phase entry while beta II-A10 cells slow S phase entry. These results suggest that PKC-beta I and PKC-beta II regulate cell proliferation bidirectionally and that PKC-beta I and PKC-beta II may have distinct and opposing functions as cell cycle check point mediators during late G1 phase and may regulate S phase entry in A10 VSMC (Yamamoto, 1998).

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis (Collas, 1999).

Oocytes from LTXBO mice exhibit defects in the regulatory mechanisms that govern the first meiotic division. There is a delayed entry into anaphase I and oocytes frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity is detected in LTXBO oocytes at prophase I and increases with meiotic maturation, with the highest activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduces M-phase-promoting factor (MPF) activity and promotes progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remains elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associates with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevents untimely entry into interphase, but only when PKC activity is reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, since suppression of PKC activity fails to prevent activation by Mos^tm1Ev/Mos^tm1Ev LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis (Viveiros, 2001).

Members of the protein kinase C family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, it has been demonstrated that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G₀. PKC activation in the IEC-18 intestinal crypt cell line results in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G₁/S cyclin-dependent kinase complexes. These events are associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G₀ as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrate that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis reveals that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium (Frey, 2000).

PKC and signaling pathways

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells were monitored using pharmacological and genetic approaches. Inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduces extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon is sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms are activated following adhesion. PKC is required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc (Miranti, 1999).

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. Dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta have been generated to determine the roles of individual isoforms of PKC in the activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicate that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE is partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activate the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells leads to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activate the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon can also induce a mutant c-fos promoter that lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation is blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf > MEK1 > ERK > TCF; MEKK1 > SEK1 > JNK > TCF, and rhoA > SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression (Soh, 1999).

In chickens, PKA and PKC are involved in intracellular signaling during feather morphogenesis. PKC immunoreactivity increases in the whole layer of developing dermis. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. Activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented (Noveen, 1995).

Induction of the p40/46 and p69/71 isoforms of the 2',5'-oligoadenylate (2-5A) synthetase by interferon-alpha (IFN-alpha) is variable among six different Burkitt lymphoma cell lines with Ramos cells expressing among the highest levels of these enzymes. Inhibitors of protein kinase C (PKC) block induction of mRNAs encoding both isoforms; however, induction of the p69/71 isoform is more sensitive to these inhibitors. Down-regulation of PKC by prolonged treatment with phorbol ester also blocks induction of 2-5A synthetase mRNAs and decreases both constitutive and IFN-alpha-induced enzymatic activity. Cotreatment of cells with phorbol and IFN-alpha increases induction of 2-5A synthetase mRNAs above that seen in cells treated with IFN-alpha alone. IFN-alpha does not directly activate PKC-alpha or PKC-delta, the two most abundant PKC isoforms present in Ramos cells, suggesting that PKC activation by another signaling pathway is necessary for maximal induction of 2-5A synthetases by IFN-alpha (Yu, 1997).

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons. The effect of the cholinergic agonist carbachol on the spontaneous release of glutamate was studied in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increases the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms are not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1-43. The carbachol-induced increase in sEPSC frequency is not mimicked by nicotine, but can be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicate that carbachol transiently increases intracellular Ca2+ concentration. Chelator experiments show that this effect cannot be attributed to the rise in intracellular Ca2+ concentration. In contrast, the protein kinase inhibitor staurosporine inhibits the carbachol effect, as well as providing a down-regulation of protein kinase C by prolonged treatment of the cells with 4beta-phorbol 12-myristate 13-acetate. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppresses the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors (Bouron, 1997).

Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. This proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells shows that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta is abrogated for Raf CAAX, which is a membrane-localized, partially active form of c-Raf1. Activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, a novel Raf desensitization induced by PKC-alpha is described, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in phorbol ester induced mitogenesis, and provide evidence for multiple PKC controls acting on this MAPK cascade (Schonwasser, 1998).

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study has shown that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induces an increase in [Ca2+]i, which is suppressed each of the following: extracellular Ca2+ removal, extracellular Na+ replacement, the blocker of L-type Ca2+ channels (nicardipine), and the secreto-inhibitory neurotransmitter dopamine. The PACAP-induced [Ca2+]i increase is mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC) and is reduced by inhibitors of PKA and PKC. Patch-clamp analysis reveals that PACAP causes inward currents with a reversal potential of -0.8 mV and facilitates voltage-dependent Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA (Tanaka, 1997).

Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). The Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. This preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. At present it is not clear which PKC isoform is responsible for down-regulation of Ral-GDS in response to NGF and EGF. The fact that the effect can be seen after PMA treatment argues that it is through the phorbol-responsive conventional PKCs alpha, beta, or gamma and/or the novel PKCs delta, epsilon, eta, or theta but not the atypical PKCs zeta and lambda. It also remains to be determined how PKC becomes activated, because multiple types of signaling molecules can enhance PKC activity in cells. For example, some PKC family members can be activated by diacylglycerol and calcium generated by NGF or EGF receptor-induced phospholipase C activation. Alternatively, Ras-Ral-regulated PLD could conceivably activate PKC isoforms, since the PLD product, phosphatidic acid, is known to be converted to diacylglycerol. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation (Rusanescu, 2001).

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), only Ser-318, but not Ser-307 phosphorylation was detected, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. Since the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, the role of Shp2 in this context was examined. Phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depends on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 are blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1 (Mussig, 2005).

A potential role of InsP3 receptors and PKC in Wingless signaling

The Drosophila gene product Wingless (Wg) is a secreted glycoprotein and a member of the Wnt gene family. Genetic analysis of Drosophila epidermal development has defined a putative paracrine Wg signaling pathway involving the zeste-white 3/shaggy (zw3/sgg) gene product. Although putative components of Wg- (and by inference Wnt-) mediated signaling pathways have been identified by genetic analysis, the biochemical significance of most factors remains unproven. In mouse 10T1/2 fibroblasts, the activity of glycogen synthase kinase-3 (GSK-3), the murine homolog of Zw3/Sgg, is inactivated by Wg. This occurs through a signaling pathway that is distinct from the insulin-mediated regulation of GSK-3, because Wg signaling to GSK-3 is insensitive to wortmannin. Wg-induced inactivation of GSK-3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31-8220 and prolonged pre-treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK-3/Zw3 during Wnt/Wg signal transduction (Cook, 1996).

In Drosophila, members of the frizzled family of tissue-polarity genes (see Drosophila Frizzled and Frizzled 2) encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane. Stimulation of a G-protein-linked receptor initiates the hydrolysis of a membrane-bound inositol lipid, generating at least two second messengers: diacylglycerol and inositol-1,4,5-trisphosphate (InsP3). Diacylglycerol stimulates protein kinase C while InsP3 promotes the release of intracellular calcium (see Drosophila InsP3 receptor). The rat protein Frizzled-2 causes an increase in the release of intracellular calcium, which is enhanced by Xwnt-5a, a member of the Wnt family. Pertussis toxin (PTX) (which is a specific inhibitor of G alpha0 and G alphai subunits of G proteins that act by preventing the catalysis of GDP-GTP exchange stimulated by receptors) inhibits rat protein Frizzled-2 modulation of calcium flux. A nonhydrolysable GDP analog that irreversibly inactives G-protein-coupled events, inhibits rat FZ-2 induced Ca2+ transients. The release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase, and hence of the phosphatidylinositol signaling pathway. This suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. These results indicate that some Wnt proteins work through specific Frizzled homologs to stimulate the phosphatidylinositol signalling pathway via heterotrimeric G-protein subunits, and that FZ-2 stimulates the phosphatidylinositol cycle through the betagamma subunits of pertussis-toxin-sensitive G proteins, leading to release of intracellular Ca2+ and diverse cellular responses. Since Gbetagamma subunits also activate protein kinase C, which may be involved in Wnt signaling, the responses by cells and embryos to signaling through Frizzled homologs could involve the stimulation of multiple cytoplasmic pathways. In early vertebrate embryos, regulation of the phosphatidylinositol pathway may be important for establishing the embryonic mesoderm and in other processes (Slusarski, 1997).

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish. Since protein kinase C (PKC) isoforms can respond to Ca2+ signals, it was asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A results in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes, does not. Rfz2 and Xwnt-5A are also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits block Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, mouse Frizzled (Mfz) homologs were tested for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulate siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulate PKC activation but not expression of siamois and Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates (Sheldahl, 1999).

Wnt signaling involves inhibition of glycogen synthase kinase-3beta (GSK-3beta) and elevation of cytoplasmic beta-catenin. This pathway is essential during embryonic development and oncogenesis. Previous studies on both Xenopus and mammalian cells indicate that lithium mimics Wnt signaling by inactivating GSK-3beta. Serum enhances accumulation of cytoplasmic beta-catenin induced by lithium in both 293 and C57MG cell lines and growth factors are responsible for this enhancing activity. Growth factors mediate this effect through activation of protein kinase C (PKC), not through Ras or phosphatidylinositol 3-kinase. In addition, Wnt-induced accumulation of cytoplasmic beta-catenin is partially inhibited by PKC inhibitors and by chronic treatment of cells with phorbol ester. Both calphostin C, a PKC inhibitor, and a dominant negative PKC exhibit partial inhibition on Wnt-mediated transcriptional activation. It is proposed that Wnt signaling to beta-catenin consists of two interactive components: one involves inhibition of GSK-3beta and is mimicked by lithium, and the other involves PKC and serves to augment the effects of GSK-3beta inhibition (Chen, 2000).

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Endogenous Wnt/ß-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. The data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/ß-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/ß-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/ß-catenin pathway upstream of ß-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/ß-catenin signaling cascade downstream of ß-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus (Kuhl, 2001).

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. The Xenopus homolog of Rho GTPase Cdc42 has been isolated and its potential role during gastrulation movements in early Xenopus embryos has been examined. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-alpha, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca ²⁺ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca ²⁺-dependent cell adhesion. Taken together, these results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca²⁺ pathway (Choi, 2002).

Transcriptional targets of Protein kinase C

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. Cultures of human astrocytes were used to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increases the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs have identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identifies a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicate that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identifies 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: (1) an initial induction by reduced cell contact, and (2) further activation, by either growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively (Moffett, 1998).

Protein kinase C and apoptosis

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of phorbol ester in the prostate epithelial cell line, LNCaP, results in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, are resistant to apoptosis. LNCaP apoptosis is initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict is prevented by serum depletion, apoptosis is suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, affords nearly total protection from protein kinase C-signaled apoptosis. This protection correlates with the total loss of caspase activity (See Drosophila Death caspase-1) as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, it is proposed that protein kinase C regulates a novel cell death pathway, initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells; the proposed killing pathway is carried out by the caspase family of cysteine proteases (Zhao, 1997).

Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC alpha is important for cellular differentiation and PKC betaII is required for proliferation. The role of the atypical PKC isoform PKC iota was assessed in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC iota in sense or antisense orientation to increase or decrease cellular PKC iota levels, respectively. Overexpression or inhibition of expression of PKC iota has no significant effect on the proliferative capacity of K562 cells nor on their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC iota does not play a critical role in these processes. Rather, PKC iota serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC iota leads to increased resistance to OA-induced apoptosis, whereas inhibition of PKC iota expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC zeta has no protective effect, demonstrating that the effect is isotype-specific. PKC iota also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC iota in leukemia cell survival (Murray, 1997).

Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by evidence that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis (Bharti, 1998).

Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological disorders, and the loss of protein kinase C (PKC) activity has been coupled to the severity of the damage. The functional relationship between stress, PKC, and cell death is, however, unknown. Using an immortalized hippocampal cell line that is particularly sensitive to oxidative stress, it has been shown that activation of PKC by the phorbol ester tetradecanoylphorbol acetate (TPA) inhibits cell death via the stimulation of a complex protein phosphorylation pathway. TPA treatment leads to the rapid activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), the inactivation of p38 mitogen-activated protein kinase (MAPK), and the downregulation of PKCdelta. Inhibition of either ERK or JNK activation blocks TPA-mediated protection, whereas p38 MAPK and PKCdelta inhibitors block stress-induced nerve cell death. Both p38 MAPK inactivation and JNK activation appear to be downstream of ERK because an agent that blocks ERK activation also blocks the modulation of these other MAP kinase family members by TPA treatment. Thus, the protection from oxidative stress afforded nerve cells by PKC activity requires the combined modulation of multiple enzyme pathways and suggests why the loss of PKC activity contributes to nerve cell death (Maher, 2001).

Protein kinase C and the cytoskeleton

The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. RGD1 acts somewhere upstream of the PKC pathway. Given that Rgd1p has been shown to have a Rho-GAP activity toward Rho3p and Rho4p, the defect of PKC pathway activation at late exponential phase observed in the rgd1 mutants might be mediated by the small GTPases Rho3p and Rho4p. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta, RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog, were were each shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. It is hypothesized that rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway (de Bettignies, 2001).

In Saccharomyces cerevisiae, the Rho family of GTPases is thought to have a central role in the polarized growth process. The main functions assigned to these GTPases involve bud formation and cell surface growth, which might occur through the involvement of the actin cytoskeleton and the secretory pathway. Genetic and functional analyses have allowed the identification of five Rho members in yeast: Cdc42 and Rho1 to Rho4. These small GTPases function as binary switches, which are turned on and off by binding to GTP or GDP, respectively. The GTP-bound form interacts with its specific target and performs its cell functions. Small GTPases are regulated by GAPs (GTPase-activating proteins), GEFs (GDP-GTP exchange factors), and a GDP dissociation inhibitor (de Bettignies, 2001).

During the sequencing of the genome of S. cerevisiae, a new gene encoding a protein with a Rho-GAP homology domain was identified. This protein, called Rgd1p (for related GAP domain), was shown in vitro to be a GTPase activating protein for the Rho3 and Rho4 proteins. Thus, in activating the hydrolysis of GTP, Rgd1p negatively regulates the action of these two Rho proteins. Rho3p and Rho4p play a role in bud formation and have some partially overlapping functions. Deletion of RHO4 does not affect cell growth, whereas deletion of RHO3 causes a severe growth delay and a decrease in cell viability. Overexpression of RHO4 suppresses the growth defect in rho3 cells. Depletion of both RHO3 and RHO4 gene products results in lysis of cells with a small bud, which can be prevented by the presence of osmotic stabilizer in the medium. In this latter condition, Rho3p- and Rho4p-depleted cells lose cell polarity as revealed by chitin delocalization and by random distribution of actin patches (de Bettignies, 2001).

Genetic interactions occur between RGD1 and the SLG1 and MID2 genes. SLG1 has also been designated HCS77 and WSC1, but for simplicity this gene is referred to here as SLG1. Slg1p and Mid2p are both plasma membrane proteins with partial overlapping functions. They act upstream of the protein kinase C (PKC) pathway and are thought to monitor the state of the cell surface and relay the information to Pkc1p. Protein kinase C is mostly regulated by the small GTPase Rho1p in vivo. Pkc1p activates a mitogen-activated protein (MAP) kinase cascade, named the PKC pathway, consisting of Bck1p, Mkk1p/Mkk2p, and the MAP kinase Mpk1p. Activation of this pathway is particularly important in response to various external stresses, including high temperature, low osmolarity, and cell wall disruption, as well as being important during mating. The protein Slg1 is linked to the PKC pathway by the finding that this MAP kinase cascade is activated by heat stress via Slg1p. A direct interaction of Slg1p with Rom2p, one of the Rho1p-GEFs, has been recently reported and this interaction is responsible for the activation of the PKC pathway through Rho1p (de Bettignies, 2001).

The loss of RGD1 function amplifies the phenotype due to the SLG1 deletion and the small-budded double-mutant cells die because of defects in cell wall structure and lysis upon bud growth. In parallel, the inactivation of MID2, the other putative sensor for cell integrity signaling in S. cerevisiae, exacerbates the specific phenotype of the rgd1Delta mutant with an increase in dead cells at late exponential phase in minimal medium. Taken together, these results suggest that Rgd1p has a regulatory role in connection with both the PKC pathway and the actin cytoskeleton organization in S. cerevisiae (de Bettignies, 2001).

To further elucidate the function of RGD1, multicopy suppressors of the viability defect of the rgd1Delta mutation were isolated in minimal medium. Phenotypic and genetic analysis has allowed the identification of several multicopy as well as monocopy suppressors of rgd1Delta: the RHO1 and RHO2 genes encoding two GTPases involved in actin cytoskeleton organization, the MID2-homolog MTL1, and the MKK1 gene coding for one of the MAP-kinase kinases of the PKC pathway. Considering the suppressor effect of additional PKC pathway components, it has been shown that activation of the PKC pathway prevents lethality of rgd1Delta cells. Analysis of the transcriptional activity of Rlm1p, one of the targets of the last kinase in the PKC pathway, and study of the PST1 transcription, which is positively regulated by Rlm1p, shows that the rgd1Delta mutation decreases the activity of this MAP-kinase pathway in minimal medium at late exponential phase. This decrease in PKC pathway activity is at least partly responsible for the rgd1Delta cell viability loss under particular growth or physiological conditions (de Bettignies, 2001).

The fertilization-competent Xenopus egg undergoes a contraction of its cortex towards the apex of the pigmented animal hemisphere within 10 min of fertilization. Evidence suggests that protein kinase C (PKC) is involved in the assembly of this contractile network; this paper shows that PKC is rapidly activated as a result of exposure of oocytes to progesterone. Xenopus oocytes contain at least five different isotypes of PKC. Three actin-binding proteins (vinculin, talin and ankyrin) appear to play an early role in the assembly of the contractile network and one of the proteins (vinculin) becomes phosphorylated shortly after progesterone treatment as the contractile network is assembling. These results indicate that progesterone acts through a phospholipase to activate PKC and that PKC participates in the remodeling of the cytoplasmic compartment as the oocyte becomes the egg (Johnson, 1997).

This study examined the modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen. Two actin microfilament networks, stress fibers and cortical actin, can be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation is stimulated by growth factors and by PMA (phorbol ester); these stimulations are blocked by tyrosine kinase inhibitors. By contrast, the cortical network occurs in quiescent cells, is unaffected by tyrosine kinase inhibitors, and is broken down after PKC activation by PMA. Spreading, migration, and actin polymerization are completely blocked, while adhesion efficacy is significantly decreased by three specific PKC inhibitors. Paxillin clustering, which is observed even in the presence of tyrosine kinase inhibitors, disappears only when actin polymerization is completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA; this indicates that focal complex formation is highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics (Masson-Gadais, 1997).

Melanophores, cells specialized for regulated organelle transport, were used to study signaling pathways involved in the regulation of transport. Immortalized Xenopus melanophores were transfected with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogs of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus that increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. It is concluded that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A (Reilein, 1998).

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations that include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations that inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor that block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha is unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers, independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta is abrogated by coexpression of DN Rac-1 N17 (Uberall, 1999).

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increases from a ratio of 1.6 +/- 0.1 in the dark to 2.1 +/- 0.1 after exposure to light. Light also causes the retraction of spinules and processes elaborated by the synaptic pedicle in the dark. Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light is mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extends up to 2.5 micrometers from the plasma membrane. Formation of F-actin occurs on the time scale of minutes and requires Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerate the growth of F-actin. Agents that inhibit PKC hinder F-actin growth in response to Ca2+ influx and accelerate F-actin breakdown on removal of Ca2+. To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D does not affect the continuous cycle of exocytosis and endocytosis that is stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. It is suggested that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions (Job, 1998).

Protein kinase C, secretion and ectodomain shedding

Signaling mechanisms that stimulate exocytosis and luteinizing hormone secretion in isolated male rat pituitary gonadotropes were studied. As judged by reverse hemolytic plaque assays, phorbol-12-myristate-13-acetate (PMA) stimulates as many gonadotropes to secrete as does gonadotropin-releasing hormone (GnRH). However, PMA and GnRH use different signaling pathways. The secretagogue action of GnRH is not very sensitive to an inhibitor of protein kinase C, but is blocked by loading cells with a calcium chelator. The secretagogue action of PMA is blocked by bisindolylmaleimide I and is not very sensitive to the intracellular calcium chelator. GnRH induces intracellular calcium elevations, whereas PMA does not. As judged by amperometric measurements of quantal catecholamine secretion from dopamine- or serotonin-loaded gonadotropes, the secretagogue action of PMA develops more slowly (in several minutes) than that of GnRH. It is concluded that exocytosis of secretory vesicles can be stimulated independently either by calcium elevations or by activation of protein kinase C (Billard, 1997).

Modulation of the size of the readily releasable vesicle pool has recently come under scrutiny as a candidate for the regulation of synaptic strength. Using electrophysiological and optical measurement techniques, it has been shown that phorbol esters increase the size of the readily releasable pool at glutamatergic hippocampal synapses in culture through a protein kinase C (PKC)-dependent mechanism. Phorbol ester activation of PKC also increases the rate at which the pool refills. The rate at which the readily releasable vesicle pool refills also increases after a high-frequency train of action potentials. This acceleration in refilling depends on the accumulation of intracellular Ca2+ during electrical stimulation. To determine whether this Ca2+-dependent increase in the refilling rate shares a common mechanism with the PKC-dependent increase, the effect of electrical stimulation was measured on the rate of refilling before and after phorbol application. For these whole-cell-recording experiments, a pair of hypertonic challenges was delivered 2.5 s apart. Trials in which there was no electrical stimulation were interleaved with trials in which a train of 14 action potentials was elicited at 9 Hz during the final 1.5 s of the first hypertonic challenge. Perforated patch recordings were used to minimize interference with endogenous calcium buffering at the terminal. The rate of refilling was 1.65 times faster for trials in which action potentials are elicited at the end of the first hypertonic solution application than for those with no electrical stimulation. After phorbol application, the rate of refilling was only 1.12 times faster for those trials in which action potentials are elicited. Thus, phorbol application and nerve impulse activity both speed the refilling, and the increased refilling rate produced by phorbol application occludes that resulting from action potentials. This occlusion is interpreted as indicating a shared pathway between the two mechanisms. These results identify two powerful ways (phorbol ester activation of the PCK pathway and neural stimulation) that activation of the PKC pathway may regulate synaptic strength by modulating the readily releasable pool of vesicles (Stevens, 1998).

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family, which encompasses a number of structurally homologous mitogens including EGF, TGF-alpha, vaccinia virus growth factor, amphiregulin, beta-cellulin and epiregulin. Like EGF, TGF-alpha and amphiregulin, HB-EGF binds to and stimulates the phosphorylation of the EGF receptor. HB-EGF is synthesized as a membrane-anchored precursor protein of 208 amino acids composed of signal peptide, heparin-binding, EGF-like, transmembrane and cytoplasmic domains. Although the membrane-anchored form of HB-EGF (proHB-EGF) is cleaved on the cell surface to yield a soluble growth factor of 75-86 amino acids, a considerable amount of proHB-EGF remains uncleaved on the cell surface. Importantly, proHB-EGF is not only a precursor of the soluble form but is also biologically active in itself; proHB-EGF forms a complex with both CD9 and integrin alpha3beta1, both localized at cell-cell attachment sites, and transduces biological signals in a nondiffusible manner to neighboring cells, as is known to occur for TGF-alpha and colony-stimulating factor. Moreover, although secreted mature HB-EGF is a potent mitogen for a number of cell types, the membrane-anchored form may act as a negative regulator of cell proliferation. Thus, the processing of the juxtamembrane domain of proHB-EGF to the soluble HB-EGF means the conversion of the mode of action of this growth factor from juxtacrine to paracrine. ProHB-EGF also acts as the specific receptor for diphtheria toxin (DT) and mediates the endocytosis of the receptor-bound toxin. Interestingly, proHB-EGF is cleaved rapidly to soluble HB-EGF by treatment with TPA, suggesting the involvement of a cellular signaling pathway involving protein kinase C (PKC). PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain (Izumi, 1998b).

PKC and Ca2+ oscillations

Glial cells in the central nervous system have traditionally been thought to provide structural, metabolic, and functional support for neurons. Astrocytes, a prominent CNS subtype of glial cells, can be stimulated by neuronal activity and they can themselves regulate the excitability of neurons. This has led to the hypothesis that astrocytes are not only helper cells but are participants in neuronal communication. Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Total internal reflection fluorescence (TIRF) microscopy has been used to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves. Glutamate stimulation of astrocytes triggers highly localized GFP-PKCgamma plasma membrane translocation events, induces rapid oscillations in GFP-PKCgamma translocation, and generates GFP-PKCgamma translocation waves that propagate across and between cells. These translocation responses are primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and are driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca2+ waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca2+ and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, leads to a marked prolongation of Ca2+ spikes, suggesting that PKC activation is involved in terminating individual Ca2+ spikes and waves and in defining the time period between Ca2+ spikes. This study suggests that cPKCs have a negative feedback role on Ca2+ oscillations and waves that is mediated by the repetitive activation of cPKCs by oscillating DAG and Ca2+ concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca2+ spikes and waves and can define the Ca2+ spike and wave frequencies (Codazzi, 2001).

PKC, synaptogenesis and neuronal maturation

Evidence is provided that astrocytes affect neuronal synaptogenesis by the process of adhesion. Local contact with astrocytes via integrin receptors elicits protein kinase C (PKC) activation in individual dissociated neurons cultured in astrocyte-conditioned medium. This activation, initially focal, soon spreads throughout the entire neuron. It was demonstrated pharmacologically that the arachidonic acid cascade, triggered by the integrin reception, is responsible for the global activation of PKC. Local astrocytic contact also facilitates excitatory synaptogenesis throughout the neuron, a process which can be blocked by inhibitors of both integrins and PKC. Thus, propagation of PKC signaling represents an underlying mechanism for global neuronal maturation following local astrocyte adhesion (Hama, 2004).

Adult neurogenesis occurs in two regions of the adult brain: the subventricular zone (SVZ) and the hippocampal subgranular zone. Mature hippocampal astrocytes regulate neurogenesis by instructing stem cells to adopt a neuronal fate. A marked increase is found in neuronal production from adult stem cells plated on a feeder layer of astrocytes. Since highly efficient neurogenesis occurs upon the plating of adult stem cells on either coated substances conditioned by primary astrocytes or directly on light-fixed astrocytes, it has been concluded that both diffusible and membrane-bound astrocyte-produced factors could promote neurogenesis. Neurogenesis in adult-derived SVZ cells is supported by astrocyte monolayers, but not by astrocyte-conditioned medium. It has been concluded that direct cell contact between SVZ precursors and live astrocytes is required for neurogenesis. Immunohistochemical experiments performed using adult rat brain tissue demonstrate that immature neurons in the hippocampal subgranular zone, which experience a high degree of astrocyte contact, stain more positively for PKC activation than mature neurons in the granular cell layer. Although there may be differences between the maturation of embryonic neurons and neurogenesis of adult neural stem cells, PKC signaling triggered by local cell-cell contact may be a general mechanism by which astrocytes regulate neuronal development (Hama, 2004).

PKC and growth cone collapse

Axonal navigation during development requires that cues present in the extracellular environment be capable of modifying the structure of the cone in a dynamic way. Protein kinase C (PKC) has long been suspected to be one of the multiple molecular relays present in the terminal structure of the developing axon and involved in the transduction of extracellular signals. The latter proposal is, however, based on the use of drugs or of protocols leading to pleiotropic and often nonspecific effects. In the present study a peptide capable of translocating across biological membranes, one that accumulates in the cytoplasm and nucleus of cells in culture, was used to internalize a highly specific peptidic inhibitor of PKC. Linking the two peptides (vector and PKC inhibitor) allows the internalization of the latter in live cells, specifically inhibits PKC and provokes a rapid modification of growth cone morphology. This set of data thus establishes that a peptidic inhibitor of PKC activity, once internalized, provokes a change in growth cone morphology, reminiscent of the collapse phenotype. The present study describes a new efficient and harmless way to introduce pharmacologically active substances into cultured neural cells (Theodore, 1995).

Protein kinase C and neural development

The expression of the genes encoding the alpha subunit of type-II calcium/calmodulin-dependent protein kinase (CAM-KII alpha) and the gamma subspecies of protein kinase C (PKC gamma) was examined throughout postnatal rat brain development by in situ hybridization histochemistry. CAM-KII alpha is found to be expressed sequentially over the different hippocampal subregions, beginning at birth with expression in the pyramidal cells of CA3, followed by expression in the external blade of the dentate gyrus and in CA1 on postnatal day (PND) 5 and finally, on PND 8, in the internal blade of the dentate gyrus. In contrast, PKC gamma expression rises simultaneously in the hippocampal subregions CA1 and CA3, with little expression over the dentate gyrus. This fashion of expression corresponds to the similar maturational state of the pyramidal cells in CA1 and CA3, whereas CAM-KII alpha expression during development of the rat hippocampus follows the time table of afferent lamination, which is delayed in CA1 compared to CA3. A temporal overexpression of CAM-KII alpha is found in the hippocampal subfields CA1 and CA3 at the end of the second postnatal week, which coincides with the development of N-methyl-D-aspartate receptor binding (Herms, 1993).

PKC gamma is highly expressed in Purkinje cells (PCs) but not in other types of neurons in the cerebellum. The expression of PKC gamma changes markedly during cerebellar development, being very low at birth and reaching a peak around the third postnatal week. This temporal pattern of PKC gamma expression coincides with the developmental transition from a multiple to a single climbing fiber innervation onto each PC. In adult mutant mice deficient in PKC gamma, 41% of PCs are still innervated by multiple climbing fibers, while other aspects of the cerebellum including the morphology and excitatory synaptic transmission of PCs appear normal. Thus, elimination of multiple climbing fiber innervation appears to be specifically impaired in the mutant cerebellum. It is suggested that the developmental role of PKC gamma may be to act as a downstream element in the signal cascade necessary for the elimination of surplus climbing fiber synapses (Kano, 1995).

Cerebellar long-term depression (LTD) is a model system for neuronal information storage that has an absolute requirement for activation of protein kinase C (PKC). It has been claimed to underlie several forms of cerebellar motor learning. Previous studies using various knockout mice (mGluR1, GluRdelta2, glial fibrillary acidic protein) have supported this claim; however, this work has suffered from the limitations that the knockout technique lacks anatomical specificity and that functional compensation can occur via similar gene family members. To overcome these limitations, a transgenic mouse (called L7-PKCI) has been produced in which the pseudosubstrate PKC inhibitor, PKC[19-31], was selectively expressed in Purkinje cells under the control of the pcp-2(L7) gene promoter. Cultured Purkinje cells prepared from heterozygous or homozygous L7-PKCI embryos show a complete blockade of LTD induction. The compensatory eye movements of L7-PKCI mice were recorded during vestibular and visual stimulation. Whereas the absolute gain, phase, and latency values of the vestibulo-ocular reflex and optokinetic reflex of the L7-PKCI mice are normal, their ability to adapt their vestibulo-ocular reflex gain during visuo-vestibular training is absent. These data strongly support the hypothesis that activation of PKC in the Purkinje cell is necessary for cerebellar LTD induction, and that cerebellar LTD is required for a particular form of motor learning, adaptation of the vestibulo-ocular reflex (De Zeeuw, 1998).

Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurs in the absence of exogenous treatments; the phosphorylation level is rapidly increased when synaptosomes are treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate also increases the level of phosphate incorporation. This occur within 10 min. PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes is reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity is produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity (Vaughan, 1997).

Protein kinase C, long term potentiation, synaptic depression, and memory

Increases in activity of both protein kinase A (PKA) and protein kinase C (PKC) contribute to short-term facilitation of Aplysia sensorimotor synapses evoked by serotonin (5-HT). Increasing levels of cAMP in sensory neurons evoke increases in both synaptic efficacy and in the number of sensory neuron varicosities contacting the major axons of motor cell L7 at intermediate times (3 hr) that persist for 24 hr. Treatment with phorbol esters results in a large transient increase in synaptic efficacy that is accompanied by a large transient increase in the number of sensory neuron varicosities with the newest varicosities most susceptible to elimination. The reversal of the synaptic facilitation and the structural changes does not appear to be the result of long-term inhibitory actions of persistent PKC activation by phorbol esters, since changes in synaptic efficacy can be evoked by additional applications of either phorbol esters or 5-HT. The short-lived changes in structure evoked by phorbol esters occur in preexisting sensory neurites and not as a result of new growth, since increases in PKC activity with phorbol esters lead to reductions in neurite extension and to retractions by sensory neuron growth cones. The action of phorbol esters on growth cone extension is reversible with washout. The results suggest that increases in PKA and PKC activities by 5-HT contribute to short (minutes) and intermediate (hours) forms of facilitation of sensorimotor synapses while increases in PKA activity also mediate long-term (days) maintenance of synaptic facilitation (Wu, 1995).

The phosphorylation state of two identified neural specific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following 32Pi labeling in the recording chamber. B-50 phosphorylation is increased from 10 to 60 min, but no longer at 90 min after induction of long term potentiation. Neurogranin phosphorylation is detected only at 60 min. Increased phosphorylation is not found when long term potentiation is blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation is applied or tetanic stimulation fails to induce long term-potentiation. Thus both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation (Ramakers, 1995).

Metabotropic glutamate receptors (mGluRs) coupled to phosphoinositide hydrolysis, desensitize in response to prolonged or repeated agonist exposure; evidence suggests that this involves activation of protein kinase C (PKC). The present studies were undertaken to determine if cloned mGluR5 undergoes similar PKC-mediated desensitization and to investigate the molecular mechanism underlying PKC-induced desensitization. In Xenopus oocytes, both mGluR5a and mGluR5b show pronounced desensitization in response to a brief activation by glutamate. Pharmacological studies clearly suggest that this desensitization requires PKC-mediated phosphorylation. Analysis of PKC consensus phosphorylation site mutants suggests that PKC phosphorylates mGluR5 at multiple sites to induce a relatively rapid form of desensitization. Because mGluRs play important roles in synaptic plasticity and in excitotoxicity, this desensitization may be involved in the dynamic regulation of these processes (Gereau, 1998).

Protein phosphorylation plays important roles in the mechanisms underlying serotonin (5-HT)-induced presynaptic facilitation of Aplysia sensory neurons. To study mechanisms involved in facilitation, the pattern of protein phosphorylation in sensory neurons was investigation as a function of different durations of 5-HT. Two minutes and 1.5 hr treatments with 5-HT altered the phosphorylation of 5 and 10 proteins, respectively. These different duration treatments with 5-HT produced unique effects on the phosphorylation of different sets of proteins. This result suggests that cells may encode and measure the duration of a stimulus by the pattern of specific proteins that are phosphorylated or dephosphorylated. Because the changes in phosphorylation produced by 2 min treatments with 5-HT were not observed after 25 min treatments with 5-HT, mechanisms must exist for the transient phosphorylation of some proteins even when the 5-HT treatment persists. Anisomycin, an inhibitor of protein synthesis, blocked the effect of 1.5 hr treatments with 5-HT on the phosphorylation of six proteins but had no effect on the phosphorylation change of four other proteins. Both CPT-cAMP (an activator of protein kinase A) and PDAc (an activator of protein kinase C) mimicked the effects of 5-HT on four proteins. Interestingly, the effect of 5-HT on these four proteins did not require protein synthesis. CPT-cAMP, but not PDAc, mimicked the effect of 5-HT on one protein (L55). The effect of 5-HT on this protein appeared to require protein synthesis. Because both activation of PKA and protein synthesis are involved in the induction of long-term facilitation, protein L55 is a good candidate for a protein that might play a key role in long-term facilitation. The effects of 5-HT on four proteins were not mimicked by either CPT-cAMP or PDAc. This finding raises the interesting possibility that some effects of 5-HT are mediated by second-messenger systems other than PKA or PKC (Homayouni, 1995).

Activity-dependent long-lasting plasticity in hippocampus and neocortex includes long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Recent studies have confirmed theoretical predictions that the sensitivity of LTP- and LTD-inducing mechanisms are dynamically regulated by previous synaptic history. In particular, prior induction of either repeated short-term potentiations or LTP lowers the threshold for induction of LTD and raises the threshold for LTP. In the current study, transient activation of protein kinase C with phorbol ester is able to substitute for synaptic activity in priming synapses to exhibit enhanced homosynaptic LTD and to suppress the induction of LTP at Schaffer collateral synapses in area CA1 of hippocampal slices. This priming lasts 30 min, but not 3 hr, following phorbol application. These data suggest that a protein kinase C-sensitive phosphorylation site may be an activity-sensitive target mediating the rapid expression of LTP and LTD (Stanton, 1995).

Ca2+-regulated protein kinases play critical roles in long-term potentiation (LTP). To understand the role of Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission better, Ca2+/CaM was injected into hippocampal CA1 neurons. Ca2+/CaM induces significant potentiation of excitatory synaptic responses, which is blocked by coinjection of a CaM-binding peptide and is not induced by injections of Ca2+ or CaM alone. Reciprocal experiments demonstrate that Ca2+/CaM-induced synaptic potentiation and tetanus-induced LTP occlude one another. Pseudosubstrate inhibitors or high-affinity substrates of CaMKII or PKC block Ca2/CaM-induced potentiation, indicating that there is a requirement for CaMKII and PKC activity in synaptic potentiation. It is thought that postsynaptic levels of free Ca2+/CaM is a rate limiting factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of LTP (Wang, 1995).

The induction of several forms of long-term potentiation (LTP) of synaptic transmission in the CA1 region of the mammalian hippocampus is dependent on N-methyl-D-aspartate receptor activation and the subsequent activation of protein kinase C (PKC), but the mechanisms that underlie the regulation of PKC in this context are largely unknown. It is known that reactive oxygen species, including superoxide, are produced by N-methyl-D-aspartate receptor activation in neurons, and recent studies have suggested that some reactive oxygen species can modulate PKC in vitro. Thus, the role of superoxide was investigated in both the induction of LTP and the activation of PKC during LTP. Incubation of hippocampal slices with superoxide scavengers inhibits the induction of LTP. The effects of superoxide on LTP induction may involve PKC, because superoxide is required for appropriate modulation of PKC activation during the induction of LTP. In this respect, superoxide appears to work in conjunction with nitric oxide, which was required for a portion of the LTP-associated changes in PKC activity as well. These observations indicate that superoxide and nitric oxide together regulate PKC in a physiologic context and that this type of regulation occurs during the induction of LTP in the hippocampus (Klann, 1998).

Relationships were examined between spatial learning and hippocampal concentrations of the alpha, beta2, and gamma isoforms of protein kinase C (PKC), an enzyme implicated in neuronal plasticity and memory formation. Concentrations of PKC were determined for individual 6-month-old (n = 13) and 24-month-old (n = 27) male Long-Evans rats trained in the water maze on a standard place-learning task and a transfer task designed for rapid acquisition. The results show significant relationships between spatial learning and the amount of PKC among individual subjects: these relationships differ according to age, isoform, and subcellular fraction. Among 6-month-old rats, those with the best spatial memory are those with the highest concentrations of PKCgamma in the particulate fraction and of PKCbeta2 in the soluble fraction. Aged rats have increased hippocampal PKCgamma concentrations in both subcellular fractions, as compared with young rats, and memory impairment is correlated with higher PKCgamma concentrations in the soluble fraction. No age difference or correlations with behavior are found for concentrations of PKCgamma in a comparison structure, the neostriatum, or for PKCalpha in the hippocampus. Relationships between spatial learning and hippocampal concentrations of calcium-dependent PKC are isoform-specific. Moreover, age-related spatial memory impairment is associated with altered subcellular concentrations of PKCgamma and may be indicative of deficient signal transduction and neuronal plasticity in the hippocampal formation (Colombo, 1997).

Activation of the mitogen-activated protein kinase (MAPK) cascade plays an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. Hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity (Roberson, 1999).

A longstanding but still controversial hypothesis is that long-term depression (LTD) of parallel fiber-Purkinje cell synapses in the cerebellum embodies part of the neuronal information storage required for associative motor learning. Transgenic mice in which LTD is blocked by Purkinje cell-specific inhibition of protein kinase C (PKC) (L7-PKCI mutants) do indeed show impaired adaptation of their vestibulo-ocular reflex, whereas the dynamics of their eye movement performance are unaffected. However, because L7-PKCI mutants have a persistent multiple climbing fiber innervation at least until 35 d of age and because the baseline discharge of the Purkinje cells in the L7-PKCI mutants is unknown, factors other than a blockage of LTD induction itself may underlie their impaired motor learning. Therefore the spontaneous discharge of Purkinje cells was investigated in alert adult L7-PKCI mice as well as their multiple climbing fiber innervation beyond the age of 3 months. It was found that the simple spike and complex spike-firing properties (such as mean firing rate, interspike interval, and spike count variability), oscillations, and climbing fiber pause in the L7-PKCI mutants were indistinguishable from those in their wild-type littermates. In addition, it was found that multiple climbing fiber innervation does not occur in cerebellar slices obtained from 3- to 6-month-old mutants. These data indicate (1) that neither PKC inhibition nor the subsequent blockage of LTD induction disturbs the spontaneous discharge of Purkinje cells in alert mice, (2) that Purkinje cell-specific inhibition of PKC detains rather than prevents the developmental conversion from multiple to mono-innervation of Purkinje cells by climbing fibers, and (3) that as a consequence the impaired motor learning as observed in older adult L7-PKCI mutants cannot be attributable either to a disturbance in the baseline simple spike and complex spike activities of their Purkinje cells or to a persistent multiple climbing fiber innervation. It is concluded that cerebellar LTD is probably one of the major mechanisms underlying motor learning, but that deficits in LTD induction and motor learning as observed in the L7-PKCI mutants may only be reflected in differences of the Purkinje cell signals during and/or directly after training (Goossens, 2001).

Cerebellar LTD requires brief activation of PKC and is expressed as a functional downregulation of AMPA receptors. Modulation of vascular smooth-muscle contraction by G protein-coupled receptors (called Ca2+ sensitization) also involves PKC phosphorylation and activation of a specific inhibitor of myosin/moesin phosphatase (MMP). This inhibitor, called CPI-17, is also expressed in brain. The hypothesis that LTD, like Ca2+ sensitization, employs a PKC/CPI-17 cascade has been tested. Introduction of activated recombinant CPI-17 into cells produces a use-dependent attenuation of glutamate-evoked responses and occludes subsequent LTD. Moreover, the requirement for endogenous CPI-17 in LTD was demonstrated with neutralizing antibodies plus gene silencing by siRNA. These interventions have no effect on basal synaptic strength but block LTD induction. Thus, a biochemical circuit that involves PKC-mediated activation of CPI-17 modulates the distinct physiological processes of vascular contractility and cerebellar LTD. These results implicate a signaling pathway that coordinates PKC with the opposing phosphatase, via inhibition by phospho-CPI-17 (Eto, 2002).

The catalytic subunit of PP1 (PP1C) can complex with many different regulatory/targeting subunits to constitute holoenzymes with varying properties, and these holoenzymes cannot be further differentiated by drugs such as okadaic acid or related compounds. In contrast, CPI-17 selectively associates with MMP, consisting of PP1C and the myosin targeting subunit, and ectopic expression of CPI-17 induces inhibition of MMP in living cells, suggesting selective recognition against MMP. Indeed CPI-17 selectively associates with MMP in a rat cerebellar extract. When a constitutively active form of CPI-17 (thio-phospho-CPI-17, 1 µM) was added to the internal saline, a dramatic attenuation of glutamate-evoked inward currents was produced and this occluded subsequent LTD. Thus, endogenous, postsynaptic CPI-17 appears to be involved in cerebellar LTD. When taken together with the experiments that have used inactivating antisera and siRNA approaches to interfere with endogenous postsynaptic CPI-17, a signaling cascade involving PKC, activation of CPI-17 appears to be required for cerebellar LTD induction. A hypothesis of the molecular mechanism for cerebellar LTD is that the phosphorylation at Ser880 of GluR2 results in internalization of AMPA receptors. Inhibition of MMP by phospho-CPI-17, together with coincident PKC activation, might increase phosphorylation of Ser880 and thereby promote the internalization of GluR2-containing AMPA receptors or block their reinsertion in the synaptic plasma membrane (Eto, 2002).

Mammals can be trained to make a conditioned movement at a precise time, which is correlated to the interval between the conditioned stimulus and unconditioned stimulus during the learning. This learning-dependent timing has been shown to depend on an intact cerebellar cortex, but which cellular process is responsible for this form of learning remains to be demonstrated. This study shows that protein kinase C-dependent long-term depression in Purkinje cells is necessary for learning-dependent timing of Pavlovian-conditioned eyeblink responses (Koekkoek, 2003).

Among the subtypes of the Ca2+-dependent protein kinase C (PKC), which play a crucial role in long-term depression (LTD), both alpha and gamma are expressed in the cerebellar floccular Purkinje cells. To reveal the functional differences of PKC subtypes, the adaptability of ocular reflexes of PKCgamma mutant mice, which show mild ataxia and normal LTD, was examined. In mutant mice, gains of the horizontal optokinetic eye response (HOKR) are reduced. Adaptation of the HOKR is not affected but its retinal slip dependency is altered in mutant mice. Sustained 1-h sinusoidal screen oscillation, which induces a relatively large amount of retinal slips in both mutant and wild-type mice, increases the HOKR gain in wild-type mice but not in mutant mice. In contrast, exposure to 1 h of sustained slower screen oscillations, which induce relatively small retinal slips in mutant and wild-type mice, increases the HOKR gain in both mutant and wild-type mice. Adaptation of the HOKR of the mutant mice to slow screen oscillation and those of wild-type mice to fast and slow screen oscillations are all abolished by local applications of a PKC inhibitor (chelerythrine) within the flocculi. Electrophysiological and anatomical studies show no appreciable changes in the sources and magnitudes of climbing fibre inputs, which mediate retinal slip signals to the flocculus in the mutant mice. These results suggest that PKCgamma has a modulatory role in determining retinal slip dependency, and other PKC subtypes, e.g., PKCalpha, may play a crucial role in the adaptation of the HOKR (Shutoh, 2003).

Induction of cerebellar long-term depression (LTD) requires a postsynaptic cascade involving activation of mGluR1 and protein kinase C (PKC). Understanding of this process has been limited by the fact that PKC is a large family of molecules, many isoforms of which are expressed in the relevant postsynaptic compartment, the cerebellar Purkinje cell. LTD has been found to be absent in Purkinje cells in which the alpha isoform of PKC has been reduced by targeted RNA interference or in cells derived from PKCalpha null mice. In both of these cases, LTD can be rescued by expression of PKCalpha but not other PKC isoforms. The special role of PKCalpha in cerebellar LTD is likely to derive from its unique PDZ ligand (QSAV). When this motif is mutated, PKC no longer supports LTD. Conversely, when this PDZ ligand is inserted in a nonpermissive isoform, PKCgamma, it confers the capacity for LTD induction (Leitges, 2004).

The sequence of molecular events involved in cerebellar LTD induction is becoming a fairly complete story, but there are some details that remain undefined. Biochemical studies have suggested that only PKCalpha in the activated state can bind PICK1. One model is that PKCalpha, activated by Ca²⁺ and diacylglycerol from conjoint activation of voltage-gated Ca²⁺ channels and mGluR1/phospholipase C, binds PICK1. PICK1-bound PKCalpha then phosphorylates GluR2 ser-880, causing GRIP/ABP to dissociate. GRIP/ABP unbinding allows PICK1 to bind GluR2. PICK1-bound GluR2 is then primed for clathrin-mediated endocytosis (through mechanisms that have yet to be uncovered). Alternatively, once GluR2 is internalized by a constitutive process, it might be stabilized in an internal pool by PICK1 interactions. However, it should be cautioned that, while PKCalpha is required for LTD and phosphorylation of GluR2 ser-880 is also required, and the two are closely associated through PICK1, there is no direct evidence at present that proves that PKCalpha phosphorylates ser-880 as opposed to some other yet-to-be discovered critical residue. In this vein, it will be interesting to determine whether the translocation of PKCalpha to the plasma membrane of Purkinje cells, which is produced by PKC-activating phorbol esters, is blocked when either the QSAV domain of PKCalpha or the PDZ domain of PICK1 are mutated. Similarly, it would be interesting to determine whether introducing the QSAV motif into an LTD-nonpermissive isoform such as PKCgamma would cause it to become translocated to the membrane upon activation (Leitges, 2004).

While the present findings argue that PKCalpha is required for LTD and that PKCß and PKCgamma are not, it is not known whether PKCalpha activation is sufficient for LTD induction. It is formally possible that one or more of the nonclassical PKC isoforms expressed in Purkinje cells (delta, epsilon, eta, zeta, and lambda) may also function in cerebellar LTD (Leitges, 2004).

Cerebellar LTD is thought to constitute a portion of the engram for certain forms of motor learning, including adaptation of the vestibulo-ocular reflex, adaptation of the horizontal optokinetic response, and associative eyelid conditioning. A transgenic mouse in which a peptide inhibitor of all PKC isoforms is expressed selectively in Purkinje cells to block cerebellar LTD shows strong deficits in both rapid adaptation of the vestibulo-ocular reflex and acquisition of associative eyelid conditioning. A PKCgamma null mouse, which has normal cerebellar LTD, is not impaired in associative eyelid conditioning. This mouse has only mild impairments in adaptation of the horizontal optokinetic response, but adaptation is completely blocked by intracerebellar injection of a broad spectrum PKC inhibitor drug (in both PKCgamma and wild-type mice). It will be useful to test the hypothesis that PKCalpha null mice will be profoundly impaired in these three motor learning tasks as a consequence of cerebellar LTD blockade (Leitges, 2004).

Neural targets of PKC

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin (see Drosophila calmodulin) and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, the gene was disrupted in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. It is concluded that MARCKS is necessary for normal mouse brain development and postnatal survival (Stumpo, 1995).

MARCKS and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin regulated by PKC phosphorylation. The kinetics of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains (PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their phosphorylation occurs with positive cooperativity. Affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD peptide yield identical patterns of tryptic phosphopeptides; as in the case of MARCKS, all of the PKC phosphorylation sites in MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic phosphopeptides reveals that the first and third (but not the second) serines in the MRP PSCBD are phosphorylated by PKC. Both MARCKS and MRP bind dansyl-calmodulin with high affinity, with a Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disrupt the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties (Verghese, 1994).

A high density of transient A-type K+ channels is located in the distal dendrites of CA1 hippocampal pyramidal neurons and these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation. Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. The effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) were investigated on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette results in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also results in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affects the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, it was found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites (Hoffman, 1998).

One of the most prominent roles of metabotropic glutamate receptors (mGluRs) in the CNS is to serve as presynaptic receptors that inhibit transmission at glutamatergic synapses. Previous reports suggest that the presynaptic effect of group II mGluRs at corticostriatal synapses can be inhibited by activators of protein kinase C (PKC). Activation of PKC inhibits the ability of group II and group III mGluRs to regulate transmission at three major synapses in the hippocampal formation. Thus, this effect may be a widespread phenomenon that occurs at glutamatergic synapses throughout the CNS. This response is not limited to PKC-activating phorbol esters but activation of A3 adenosine receptors induces a PKC-dependent inhibition of group III mGluR function at the Schaffer collateral-CA1 synapse. In addition to inhibiting mGluR modulation of excitatory synaptic transmission, activation of PKC reduces inhibition of forskolin-stimulated cAMP accumulation by group II and group III mGluRs, suggesting that the effect of PKC on mGluR signaling is not specific to their effects on neurotransmitter release. This led to the hypothesis that PKC acts upstream from effector proteins regulated by mGluRs and acts at the level of the receptor or GTP-binding protein. Interestingly, PKC inhibits mGluR-induced increases in [35S]-GTPgammaS binding in cortical synaptosomes. These data suggest that PKC-induced inhibition of mGluR signaling may be mediated by the inhibition of coupling of mGluRs to GTP-binding proteins (Macek, 1998).

Syntaxin 1A (see Drosophila Syntaxin) inhibits GABA uptake of an endogenous GABA transporter in neuronal cultures from rat hippocampus and in reconstitution systems expressing the cloned rat brain GABA transporter GAT1. Evidence of interactions between syntaxin 1A and GAT1 comes from three experimental approaches: botulinum toxin cleavage of syntaxin 1A, syntaxin 1A antisense treatments, and coimmunoprecipitation of a complex containing GAT1 and syntaxin 1A. Protein kinase C (PKC), which modulates GABA transporter function, exerts its modulatory effects by regulating the availability of syntaxin 1A to interact with the transporter, and a transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC. These results suggest a new target for regulation by syntaxin 1A and a novel mechanism for controlling the machinery involved in both neurotransmitter release and reuptake (Beckman, 1998).

Protein kinase C (PKC) positively modulates NMDA receptor (NMDAR) currents. In contrast to previous reports, this study determines the importance of individual exons in the mechanism underlying the potentiation process by examining the complete set of eight naturally occurring splice variants expressed in Xenopus oocytes, both as homomers and as heteromeric NR1/NR2A or NR1/NR2B complexes. After PKC stimulation, homomeric currents demonstrate a high level of potentiation (approximately 500% of untreated baseline currents) that is reduced to a lower level (approximately 300% of baseline) in variants containing the first C-terminal exon (C1). An ANOVA shows that only C1 and no other exon or interaction of exons determine the degree of NMDAR current modulation by PKC. When recordings are performed in solutions in which barium replaces calcium, only the lower form of potentiation is observed, regardless of the splice variant exon composition. This suggests an important role for calcium in the PKC modulation of homomeric NMDA splice variant currents in which the C1 exon also participates. The effectiveness of the C1 exon to reduce the higher form of potentiation is modulated by heteromeric assemblies with NR2A heteromers yielding smaller levels of potentiation and a larger C1 exon effect, when compared with NR2B heteromers. The heteromers demonstrate the higher form of potentiation even in the absence of calcium. Furthermore, calcium has different effects in the potentiation of the heteromers depending on the NR2 subunit. This study refines the region of the NR1 subunit involved in a modulation crucial to the function of NMDA receptors and provides evidence that the NR2A and NR2B subunits realize this modulation differentially (Logan, 1999).

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits, yet the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. Whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices were used to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs are differentially affected by PKA and PKC. In pyramidal cells, PKA reduces mIPSC amplitudes and enhances the fraction of events decaying with a double exponential, whereas PKC has no effect. In contrast, in GCs PKA is ineffective, but PKC increases the peak amplitude of mIPSCs and also favors double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin reveals that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation (Poisbeau, 1999).

The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process have previously been unknown. The bHLH transcriptional repressor HES-1 has now been shown to be a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling. The MASH1 bHLH activator protein is a likely target for direct repression by HES-1. Previous studies have shown that NGF signaling induces the activation or localization of both cytoplasmic and nuclear PKC isoforms in PC12 and other cells. Given that both PKCs and at least some ribosomal S6 kinases are activated during NGF signaling, HES-1 may be a target for multiple kinases activated or functioning during NGF signaling (Strom, 1997).

Expression of specific protein kinase C isoforms correlates with cell fate in neural chicken embryo cells. Consequently, the effects of PKC activation by phorbol esters were tested on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes derived from 15-day-old chick embryos (E15CH). Short term treatment with the phorbol ester (TPA), which activates PKC-alpha/beta in E15CH, causes association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrates phosphorylation of many cytoskeletal proteins. Phosphorylation is blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increases cotranslational assembly of vimentin. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, is mitogenic and causes dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes have decreased activity of the astrocyte specific enzyme, glutamine synthetase, but have increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes (Mangoura, 1995).

AMPA receptor subunits interact with a PDZ domain-containing protein called PICK1, which is known to bind protein kinase C alpha (PKC alpha). PICK1 interacts with sequences within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif. The PDZ domain of PICK1 is required for the interaction; the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1 and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha, indicating that the same domain of PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2; short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals colocalization and an altered pattern of distribution for each protein, in comparison to the expression patterns when expressed individually. This novel interaction provides a possible regulatory mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha (Dev, 1999).

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuate LTD induction by glutamate/depolarization pairing, as do antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins (Xia, 2000).

Four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, have been identified as interactors with the kainate receptor (KAR) subunits GluR5_2b, GluR5_2c, and GluR6. Of these, it is shown that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCalpha can phosphorylate ct-GluR5_2b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. It is proposed that PICK1 targets PKCalpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses (Hirbec, 2003).

The finding that KARs and AMPARs can bind to a common pool of PDZ proteins suggests that these proteins may play important general roles in the regulation of glutamatergic synapses. Based on the present findings and previous work on AMPARs, it is possible to speculate on the molecular mechanisms that mediate the differential regulation of AMPARs and KARs by these PDZ proteins. In this scheme, AMPARs are secured in intracellular pools via association of the GluR2 subunit with GRIP and/or ABP. These 'gripped' receptors are immobile over the time course of the electrophysiology experiments. PICK1 exchanges for GRIP and targets PKCalpha, which then phosphorylates S880 of GluR2, thereby preventing the rebinding of GRIP. The S880-phosphorylated AMPARs are mobile and available for surface expression. It is proposed that KARs are also 'gripped' by GRIP, but in this case, PICK1-targetted, PKC-dependent phosphorylation stabilizes the GRIP interaction with GluR5/6 and anchors the receptors at the postsynaptic membrane. These data are entirely consistent with the observations that blockade of either GRIP or PICK1 binding, or inhibition of PKC, results in a rapid decrease in KAR-mediated synaptic currents. It is speculated that, whereas phosphorylation of S880 of GluR2 prevents GRIP binding, phosphorylation of S880 and/or S886 of GluR52b (and/or equivalent residues of GluR6) stabilizes GRIP binding and anchors the receptors at the synapse (Hirbec, 2003).

These differences in the molecular consequences of PKC-mediated phosphorylation of AMPARs and KARs can explain the differential regulation in opposite directions of the functional synaptic responses. The results showing that, at the same population of synapses, disruption of PDZ protein interactions results in an increase in EPSC_A and a simultaneous decrease in EPSC_K suggests that these proteins may act to regulate the relative proportions of AMPARs and KARs at synapses. Physiologically, given the distinct biophysical and functional profiles of AMPARs and KARs, the dynamic regulation of these interactions will play important roles in the modulation of basal glutamatergic synaptic transmission. Furthermore, it has been reported previously that some forms of developmental and activity-dependent synaptic plasticity involve a switch from functionally expressed KARs to AMPARs. The differential effects of PDZ-interacting proteins demonstrated here on these two receptor types provide an attractive molecular mechanism to account for these developmental and activity-dependent changes in the AMPAR and KAR complement at synapses (Hirbec, 2003).

Spine morphology is regulated by intracellular signals, like PKC, that affect cytoskeletal and membrane dynamics. This study investigated the role of MARCKS (myristoylated, alanine-rich C-kinase substrate) in dendrites of 3-week-old hippocampal cultures. MARCKS associates with membranes via the combined action of myristoylation and a polybasic effector domain, which binds phospholipids and/or F-actin, unless phosphorylated by PKC. Knockdown of endogenous MARCKS using RNAi reduces spine density and size. PKC activation induces similar effects, which are prevented by expression of a nonphosphorylatable mutant. Moreover, expression of pseudophosphorylated MARCKS is, by itself, sufficient to induce spine loss and shrinkage, accompanied by reduced F-actin content. Nonphosphorylatable MARCKS causes spine elongation and increases the mobility of spine actin clusters. Surprisingly, it also decreases spine density via a novel mechanism of spine fusion, an effect that requires the myristoylation sequence. Thus, MARCKS is a key factor in the maintenance of dendritic spines and contributes to PKC-dependent morphological plasticity (Calabrese, 2005).

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gα_s-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC₁R) also signals via Gα_q subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC₁R, Gα_q, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gα_q pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca²⁺, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).

Syndecan-4 (Syn4) is a heparan sulphate proteoglycan that is able to bind to some growth factors, including FGF, and can control cell migration. This study describes a new role for Syn4 in neural induction in Xenopus. Syn4 is expressed in dorsal ectoderm and becomes restricted to the neural plate. Knockdown with antisense morpholino oligonucleotides reveals that Syn4 is required for the expression of neural markers in the neural plate and in neuralised animal caps. Injection of Syn4 mRNA induces the cell-autonomous expression of neural, but not mesodermal, markers. Two parallel pathways are involved in the neuralising activity of Syn4: FGF/ERK, which is sensitive to dominant-negative FGF receptor and to the inhibitors SU5402 and U0126, and a PKC pathway, which is dependent on the intracellular domain of Syn4. Neural induction by Syn4 through the PKC pathway requires inhibition of PKCdelta and activation of PKCalpha. PKCalpha inhibits Rac GTPase and c-Jun is a target of Rac. These findings might account for previous reports implicating PKC in neural induction and suggest a link between FGF and PKC signalling pathways during neural induction (Kuriyama, 2009).

Syn4 modulates FGF signalling through its extracellular domain (containing the GAG-binding region, which will present heparin sulphates to which FGF is expected to bind) and by an effect on the transduction of intracellular signals. The data support the idea that FGF is required for neural induction and that Syn4 is a likely modulator, by showing that the inhibition of FGF receptor and of MAPK activity impair neural induction by Syn4. Syn4 could act as a co-receptor of the FGF receptor or as a presenter of the FGF ligand, through binding of FGF to the GAG side-chains, to facilitate the activation of FGF receptor (Kuriyama, 2009).

However, Syn4 also plays a separate role in neural induction involving PKC. It is proposed that this involves inhibition of PKC{delta} and activation of PKC{alpha}, and that PKC{alpha} is an inhibitor of the small GTPase Rac. Since the BMP-inhibiting effects of FGF act through MAPK, this pathway could account for the BMP-inhibition-independent role of FGF signalling in neural induction. Rac is a well-known regulator of cell migration that acts by controlling actin polymerisation, but has not previously been implicated in neural induction. Evidence that Rac can control JNK activity suggested the hypothesis that Syn4/PKC{alpha} might inhibit Rac activity by an increase in AP-1 (c-Fos/c-Jun) activity that is mediated through inhibition of JNK (Kuriyama, 2009).

PKC{alpha} has never been connected with the signalling pathways now known to be involved in neural induction. It was originally shown that PKC{alpha} is activated and translocated to the membrane during neural induction, and it was suggested that this is required to confer neural competence on the ectoderm. This study has confirmed and extended these observations by showing that expression of PKC{alpha} in ventral ectoderm or in animal caps can act as a neuralising signal and that PKC{alpha} activity is regulated by interactions with Syn4 and PKC{delta}. PKC{delta} appears to work as a repressor of PKC{alpha}, whereas Syn4 appears to be required for PKC{alpha} activity; however, it was also shown that PKC{alpha} is required for the neuralising activity of Syn4. Thus, this finding allows proposal of a link between the PKC and FGF pathways, both of which have been identified as being involved in neural induction (Kuriyama, 2009).

These observations have parallels in studies of migrating cells. Syn4 interacts with PIP2, and this stabilises the oligomeric structure of Syn4 and promotes the association of PKC{alpha} and Syn4; the catalytic domain of PKC{alpha} binds to the cytoplasmic domain of Syn4, and PKC{alpha} is 'superactivated'. This interaction between PKC{alpha} and Syn4 provides a satisfactory explanation for the observation that neural induction by Syn4 requires PKC{alpha} and vice versa. In addition, during cell migration, PKC{delta} phosphorylates Syn4, decreases its affinity for PIP2 and abolishes its capacity to activate PKC{alpha}. This study has found a similar negative regulation between PKC{alpha} and PKC{delta} during early neural plate development (Kuriyama, 2009).

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