InteractiveFly: GeneBrief

CASK ortholog : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The ability of CaMKII to act as a molecular switch, becoming Ca²⁺ independent after activation and autophosphorylation at T287, is critical for experience-dependent synaptic plasticity. This study shows that Drosophila CASK, the homolog of mammalian CASK, also known as Camguk or Caki, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's Calmodulin (CaM) binding domain. Phosphorylation of T306 blocks Ca²⁺/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch (Hodge, 2006).

Autophosphorylation of CaMKII at a site in the N terminus of its autoregulatory domain (T287 in Drosophila and T286 in mammalian αCaMKII) confers Ca²⁺-independent activity on the enzyme. This switch-like property of the kinase is crucial to its role in long-term potentiation and memory formation in mammals and flies, and generation of the autonomous form of the kinase can be stimulated by a number of different behaviors and activity paradigms. Interestingly, in mice, both mutations that prevent T286 phosphorylation, and transgenes that increase constitutive activity have been shown to block plasticity. This implies that the level of constitutive activity needs to be tightly controlled to remain in a range optimal for learning. This balance has been believed to be exerted by the positive effects of Ca²⁺/CaM and the negative effects of phosphatases. To date, no proteins other than calmodulin have been shown to influence the development of autonomous activity via T287 autophosphorylation (Hodge, 2006).

The function of autophosphorylation in the C terminus of the regulatory domain within the CaM binding region (T306 in Drosophila and T305 in mammalian αCaMKII) has been more mysterious, but also has consequences for plasticity. In the test tube, with purified kinase, this phosphorylation occurs only after T287 phosphorylation and removal of CaM with EGTA because the site is protected from phosphorylation by bound CaM. With purified kinase, this means that pT306 is only found in doubly phosphorylated pT287, pT306 enzyme. Recently, a novel mechanism for phosphorylation of T306 in the absence of pT287 has been described (Lu, 2003). dCASK interacts with the regulatory domain of the kinase and, when the CaM binding site of the kinase is unoccupied (e.g., when synaptic calcium is low), stimulates the kinase to autophosphorylate at T306. This reaction releases CaMKII from the dCASK complex in a form that is incapable of binding Ca²⁺/CaM and has been suggested to provide a mechanism for downregulation of the activatable kinase pool at quiescent synapses. The effects of dCASK on T287 phosphorylation have not been addressed, and modulation of this site would have important consequences for plasticity (Hodge, 2006).

This study presents evidence that dCASK can also act as an activity-dependent modulator of the levels of constitutively active CaMKII in vivo. CaMKII pT287 autophosphorylation occurs within the dodecameric holoenzyme and requires Ca²⁺/CaM to be bound to both the subunit doing the catalysis and the subunit that contains the substrate threonine. This cooperativity implies that modifications that alter the ability of Ca²⁺/CaM to bind would affect the ability of the kinase to become Ca²⁺ independent. Because it promotes phosphorylation of T306, interaction with dCASK regulates T287 phosphorylation by altering the occupancy of CaM binding sites in the holoenzyme. In vivo this postsynaptic interaction provides a synapse-specific mechanism to alter the probability of generating autonomous activity that is controlled by the activity history of the synapse. This makes dCASK an important gain controller for the CaMKII molecular switch (Hodge, 2006).

dCASK, the Drosophila homolog of the mammalian MAGUK scaffold protein CASK and C. elegans Lin-2, is a synapse-specific regulator of the ability of CaMKII to become Ca²⁺ independent via T287 autophosphorylation. The transition to Ca²⁺ independence has been shown to be a critical part of CaMKII's role in neural plasticity. The plasticity-inducing effects of constitutively active CaMKII have been documented at the cellular and behavioral levels in both vertebrates and invertebrates. This feature of CaMKII's activity is fundamental to its role in the neuron, and both too much and too little constitutive activity have deleterious effects on learning. Regulation of both the transition to the Ca²⁺-independent state and the maximum achievable level of constitutive activity is therefore likely to be important for brain function. dCASK is the first protein other than CaM to be shown to modulate this process (Hodge, 2006).

The mechanism underlying dCASK's ability to impose gain control on CaMKII T287 autophosphorylation is based on its ability to stimulate autophosphorylation of the kinase's CaM binding domain at T306 in low-calcium/low-activity conditions. Phosphorylation of T306 (T305 in mammalian αCaMKII) was first seen in vitro with purified kinase and only occurs when the kinase is first rendered constitutively active by T287 phosphorylation and then has Ca²⁺/CaM stripped by addition of EGTA. This results in a doubly phosphorylated (pT287 + pT306) enzyme that is active but cannot bind CaM. In contrast, dCASK-stimulated T306 phosphorylation does not require previous T287 phosphorylation and can therefore produce a monophosphorylated (pT306) enzyme that is inactive and cannot bind CaM (Lu, 2003; Hodge, 2006).

Monophosphorylation of T306 would have two major consequences for CaMKII activity. The first is that individual pT306 subunits within a holoenzyme cannot be activated by Ca²⁺/CaM. In a neuron, this will produce a linear decrease in the level of CaMKII activity elicited by a calcium pulse since each subunit is an independent kinase. The second consequence is more subtle, but perhaps more important because of the special role of autonomously active CaMKII in plasticity. T306 monophosphorylation decreases T287 phosphorylation, because T287 phosphorylation obligatorily occurs between subunits within a holoenzyme and requires CaM binding to both catalytic and substrate subunits. The cooperativity of T287 phosphorylation with respect to CaM binding means that there is a greater than stoichiometric disruption of T287 phosphorylation with increasing pT306. Thus, T306 phosphorylation has a greater impact on T287 autophosphorylation than on Ca²⁺-stimulable activity for intermediate levels of pT306 within a holoenzyme (Hodge, 2006).

In Drosophila, there is evidence for behavioral defects that may be a result of alterations in CaMKII activity. Long-term memory of odor-shock conditioning requires CaMKII and is associated with large increases in the amount of total protein in specific brain areas (Ashraf, 2006). Associative courtship suppression has been shown to require antennal lobe CaMKII activity and to be enhanced by overexpression of constitutively active CaMKII in the antennal lobes. These studies suggest that both the total amount of CaMKII and the level of Ca²⁺-independent activity can be altered by plasticity-inducing events (Hodge, 2006).

These two modifiable parameters operate on different time scales and may have different roles. Autophosphorylation is very fast while new protein synthesis can take minutes to hours and is likely to be more important in long-term memory. In the retina, changes in total CaMKII levels were evident with the chronic exposure to a light:dark cycle, but changes in CaMKII autophosphorylation that were independent of kinase level also occurred. In the deeper reaches of the optic system, changes in autophosphorylation appeared to be the dominant effect. The insignificant change in CaMKII levels that were observed between mated and virgin males may be due to the very fast processing of the males after copulation or to the fact that pT287 was averaged over the entire antennal lobe. It is possible that there are glomerulus-specific changes in pT287 or total CaMKII that were not detected (Hodge, 2006).

There may be some short-term behaviors that are driven primarily by changes in autophosphorylation, such as suppression of courtship during training with a mated female. In this behavior, overexpression of a Ca²⁺-dependent form of the kinase has no effect, implying that it is the Ca²⁺-independent kinase rather than total kinase activity that is important. The enhanced courtship suppression caused by constitutively active kinase is dose dependent and is manifested by a reduction in initial courtship index that parallels the level of autonomous kinase during the 1 hr training period. At extremely high doses, such as those achieved by the strong GAL4 driver GH146, courtship is completely suppressed and therefore no longer plastic. This indicates that there is a limited range over which CaMKII constitutive activity supports plasticity. Cellular mechanisms that regulate T287 phosphorylation are therefore critical to maintaining animals within that range (Hodge, 2006).

When activity-dependent autophosphorylation is examined in dCASK null animals, the in vivo role of the dCASK/CaMKII interaction can be dissected. Basal levels of pT306 in dCASK-deficient animals are decreased. Presumably, the residual pT306 in these animals is a result of autophosphorylation secondary to T287 phosphorylation, and most of the pT306 therefore would be expected to be found in doubly phosphorylated pT287 + pT306 kinase. The profound activity-dependent decrease in pT306 in null animals is likely due to this dependence on primary T287 phosphorylation (Hodge, 2006).

Is the dCASK/CaMKII interaction important for behavior? Basal levels of pT287 are increased in dCASK null animals, presumably as a secondary effect of the decrease in monophosphorylated pT306 subunits. For neurons in which the absolute level of constitutive CaMKII activity is important for behavioral or cellular plasticity, this basal elevation of pT287 would be expected to have consequences since neurons would be closer to their threshold for the plasticity process. Given the dependence of suppression on constitutive CaMKII activity, the elevation of pT287 in dCASK-deficient males would therefore be expected to reduce initial courtship. Consistent with this, a slight reduction weas observe in courtship of anesthetized mature virgin females by dCASK-deficient males compared to wild-type. Since dCASK has many roles in the cell, it is difficult to assign this reduction solely to the change in pT287, but it is consistent with the robust effects of dCASK on CaMKII and the role of CaMKII in courtship behavior. Genetic rescue with a UAS-linked transgene (which is unlikely to provide exactly the correct amount of dCASK) would be uninformative on this issue since there appears to be a directly dose-dependence of dCASK level with pT306, and both decreases and increases in CaMKII activity can affect behavior (Hodge, 2006).

When neuronal circuits are activated in the context of this higher basal level of pT287, the response to activity in primary cells of the circuit is blunted, as if there is a ceiling effect. The inability to increase the level of constitutive activity over this elevated baseline might also affect plastic processes in neurons where the biochemical processes subserving plasticity rely on the incremental change over baseline rather than on some absolute level of constitutive activity. There is evidence in Drosophila for such mechanisms in associative memory formation of courtship conditioning and in hippocampus where it has been shown that LTP is occluded by overexpression of active CaMKII. Changes in both the baseline and stimulable levels of T287 phosphorylation could disrupt the ability of neurons to respond to plasticity-inducing signals (Hodge, 2006).

Alterations in CaMKII autophosphorylation also appear to have consequences for circuit function. Mushroom bodies in Drosophila receive information about visual stimuli indirectly. In wild-type animals, light does not stimulate phosphorylation of T287 in this neuropil. In dCASK-deficient animals, there is inappropriate activation of the kinase in the mushroom body calyx. This suggests that dCASK also has a role in controlling spread of information within neuronal circuits, perhaps by dampening CaMKII activation (Hodge, 2006).

The results from biochemical assays and intact animal studies support a model in which the normal function of the dCASK/CaMKII interaction is to allow the activity history of the synapse to alter the probability with which CaMKII can become Ca²⁺ independent. At synapses where activity has been low, the dCASK/CaMKII interaction will decrease CaM binding in the holoenzyme, making it less able to initiate T287 phosphorylation even after a strong calcium pulse. In synapses that have been active, Ca²⁺/CaM protects CaMKII T306 from autophosphorylation, and the kinase can robustly respond to calcium influx. This makes dCASK an important regulator of plasticity. How this protein is localized to synapses and how its availability for interaction with CaMKII might be regulated will be important to determine (Hodge, 2006).

Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, previous work has shown that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). The present study shows that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the alpha'/beta' Kenyon cells. Using a biochemical approach, this study identified the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, this study shows that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the alpha'/beta' neurons. Finally, using a double RNAi approach, it was demonstrated that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, these results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species (Silva, 2020).

AD is the principal neurodegenerative disorder affecting the elderly, and it is characterized by amyloid β (Aβ) deposition derived from proteolytic processing of amyloid precursor protein (APP). A pathological hallmark of AD is a progressive memory decline that correlates intimately with synaptic loss. One of the main hypotheses for the cognitive deficits observed in AD is thus a dysfunction of synapses leading ultimately to synaptic loss and alteration of neural network activity. Therefore, it is essential to understand the physiological role of APP at the synapse. APP is a transmembrane protein expressed on both sides of the synapse (Wang, 2009). The APP extracellular domain can mediate dimerization across the synapse or interact with extracellular matrix components, growth factors and receptor-like proteins. These interactions are involved in synapse stabilization during development and also in regulating synapse plasticity in mature neuronal networks (Montagna, 2017). APP can undergo two types of proteolytic processing, including the non-amyloidogenic pathway, which is initiated by α-secretase and produces a secreted form of APP (sAPPα), and the amyloidogenic pathway, which successively involves β- and then γ-secretase to release Aβ peptide and an APP intracellular C-terminal domain (AICD). Although the manner in which proteolytic processing of APP and its derivatives interferes with neuronal physiology has been extensively studied, little is known about the function of APP intracellular domain at the synapse or its synaptic partners (Silva, 2020).

Studies in mammals suggest that APP can interact via its intracellular domain with synaptic MAGUK proteins such as X11, CASK, or PSD-95. MAGUK proteins are involved in the assembly, maintenance and remodeling of the scaffolding in synaptic compartments mainly via regulation of the targeting of receptors and ion channels to the synapse. Therefore, understanding the interactions between APP and MAGUKs should help decipher the synaptic function of APP (Silva, 2020).

The three mammalian orthologs APP, APLP1 and APLP2 are partially functionally redundant, whereas Drosophila expresses a single APP homolog named APP-like (APPL) that has been implicated in olfactory memory (Bourdet, 2015; Goguel, 2011) and visual memory (Rieche, 2018). APPL is strongly expressed in the adult mushroom body (MB), the main olfactory memory center in insects. Previous work has investigated the function of APPL in Drosophila aversive olfactory memory (Bourdet, 2015; Goguel, 2011; Preat, 2016). However, whether the APP synaptic partners and their interactions might contribute to memory is still unexplored. Several MAGUK homologs in Drosophila have been identified such as dX11, CASK/Caki, and Discs-large (Dlg). Similar to its mammalian counterpart, dX11 binds APPL, and both are necessary for synaptic remodeling at the Drosophila neuromuscular junction (Ashley, 2005). Drosophila CASK regulates CaMKII activity, interacts with dX11. Mammalian Dlg1/SAP97, Dlg2/PSD-93, Dlg3/SAP102 and Dlg4/PSD-95 share similarities with the fly Dlg proteins DlgA and DlgS97), which are encoded by a single dlg gene. Both CASK and Dlg play key roles in neurotransmission, synaptogenesis and plasticity (Silva, 2020).

This study has aimed to decipher the role of APPL and its synaptic partners in appetitive olfactory memory. Additionally, to investigate the AICD interactome, this study used a proteo-liposome recruitment method and found that AICD interacts with the MAGUK synaptic proteins X11, CASK and Dlg. It was then found in flies that APPL, CASK and Dlg are required specifically in the same neuronal subpopulation (the α'/β' KCs) for appetitive LTM. Finally, a double RNAi strategy was used to demonstrate that genetic interaction between APPL and MAGUKs is critical for appetitive LTM. To determine whether this memory deficit could be due to a major disorganization of the synaptic structure, both the pre-synaptic and the post-synaptic sites of MB α'/β' neurons were investigated using confocal immuno-labeling of synaptic proteins (Silva, 2020).

Previous work has showed that APPL is required in the α/β and γ KCs for aversive LTM. Additional work demonstrated that APPL is required for aversive LTM and MTM in the DPM neurons (Turrel, 2017), a pair of serotonergic and GABAergic neurons that project to each of the MB lobes, where they connect both pre- and post-synaptically to the KCs. This study has found that APPL is also required in another form of long-lasting protein synthesis-dependent memory, appetitive LTM, albeit surprisingly in a different subpopulation of KCs, the α'/β' neurons. The requirement of APPL in α'/β' but not in the other KCs for appetitive LTM suggests that it has a specific role in appetitive LTM consolidation, as consolidation of appetitive LTM requires synaptic neurotransmission from α'/β'. Interestingly, recurrent activity of the α'/β'-DPM loop has been described as necessary to consolidate appetitive memories, with LTM eventually being stored in the α/β neurons. An involvement of APPL in memory consolidation may rely on transsynaptic APPL interactions and may also contribute to the molecular support of the α'/β'-DPM loop. Eventually, such a role of APPL in memory consolidation through transsynaptic interaction would be consistent with published research in mammals. Indeed, at the cellular level APP is expressed in pre- and postsynaptic compartments and can form trans-dimers that have been suggested as necessary for synaptic function (Wang, 2009). In addition, perturbation of APP function by intraventricular infusion of an antibody against APP induced memory impairments only when it was performed during the memory consolidation phase of a passive avoidance task. As shown for other synaptic cell adhesion molecules, the regulation of APP expression at the neuronal membrane is critical for hippocampal-dependent memory consolidation in the dentate gyrus, suggesting a potential involvement of APP in synaptic remodeling. Altogether, the present findings combined with research on mammalian models suggest that APP might have a conserved function across species in memory consolidation processes via transsynaptic interactions (Silva, 2020).

This study has shown that in addition to the previously known X11 proteins, the intracellular domain of APP interacts with other scaffolding proteins (CASK, Dlg2/PSD-93 and Dlg4/PSD-95). However, it is not clear whether these proteins interact directly with APP or if their interactions are mediated by the X11 adaptor proteins, as it has been described for CASK. This study demonstrates that the Drosophila homologs of these proteins, i.e. CASK and Dlg, are required in the same neuronal subpopulation as APPL for proper long-term memory. Several arguments in favor of an APPL/X11/CASK/Dlg macromolecular complex can be found in previous studies on mammals or Drosophila. In the Drosophila visual system, the MAGUK complex Lin-7/Dlg/CASK is involved in synaptic stabilization and the interaction between CASK and Dlg proteins has been described as direct (Soukup, 2013). The role of CASK in the recruitment of Dlg1 to the membrane in various cell types has been confirmed in a recent study showing that the N-terminal domain of Dlg1 is critical for its interaction with CASK (Porter, 2019). In mammals, the existence of the APP/X11/CASK ternary complex has also been documented, and the regulation of neuronal excitability through potassium Kir2 channels involves a macromolecular complex consisting of Lin-7/SAP97/CASK/X11. Altogether these studies demonstrate that the interaction between the Dlg, CASK and X11 proteins is both possible and functionally relevant for neuronal physiology. Finally, CASK binds to the Dlg protein SAP97 in mammalian hippocampal neurons to regulate its conformation state and thus its role in glutamate receptor trafficking and insertion at the synapse. Thus, the existence of the APPL/X11/CASK/Dlg macromolecular complex is consistent with previously published reports, and even if this study only demonstrate the genetic interaction between APPL, CASK and Dlg for appetitive LTM, it is likely that such a complex exists in Drosophila α'/β' MB neurons. Complementary studies using genetic tools to impair the interactions between these proteins such as overexpression of an interfering peptide corresponding to the N-terminus of Dlg as in Porter (2019) could bring the confirmation that protein-protein interactions between APPL/X11/CASK/Dlg are required for appetitive LTM via synaptic stabilization of the α'/β' neurons. The present study looked at synaptic organization using confocal microscopy and immuno-labeling of either pre-synaptic proteins of the active zone or post-synaptic scaffold proteins. The levels of the analyzed pre- or post-synaptic proteins were not affected by the concomitant knockdown of APPL and MAGUKs in the adult α'/β' neurons, indicating no obvious alterations in MB synaptic structure. It is noted that as expected this study observed a significant decrease in Dlg levels in the MB calyx upon knockdown of APPL;Dlg in α'/β' neurons, demonstrating the efficacy of the knockdown. To determine the requirement of these proteins for pre- or post-synaptic subtle organization, higher-resolution imaging studies would be required. However, such modification might be difficult to observe if, as suggested by the role of CASK and Dlg proteins, the APPL/X11/CASK/Dlg macromolecular complex is involved in synaptic stabilization specifically when synapses are modified during a plastic event, and not as a basal mechanism for synaptic organization and formation as described for APPL and FasII interactions at the NJM (Silva, 2020).

Interestingly, the Dlg protein SAP97 is involved in trafficking the α-secretase ADAM10 to the synapse through direct interaction, consequently regulating APP processing and the production of the neurotrophic and neuroprotective secreted-APPα fragment. Previous work has shown in Drosophila that a secreted fragment of APPL is involved in aversive memory, as well as KUZBANIAN, the Drosophila homolog of ADAM10 (Bourdet, 2015). Therefore, an APPL/X11/CASK/Dlg supramolecular complex could also be involved in recruiting α-secretase at the synaptic site as well as generating sAPPα (Silva, 2020).

The subcellular localization of APP and MAGUK interactions in the KCs is still an open question. In Drosophila, Dlg and CASK are known to be present at both the pre- and postsynaptic compartments. However, APPL has been described mainly in the neuropil of KCs. Furthermore, the APPL binding protein X11 targets APPL to the axonal compartments and excludes it from MB dendrites via endocytosis. These data suggest that the APPL-MAGUKs complexes would be localized in the α'/β' KC axonal compartment (i.e. the α'/β' lobes), which is importantly also the site of the DPM/KC dialog for appetitive LTM consolidation (Silva, 2020).

In conclusion, this work highlights a novel role of APPL and its synaptic partners in appetitive long-term memory in Drosophila. Genetic interactions between APPL and the MAGUKs are critical for appetitive LTM in the α'/β' KCs, a neuronal sub-population known to be involved in the consolidation of appetitive LTM. A model is proposed in which the role of the interactions between APPL, CASK and Dlg might be the synaptic stabilization of the α'/β'-DPM loop through transsynaptic interactions (Silva, 2020).

REGULATION

Regulation of dopamine release by CASK- modulates locomotor initiation in Drosophila

CASK is an evolutionarily conserved scaffolding protein that has roles in many cell types. In Drosophila, loss of the entire CASK gene or just the CASK-β transcript causes a complex set of adult locomotor defects. This study shows that the motor initiation component of this phenotype is due to loss of CASK-β in dopaminergic neurons and can be specifically rescued by expression of CASK-β within this subset of neurons. Functional imaging demonstrates that mutation of CASK-β disrupts coupling of neuronal activity to vesicle fusion. Consistent with this, locomotor initiation can be rescued by artificially driving activity in dopaminergic neurons. The molecular mechanism underlying this role of CASK-β in dopaminergic neurons involves interaction with Hsc70-4, a molecular chaperone previously shown to regulate calcium-dependent vesicle fusion. These data suggest that there is a novel CASK-β-dependent regulatory complex in dopaminergic neurons that serves to link activity and neurotransmitter release (Slawson, 2014: PubMed).

Protein Interactions

A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. A protein-protein interaction domain, called the L27 domain, has been identified which is present twice in mLin-2/CASK. This study reports the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identifies the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. A novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, contains a region highly related to the SAP97 N terminus and binds Camguk, a Drosophila orthologue of mLin-2/CASK. These data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice (Lee, 2002; full text of article).

A schematic portrayal of the recognized protein interaction domains in mLin-2/CASK shows the two L27 domains between the CKII domain and PDZ domain; an alignment of L27 domains is also portrayed. Included are the deduced amino acid sequences of MAGUKs, including mLin-2/CASK, Drosophila Camguk, PALS2/VAM-1, and mLin-7. This alignment revealed two adjacent regions of high homology, which were named the L27N and L27C domains and has been previously reported as L27A and L27B (Doerks, 2000), respectively. After examining its sequence similarity to the adjacent L27C domain, which mediates interaction with mLin-7, it was postulated that the L27N domain was also a novel protein-protein interaction domain that mediates homo- or heterodimerization. To test this hypothesis, several proteins consisting of fusions of GST and the L27N and L27C domains of mLin-2/CASK, mLin-7, and PALS2 were constructed and an in vitro affinity binding assay was performed to examine each domain's ability to bind either Myc-tagged mLin-2/CASK or mLin-7. GST fusion proteins containing both L27N and L27C were used as positive controls. The GST fusion protein containing the minimal L27C domain of mLin-2/CASK interacted with mLin-7 while the L27N domain, previously considered to be part of the mLin-7 interaction domain, did not. In addition, the minimal L27 domain of mLin-7 interacted with mLin-2/CASK. The assay also revealed that the L27N domain of mLin-2/CASK does not function as a homodimerization domain. Next, tests were performed to see whether the L27N domain of mLin-2/CASK interacts with another protein. The GST-mLin-2/CASK L27N domain bound to a protein doublet with a molecular mass of 120 to 130 kDa, identified as SAP97 (Lee, 2002).

It was of interest to determine whether or not the interaction between the N terminus of SAP97 and the L27N domain of mLin-2/CASK was conserved in other organisms. It was postulated that, if the L27N domain-mediated interaction between MAGUK proteins is one of the mechanisms that govern the targeting of membrane proteins, it should be evolutionarily conserved. To test the hypothesis, other proteins containing sequences that are similar to the first 75 aa residues of SAP97 were sought using BLAST analysis. As a result, a small Drosophila gene product, called CG1730 (accession no. AAF48037) was identified, which contained N-terminal sequences that are highly homologous to the N-terminal domain of SAP97. After analyzing the genomic sequence encoding CG1730 and its chromosomal location, it was realized that the sequence was located near the beginning of the gene encoding Drosophila Dlg (CG1725) . Subsequently the full-length CG1730 protein, containing 198 aa, was cloned by PCR using an EST (LP07807) as a template. The protein CPD, component protein of Dlg, was named for its potential role as a missing N-terminal domain of the Drosophila Dlg protein, which is also present in other orthologues such as C. elegans DLG-1. A sequence alignment of different SAP97 homologues reveals the high degree of sequence similarity within the first 65 aa (Lee, 2002).

The possibility was examined that a splice variant of the Drosophila Dlg gene might contain regions of the CPD gene due to their close genomic location. To identify the transcript, RNA from two different mixed stages of Drosophila embryos (0 to 8 h and 8 to 16 h) was isolated and reverse transcribed. The CPD gene-Dlg gene hybrid 5' end was amplified by reverse transcription-PCR from cDNA using primers specific for CPD gene sequences, and the 3' end was amplified by using primers specific for the Dlg (aa 1 to 493) coding sequence. When primers specific for sequences generating full-length CPD (aa 1 to 198) and full-length DLG (aa 1 to 978) were used as internal controls, all three fragments with the predicted sizes were amplified. Subsequent sequencing of those DNA fragments revealed the existence of a unique CPD-Dlg hybrid transcript consisting of the first 146 aa of CPD fused to aa 30 of Dlg, resulting in a protein of 1,094 amino acids (Lee, 2002).

Since the SAP97 N terminus mediates interaction with mLin-2 via the L27N domain, whether regions of CPD interacted with the L27N domain of mLin2/CASK as well as with Drosophila Camguk, the orthologue of mLin-2/CASK, was tested by using a GST fusion protein affinity binding assay. Bacterially expressed GST fusion proteins were incubated with lysate from MDCK cells stably expressing either Myc-mLin-2/CASK or HEK 293 cell lysate transiently expressing Myc-Camguk proteins (aa 1 to 500). Full-length CPD interacted with both mammalian and Drosophila proteins. The binding region was narrowed to the first 65 aa of CPD in agreement with the alignment. It was also determined that the first 26 aa residues were necessary for the interaction. Upon deletion of these residues, the interaction between CPD and either Camguk or mLin-2/CASK was significantly reduced or abolished, respectively. It was also found that the L27N domain of mLin-2/CASK is conserved in Camguk by demonstrating that the L27N domain of Camguk can bind CPD (Lee, 2002).

This study identified a novel interaction between the L27N domain of mLin-2/CASK and SAP97, a mammalian homologue of Drosophila Dlg tumor suppressor protein and another MAGUK family member. Recently, the SH3 domain of SAP97 was reported to associate with the GUK domain of mLin-2/CASK (Nix, 2000). However, these experiments showed that association between the L27N domain of mLin-2/CASK and the N terminus of SAP97 was stronger than the interaction between the GUK and SH3 domains of these proteins. It is possible that there is more than one point of attachment between these proteins. Furthermore, their association site may vary upon binding to other interaction partners since, like other MAGUK proteins, SAP97 is also a multidomain scaffolding protein that recruits transmembrane proteins and signaling molecules to the plasma membrane sites (Lee, 2002).

SAP97 contains three PDZ domains as well as one SH3 domain, one Hook domain, and one GUK domain. In neuronal cells, SAP97 is expressed in presynaptic membranes and mediates the clustering of receptor molecules, such as the voltage-gated Shaker K+ channels and the NR2 subunits of the N-methyl D-aspartate receptor, by interacting with PDZ domains. It is also expressed at the basolateral membrane of epithelial cells and at the lymphocyte immune synapse, but its precise role in epithelia remains to be characterized. The PDZ domains of SAP97 interact with adenomatous polyposis coli, PTEN tumor suppressor protein (1), and several viral oncoproteins, while the GUK domain interacts with the GKAP/SAPAP1/DAP1 family. There have been reports suggesting the possibility of oligomerization of SAP97 through the N-terminal protein sequences (aa 106 to 120), which are also conserved in other SAP proteins such as PSD-95 and SAP102. In addition, the functional significance of the N termini of these MAGUK proteins in targeting and receptor clustering has been previously demonstrated. The first 65 aa residues of SAP97 have been reported to direct selective subcellular localization and the attachment to the cytoskeleton assembly at sites of cell-cell contact. However, the protein that interacts with the N terminus of SAP97 and that is responsible for its targeting to the lateral membrane was unknown. This report using dominant-negative mLin-2/CASK revealed that the membrane localization of SAP97 was dependent on its association with mLin-2/CASK. When dominant-negative protein mLin-2/CASK(1-612) was overexpressed, SAP97 failed to properly localize to the lateral membrane, while the dominant-negative effect was removed upon deletion of the interacting L27N domain. The hypothesis that SAP97 basolateral membrane localization was mediated through its interaction with the mLin-2/CASK protein was further supported by demonstrating that expression of a dominant-negative mLin-2/CASK missing the Hook domain also results in mislocalization of SAP97 in the epithelia. However, it was difficult to show complete mislocalization of all SAP97 proteins due to high expression of the endogenous full-length mLin-2/CASK protein present in cells (Lee, 2002).

Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation

CaMKII is critical for structural and functional plasticity. Camguk (Cmg, also known as Caki, the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca2+/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca2+/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca2+/CaM, can provide a localized source of active kinase. When Ca2+/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses (Lu, 2003).

Cmg, mammalian CASK, and C. elegans Lin-2 form a MAGUK subfamily that is defined by an N-terminal CaMKII-like domain in addition to the SH3, PDZ, and guanylate kinase domains typical of the MAGUK family. In mammalian neurons, CASK has been shown to be localized to synapses both pre- and postsynaptically where it associates with cell surface molecules like neurexins, calcium channels, and syndecans, the cytoskeletal protein 4.1, PSD95, and other adaptor molecules. CASK may also participate in signaling to the nucleus by directly interacting with a transcription factor (Lu, 2003 and references therein).

Cmg null animals have impaired locomotion, but their ability to learn had not been tested. Modification of courtship behavior in Drosophila has been used to test both associative and nonassociative memory formation; CaMKII is required for both. Exposure of a male to a mated female leads to decreased courtship of a subsequently presented virgin. This suppression reflects formation of an associative memory that requires mushroom body and central complex circuits. Courtship suppression also occurs during the training period via a non-mushroom body central circuit. Habituation to a courtship stimulating cue can be demonstrated after training with immature males. In these behavioral assays, the locomotor defects of the Cmg null flies could only cause an underestimate of behavioral defects since 'failure' in the assay is signified by an increase in courtship activity. Wild-type, Cmg null, and cmg deficiency heterozygotes were tested for mated female conditioning. All genotypes were normal for associative memory formation when compared to males that had been sham conditioned in an empty chamber. Behavior during the training period, however, was abnormal; Cmg null males fail to decrease courtship during training. Deficiency heterozygotes are intermediate. To determine if the Cmg null has a habituation defect, the response to immature males, who emit courtship promoting pheromones distinct from the female type, was examined. Cmg null animals fail to habituate. Courting Cmg null males have normal initial courtship levels and the ability to discriminate between virgin and mated females, indicating that peripheral sensory pathways are intact (Lu, 2003).

Association of Cmg with CaMKII is stimulated by ATP bound to the CaMKII catalytic domain. At physiological levels of ATP, Cmg and CaMKII are likely to be associated. ATP hydrolysis in the presence of Ca2+/CaM leads to autophosphorylation of CaMKII on T287 and production of a Cmg-localized constitutively active kinase. Cmg can also promote ATP hydrolysis by CaMKII in the absence of Ca2+/CaM. Under these conditions and without prior T287 autophosphorylation, CaMKII preferentially autophosphorylates its own CaM binding domain. This reaction leads to the fast release of the CaMKII in a form that cannot be activated by Ca2+/CaM. The ability of genetic manipulation of Cmg, PP2A, and synaptic activity levels in vivo to alter the amount of postsynaptic CaMKII that is in the inactivated state argues strongly that these reactions are physiologically relevant and define a new mechanism for the regulation of CaMKII activity (Lu, 2003).

What is the function of this interaction in vivo? One possible role is to provide a basis for maintaining differences between active and inactive synapses. The effect of cmg on courtship conditioning, a behavior known to require CaMKII, is suggestive of a role for this interaction in behavioral plasticity. Differences in synaptic strength can fine tune networks and are crucial to most memory models. Local CaMKII inactivation by Cmg could serve to decrease the gain on CaMKII-mediated calcium signaling at synapses that have been inactive. At low Ca2+, T306 autophosphorylation would occur, and inactive kinase would be released from Cmg. This provides a 'use it or lose it' mechanism for preserving synapse-specific strength differentials, since CaMKII tethered to Cmg at synapses that see higher Ca2+ levels would be protected from inactivating autophosphorylation by bound CaM. This complexed kinase can undergo T287 autophosphorylation, resulting in constitutive kinase activity and a slowed CaM dissociation rate that would further prolong the lifetime of the complex. Whether the Cmg-mediated inactivation of CaMKII operates locally or globally remains to be determined and likely depends on the ability of the inactivated kinase to escape from complexes with its other binding partners in the postsynaptic density (Lu, 2003).

Establishment of a pool of autophosphorylated CaMKII that cannot be directly stimulated by Ca2+/CaM also provides a mechanism for phosphatase activity to regulate CaMKII. In vitro, both PP1 and PP2A are able to dephosphorylate the CaM binding domain of CaMKII. In Drosophila, PP2A is the relevant in vivo phosphatase for postsynaptic pT306. The opposing actions of Cmg and PP2A provide machinery for dynamic regulation of the maximum level of calcium-activatable CaMKII at synapses (Lu, 2003).

In mouse, knockin of a CaMKII T305D mutation blocks LTP and learning and decreases postsynaptic localization of the kinase, suggesting that this mutant is unable to interact with one or more of its normal synaptic binding partners. Inability to undergo inactivating autophosphorylation (T305VT306A knockin) is associated with a lower threshold for LTP and loss of behavioral fine tuning. These data argue that, in mammals, autophosphorylation of the CaM binding domain of CaMKII is also an important regulatory mechanism for both function and localization of CaMKII. Whether in mammals CASK (the Cmg homolog) is the critical synaptic binding partner and regulator of synaptic T305 autophosphorylation is unknown (Lu, 2003).

This study also demonstrates a novel binding mechanism for CaMKII on a MAGUK scaffolding protein. It is proposed that this interaction consists of two discrete steps. First, binding of ATP to the catalytic domain of CaMKII reveals an interaction surface on the kinase that forms an initial bond to the Cmg protein. This initial interaction can occur either in the presence or absence of Ca2+/CaM, implying that it does not involve the C-terminal CaM binding sequences of the autoinhibitory domain. The ability of a peptide corresponding to the N terminus of the autoinhibitory domain to partially block complex formation suggests that this region, which is known to be involved in regulation of ATP binding or the catalytic domain surface that the N terminus of the autoinhibitory domain binds to, is the site of this initial interaction (Lu, 2003).

Once an initial interaction is established, bound ATP is no longer required. This could be due to a stabilization of the ATP-bound conformation by Cmg binding. Stabilization of the open form of the kinase autoregulatory domain is postulated to occur after Ca2+/CaM-dependent NR2B binding, since this interaction is insensitive to CaM stripping. Another possibility for the loss of ATP requirement is that a secondary interaction occurs that abrogates the need for the initial interaction. Such a secondary interaction does occur, but this does not rule out a concurrent stabilization of the ATP-bound conformation (Lu, 2003).

The secondary interaction that forms after ATP-dependent binding of Cmg involves the C-terminal region of the autoinhibitory domain, including the CaM binding sequences. Three lines of evidence suggest that this secondary interaction provides the most stable form of the complex. (1) N-terminal peptides only partially block complex formation, while peptides that contain the CaM binding domain completely block complex formation. (2) CaMKIIs with mutations of T306/7 do not bind to Cmg at all. These mutants are fully capable of binding ATP, but the exposure of the initial binding domain is insufficient to maintain a salt-stable interaction in the absence of a normal CaM binding domain. The failure of the relatively subtle threonine to serine mutant to bind suggests a direct interaction of Cmg with T306/7. (3) The natural release mechanism of this complex is via autophosphorylation of the CaM binding domain. Protection of this region by Ca2+/CaM can prevent both the autophosphorylation and dissociation of CaMKII (Lu, 2003).

The two-step association mechanism and the autophosphorylation-regulated dissociation of CaMKII from Cmg provide a new way in which CaMKII can be localized and regulated using its autoinhibitory domain. As defined for catalytic activation, the regulatory region of CaMKII is a small but complicated protein interaction domain. It is becoming clear that this region can support multiple intra- and intermolecular interactions that have profound effects on local CaMKII activity and synaptic plasticity (Lu, 2003).

Camguk/CASK enhances Ether-a-go-go potassium current by a phosphorylation-dependent mechanism

Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. This study demonstrates that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-a-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II. CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. These findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel (Marble, 2005; full text of article).

Increased transmitter release and aberrant synapse morphology in a Drosophila Calmodulin mutant; potential interaction of Calmodulin and Caki

Mutations have been generated in the single Drosophila Calmodulin gene and the effects of these mutations then examined on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam^3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. The effects on motor neuron function and transmitter release of many behavioral mutations, particularly those affecting ion channels, are enhanced by application of the K+ channel-blocking drug quinidine. This drug, when applied at a concentration of 0.1 mM, completely and specifically blocks the delayed rectifier K+ current in the Drosophila larval muscle. Application of quinidine enhances the effects of ion channel mutations, such as Shaker and Hyperkinetic, on the duration of motor nerve terminal depolarization and transmitter release. The phenotypes of other excitability mutants, such as inebriated and pushover, are also enhanced by quinidine application. Application of quinidine has no significant effect on excitatory junctional current (EJC) amplitude in the wild-type control larvae or in larvae heterozygous for Cam mutations. In contrast, quinidine application to Cam^3c1/Cam^null at the three lowest external [Ca2+] levels tested causes an approximately threefold increase in EJC amplitude. The muscle responsiveness to neurotransmitter is shown to be normal, and that the increased amplitude EJCs observed in Cam^3c1/Cam^null larvae reflect increased transmitter release. One might imagine that impaired activation of a K+ channel in Cam^3c1/Cam^null might have no observable phenotypic consequences under otherwise normal conditions; however, in combination with quinidine, which could block a distinct, functionally redundant K+ channel, this effect could lead to increased nerve terminal depolarization and increased Ca2+ influx into the nerve terminal. The observation that the effects of Cam^3c1/Cam^null occur only at low external [Ca2+] is consistent with this view: the effects of most excitability mutations, including Shaker, Hyperkinetic, and inebriated, are also revealed only at low external [Ca2+]. This observation has been proposed to result from the activation of a Ca2+-activated K+ current at higher external [Ca2+]. Alternatively, Cam^3c1/Cam^null could be defective in Ca2+ buffering. In this view, a broadening of the action potential conferred by quinidine application combined with reduced Ca2+ buffering as a consequence of Cam3c1/Camnull could lead to increased transmitter release (Arredondo, 1998).

Synaptic bouton structure at motor nerve terminals is altered. Structural abnormalities are present in the nerve terminals of Cam^3c1/Cam^null larvae in muscle 13 of abdominal segments 3 - 5. In particular, rather than cascading into a string of distinct type I and II boutons as in the control larvae, the terminal arbor of the Cam^3c1/Cam^null larvae forms a thickened, or large, misshapen structure with few distinct boutons. The large structure results in a reduced number of boutons and a nearly complete lack of terminal branching in muscle 13. In comparison to control larvae, no abnormalities in the structure of nerve terminals on muscles 6, 7, or 12 have been observed. Despite this altered synapse morphology, muscle 13 synaptic transmission in Cam^3c1/Cam^null larvae resembles the muscle 6 properties. This observation raises the possibility that this defective bouton might still be functional. Although mutations in several neuronal signaling genes confer defects in the pattern of motor neuron innervation of the target muscle, the morphological defects of Cam^3c1/Cam^null at the nerve terminal differ from any that have been reported previously and do not appear to result from the same mechanisms as the "activity-dependent" increases in synaptic bouton number or axonal branching Rather, the phenotype observed appears to result from defects in the formation of distinct boutons at the proper locations along the muscle surface. It is unclear why this defect in bouton formation is observed only in muscle 13. Proper synapse formation on muscle 13 may be more sensitive to altered CaM function, or perhaps distinct mechanisms control bouton formation in muscle 13 vs. other muscles. Analysis of the genes required for proper bouton formation is less characterized than for axon pathfinding or growth cone guidance. The results presented here suggest a role for CaM in this process (Arredondo, 1998).

These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC or Rutabaga). Mutations in rutabaga reduce facilitation and post-tetanic potentiation at the larval neuromuscular junction, whereas activation of an inhibitory domain of CaMKII confers a number of behavioral, electrophysiological, and anatomical defects, including defects in courtship conditioning, an abnormal spontaneous firing of motor axon action potentials, increases in axon branching and transmitter release, and a reduction in facilitation and augmentation. Cam3c1/Camnull larvae do not show these phenotypes; e.g., Cam^3c1/Cam^null larvae possess a normal number of axon branches and display normal EJC amplitude (in the absence of quinidine) and normal paired pulse facilitation. Similarly, whereas application of quinidine to Cam^3c1/Cam^null substantially increases evoked transmitter release, quinidine application has little or no effect on CaMKII-inhibited larvae. Thus, it is unlikely that the Cam^3c1 mutation is exerting its effects via either CaMAC or CaMKII, but rather via an alternative target. One alternative target, the Drosophila CaM-activated protein kinase Caki, is expressed in the central nervous system; mutants defective in caki exhibit behavioral defects related to those described here (Martin, 1996). The catalytic domain of Caki shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm'. Thus, the Cam^3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target, distinct from CaMKII or CaMAC (Arredondo, 1998).

Neuron-specific protein interactions of Drosophila CASK-beta are revealed by mass spectrometry

Modular scaffolding proteins are designed to have multiple interactors. CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, has been shown to have roles in many tissues, including neurons and epithelia. It is likely that the set of proteins it interacts with is different in each of these diverse tissues. This study asked if within the Drosophila central nervous system, there were neuron-specific sets of CASK-interacting proteins. A YFP-tagged CASK-beta transgene was expressed in genetically defined subsets of neurons in the Drosophila brain known to be important for CASK function, and proteins present in an anti-GFP immunoprecipitation were identified by mass spectrometry. Each subset of neurons had a distinct set of interacting proteins, suggesting that CASK participates in multiple protein networks and that these networks may be different in different neuronal circuits. One common set of proteins was associated with mitochondria, and it was shown that endogenous CASK-beta co-purifies with mitochondria. CASK-beta posttranslational modifications were also determined for one cell type, supporting the idea that this technique can be used to assess cell- and circuit-specific protein modifications as well as protein interaction networks (Mukherjee, 2014).

DEVELOPMENTAL BIOLOGY

Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. A new Drosophila CaM kinase gene named caki has been cloned. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active since it causes a 3-fold increase in the level of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter in a co-transfusion assay. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm' (Martin, 1996; full text of article).

Drosophila Camguk (Cmg) is a member of the CAMGUK subfamily of the MAGUK family of proteins which are localized at cell junction and other plasma membrane specialized regions, from worms to mammals. The protein structure of Cmg, as the other CAMGUK proteins, is characterized by only one PDZ domain and an additional CaM kinase domain, similar to CaMKII. While the mammalian ortholog CASKs play an important role in synaptic protein targeting and in synaptic plasticity, the Drosophila Cmg role is unknown. To study its potential role, a detailed analysis was performed of mRNA distribution of the Drosophila cmg gene at cellular and developmental level, during embryonic, larval, pupal and adult stages. The transient cmg transcription in midgut and Malpighian tubules may suggest a potential function in cell junction formation and in epithelial tissue patterning. Interestingly, cmg transcription increases substantially during embryonic neuroblast proliferation, becoming predominant in the developing central nervous system (CNS) during embryonic and postembryonic development stages and in the mature brain. In addition, a high transcriptional level was detected in the eye imaginal discs and in the adult retina, demonstrating a specific and continuous expression of cmg in neuroblasts and photoreceptor neurons, from the onset of cytodifferentiation. These findings suggest that Cmg could play a potential role in transmembrane protein targeting, particularly in synapses. These observations suggest the existence of a common highly conserved mechanism involved in forming and maintaining proper synaptic protein targeting, which are fundamental features of synaptic plasticity, learning and memory. Through its function, the CaM kinase domain-containing Cmg may be involved in signal transduction cascade. Its potential relation to Calmodulin and CaMKII is discussed (Lopes, 2001).

Autophosphorylation of T287 and T306 on CaMKII is regulated independently in different adult brain areas

Assessment of NMJ autophosphorylation gave openned the opportunity to probe the CaMKII/dCASK interaction under very defined conditions at a well-characterized synapse to demonstrate that dCASK is a synapse-specific and activity-dependent modulator of T287 phosphorylation. Changes in dCASK function (Lu, 2003), CaMKII levels (Ashraf, 2006), and CaMKII autophosphorylation (Mehren, 2004) have been shown to be important for learning-related neuronal plasticity of the adult Drosophila brain. To determine if regulation of CaMKII autophosphorylation could be mediated by dCASK in adults, sections of wild-type male brains were double-stained with antibodies that recognized total CaMKII protein or were specific to CaMKII phosphorylated at either T287 or T306. CaMKII protein is found in all neuropil regions and at lower levels in somatic regions surrounding them (Takamatsu, 2003). Autophosphorylation of CaMKII at T287 and T306 occurred primarily in synaptic areas. dCASK has previously been shown to be expressed relatively uniformly (Lopes, 2001; Martin, 1996) in all synaptic regions in adult Drosophila brain (Hodge, 2006).

Although staining with the two phosphospecific antibodies occurred in all the regions containing CaMKII, the absolute intensity of staining did not directly parallel total kinase levels. Regions in which total CaMKII appeared similar could have very different levels of autophosphorylation. It was also clear that the patterns of staining seen with the anti-pT306 and anti-pT287 antibodies were distinct. Obvious examples of this are the retina and the lamina where the R1-6 photoreceptors synapse. In retina, pT306 is very high, but there is less pT287. In the laminar synaptic region, the opposite is true. In other regions such as the antennal lobe (AL) and mushroom body (MB), calyx phosphorylation levels were roughly equivalent. This is made clear by examination of the ratios of phosphospecific antibody CaMKII staining in various brain regions. While these numbers have no intrinsic meaning in terms of site occupancy for either phosphorylation, the ratio does reflect relative occupancy within a region. These data strongly suggest that autophosphorylation of T287 and T306 are regulated independently and that properties of particular circuits or subcellular locations can modulate autophosphorylation (Hodge, 2006).

EFFECTS OF MUTATION

dCASK alters the ability of CaMKII to autophosphorylate at T287

dCASK, the Drosophila homolog of C. elegans Lin-2 and mammalian CASK, is a member of the MAGUK family of scaffolding proteins that contains an N-terminal CaMKII-like domain along with the canonical MAGUK PDZ, SH3, and GUK domains. dCASK has been shown to physically interact with CaMKII and regulate phosphorylation of CaMKII T306 postsynaptically in Drosophila (Lu, 2003). The levels of pT287 CaMKII were examined in wild-type and dCASK-deficient fly head extracts by Western blotting with a phosphospecific antibody to T287 to determine if dCASK affected autophosphorylation of this site in vivo. Animals carrying a deletion of the dCASK locus have very little pT306 compared to wild-type animals, but total CaMKII levels are unchanged (Lu, 2003). Phosphorylation of T287 is also sensitive to dCASK levels but is modulated in opposite direction from T306; deficiency animals with no dCASK protein have significantly higher pT287 levels than wild-type (Hodge, 2006).

Acute activity modulates CaMKII autophosphorylation in a dCASK-dependent manner

Synaptic phosphorylation of T286 in mammals is activity regulated. To determine if CaMKII autophosphorylation is regulated by acute changes in activity in Drosophila and whether this is modulated by dCASK, the third-instar larval neuromuscular junction (NMJ) was examined. CaMKII pT306 levels are regulated by both dCASK and chronic changes in neuronal activity at this synapse (Lu, 2003). To find out if acute activity could regulate CaMKII autophosphorylation, a strong stimulus was applied to the motor axon on one side of the animal and staining was performed with an antibody that recognizes total kinase and an antibody that recognizes either pT287 or pT306. CaMKII and pT306 staining were examined of two muscle 12 NMJs from the same animal (the unstimulated side, and the stimulated side). A small decrease in pT306 is can be seen with stimulation, suggesting that synaptic activity may activate a pT306 phosphatase. NMJs from an animal stained with anti-CaMKII and anti-pT287 show a significant increase in pT287 on the stimulated side (Hodge, 2006).

To quantify these changes, immunoreactivity of phosphospecific antibodies was normalized to total synaptic kinase. Comparison of the stimulated side to the unstimulated side in wild-type animals demonstrates that acute activity slightly decreases pT306 in wild-type and dCASK-deficient animals. In animals that overexpress dCASK postsynaptically, activity causes a much more significant percent decrease in pT306. This may be due to the fact that these animals have elevated baseline levels of pT306 (Lu, 2003). Alternatively, dCASK might directly or indirectly regulate activity of a phosphatase in periods of high activity (Hodge, 2006).

The effect of acute activity on pT287 levels is qualitatively different than its effects on pT306. Wild-type flies have a modest but significant increase in pT287, and postsynaptic overexpression of dCASK blocks this effect. The NMJs of animals that lack dCASK have an exaggerated response to activity, which quadruples the pT287 content of CaMKII compared to wild-type. These data support the idea that dCASK acts as a gain control on activity-dependent T287 phosphorylation (Hodge, 2006).

Modulation of CaMKII T287 autophosphorylation by activity and dCASK is synapse specific

The ability to independently regulate the strength of individual synapses on a neuron confers computational strength to the nervous system. CaMKII autophosphorylation within the postsynaptic apparatus is believed to be synapse specific in mammalian neurons and to contribute to synapse-specific plasticity (for review see Merrill, 2005). For dCASK modulation of CaMKII autophosphorylation to be useful for such processes it must also be synapse specific. To investigate this in Drosophila, electrical activity or dCASK levels were altered selectively in muscle 12 and the phosphorylation of CaMKII at synapses on muscles 12 and 13 that are made by a single type II motor neuron was examined (Hodge, 2006).

Phosphorylation of T306 is increased at muscle 12 synapses containing more postsynaptic dCASK relative to synapses made by the same neuron onto muscle 13. Decreasing electrical activity in the postsynaptic cell by expressing dORKΔC, a hyperpolarizing potassium channel, had little effect on T306. This may be due to the low levels of endogenous dCASK at type II synapses (Hodge, 2006).

Phosphorylation of T287 was decreased in manipulated synapses compared to synapses made by the same neuron onto a normal postsynaptic cell. Both electrical silencing with dORKΔC and postsynaptic overexpression of dCASK led to lower pT287 levels specifically in muscle 12 synapses. None of the manipulations changed total CaMKII levels (Hodge, 2006).

In adult brain, dCASK modifies the effect of sensory input on T306 levels

To determine if a natural stimulus that alters the level of activity within a neural pathway could affect CaMKII autophosphorylation specifically within that circuit, pT306 levels in sections of animals that had been dark reared were compared to those found in animals raised in a 12 hr:12 hr light:dark cycle. Levels of pT306 measured at boutons of adult muscles were unaffected by light conditions but were decreased by dCASK deficiency, consistent with previous work at the larval NMJ (Lu, 2003). In the CNS of wild-type animals, phosphorylation of T306 was decreased by light input in the retina and in the medulla where R8 photoreceptor axons terminate in neuropil layer M3. Light did not significantly affect the level of pT306 at R1–R6 or R7 photoreceptor synapses in the lamina and medulla of wild-type animals (Hodge, 2006).

In the retina of dark-reared dCASK mutant animals, pT306 levels were decreased in comparison to wild-type. In lamina and medulla, pT306 levels at the R1–R6 and R7 synapses in dark-reared animals were not significantly affected, but at medullar R8 photoreceptor synapses it was decreased. For dCASK-deficient animals, light profoundly decreased pT306, even below levels seen in wild-type. This may reflect a difference in the “set point” of multiple kinase autophosphorylation sites in these animals (Hodge, 2006).

dCASK regulates the dynamic range of the T287 switch

Increases in neuronal activity are known to stimulate autophosphorylation of CaMKII at T287 and convert it into a Ca²⁺/CaM-independent enzyme. To test the ability of light to stimulate T287 phosphorylation in the adult optic system, head sections stained with anti-pT287 were examined. In wild-type adult fly heads, light significantly increased pT287 in the retina and at synaptic regions in the optic lobes corresponding to R7 and R8 photoreceptor termini. The light-dependent increase in pT287 in the axon terminal fields of R1–R6 photoreceptors was not statistically significant (Hodge, 2006).

In the optic system of dark-reared animals containing no dCASK protein, pT287 levels were in general higher than wild-type (for R8 and R7 synaptic terminals p < 0.001). Increased neuronal activity due to light stimulus was able to increase pT287 level to that found in wild-type animals, but no higher. Since basal pT287 levels are elevated in this genotype, this suggests that the dynamic range of T287 phosphorylation is blunted in dCASK-deficient animals (Hodge, 2006).

The retina, lamina, and the medulla are areas of the brain specialized to receive and process light information, and as such it makes sense that they show activity-induced changes in CaMKII autophosphorylation. To test the specificity of the light-induced increases in pT287, levels of phosphorylation were measured in nonoptic areas. Areas of the nervous system such as the NMJ or antennal lobes that have no optic inputs and areas in the CNS that only indirectly receive information about light such as the mushroom bodies might be expected to have a smaller response. Quantification of bouton pT287 at the adult NMJ shows that there is no difference between light- and dark-reared animals. Likewise, in wild-type animals, there is no enhancement of T287 autophosphorylation in the antennal lobes or the mushroom bodies (Hodge, 2006).

dCASK regulates the set point and fidelity of the T287 switch

While light does not affect CaMKII autophosphorylation at nonoptic synapses, the level of dCASK protein does. Multifactoral ANOVA of the entire data set indicates that there is a significant genotype effect. Animals with no dCASK protein have significantly higher levels of pT287 in both light- and dark-reared conditions at the NMJ, a synapse which does not get either direct or indirect information about light levels. Similarly, levels of pT287 are slightly, but not significantly, higher in antennal lobes of dCASK-deficient animals compared to wild-type. In neither of these synaptic regions does light alter pT287 in dCASK-deficient animals (p > 0.05) (Hodge, 2006).

The situation in the mushroom bodies is more interesting. This brain region may receive inputs from optic centers via a polysynaptic pathway. In mushroom body calyx, light is able to evoke an increase in pT287 in animals that do not have dCASK even though in wild-type pT287 levels in this region are insensitive to light. This suggests that the lack of dCASK is allowing downstream neurons to become hypersensitive to light, supporting a role for dCASK in activity-dependent gain control of CaMKII T287 autophosphorylation at the circuit level as well as the subcellular level. It cannot be distinguish from these data if the circuit level effects are realized via the effects of CaMKII on neuronal activity or are a consequence of loss of other functions of dCASK (Hodge, 2006).

Increases in CaMKII protein levels contribute to light-induced changes in CaMKII in the retina

Activity-driven autophosphorylation is not the only mechanism for altering CaMKII activity in vivo. In mammals, the mRNA for CaMKII is locally translated in response to light stimulation, and this translation is required for some forms of plasticity. CaMKII protein can also translocate between subcellular compartments in the mammalian CNS. Activity-dependent control of CaMKII levels has also been demonstrated in the Drosophila CNS (Ashraf, 2006). At NMJ of muscles 12 and 13, synaptic levels of CaMKII were not altered significantly by manipulation of activity or dCASK, indicating that changes in autophosphorylation were the predominant effector of activity-dependent CaMKII modulation at these synapses. To address this issue in the CNS, CaMKII immunoreactivity was quantified in the optic system in dark-reared and light-reared wild-type males. In the retina of animals reared in a 12 hr:12 hr light:dark cycle, CaMKII levels were 122% ± 6% of dark-reared animals. CaMKII levels in the lamina at the R1–R6 synapses and in the medulla were not significantly higher. When the pT306 and pT287 changes in the retina are normalized to account for the change in CaMKII, there is still a significant increase in pT287 (light-reared pT287/total CaMKII is 149% of dark-reared) and an even greater decrease in pT306 as a percentage of total kinase (light-reared pT306/total CaMKII is 39% of dark-reared). These data suggest that modulation of both CaMKII levels and autophosphorylation occur in the sensory cells of the optic system, but that at synapses within the CNS, the primary activity-driven changes are in autophosphorylation (Hodge, 2006).

Complex behavior can alter T287 phosphorylation

The data has indicated that light, a single channel sensory input, regulates CaMKII autophosphorylation in the optic system. It was also of interest to determine if more complex experiences could also alter T287 phosphorylation. Drosophila courtship is a stereotyped behavior that is driven by visual, olfactory, gustatory, and tactile cues. Males can learn to suppress courtship during training with a mated female, and this suppression is blocked by inhibition of CaMKII in antennal lobes and enhanced by expression of Ca²⁺-independent CaMKII in the same region. To determine if mating experience acutely altered pT287 levels, antennal lobe pT287 in head sections of males that had been raised in isolation was compared to males that were raised in isolation but allowed to copulate with a mature virgin female immediately before they were sectioned. In wild-type males, mating caused a significant increase in pT287. Total CaMKII levels were not significantly increased. Animals lacking dCASK had a higher basal level of pT287 but were not able to significantly increase it with mating. These results are consistent with dCASK functioning in this brain region to regulate the level and dynamics of CaMKII constitutive activity (Hodge, 2006).

Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, a caki null mutant was studied in Drosophila. By means of electrophysiological methods, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system were analyzed in adult flies. In caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ is increased, the response of the giant fiber pathway to continuous stimulation is impaired, and electroretinographic responses to single and continuous repetitive stimuli is altered and optomotor behavior is abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function (Zordan, 2005; full text of article).

Genetic interaction between Neurexin and CAKI/CMG is important for synaptic function in Drosophila neuromuscular junction

Neurexins are neuron-specific cell surface molecules thought to localize to presynaptic membranes. Recent genetic studies using Drosophila have implicated an essential role for the single Drosophila Neurexin in the proper architecture, development and function of synapses in vivo. However, the precise mechanisms underlying these actions are not fully understood. To elucidate the molecular mechanism of Neurexin in vivo, dnrx and caki mutant flies, combined with various methods, were used to analyze locomotion, synaptic vesicle cycling and neurotransmission of neuromuscular junctions. Dneurexin (DNRX) was found to be important for locomotion through a genetic interaction with the scaffold protein, CAKI/CMG, the Drosophila homolog of vertebrate CASK. Similar to its mammalian counterparts, DNRX is essential for synaptic vesicle cycling, which plays critical roles in neurotransmission at neuromuscular junctions (NMJ). However, this interaction appears not to be required for the synaptic targeting of DNRX, but may instead be needed for proper synaptic function, possibly by regulating the synaptic vesicle cycling process (Sun, 2009).

Central regulation of locomotor behavior of Drosophila melanogaster depends on a CASK isoform containing CaMK-like and L27 domains

Genetic causes for disturbances of locomotor behavior can be due to muscle, peripheral neuron, or central nervous system pathologies. The Drosophila homolog of human CASK (also known as Caki or Camguk) is a molecular scaffold that has been postulated to have roles in both locomotion and plasticity. These conclusions are based on studies using overlapping deficiencies that largely eliminate the entire CASK locus, but contain additional chromosomal aberrations as well. More importantly, analysis of the sequenced Drosophila genome suggests the existence of multiple protein variants from the CASK locus, further complicating the interpretation of experiments using deficiency strains. This study generated small deletions within the CASK gene that eliminate gene products containing the CaMK-like and L27 domains (CASK-β), but do not affect transcripts encoding the smaller forms (CASK-α), which are structurally homologous to vertebrate MPP1. These mutants have normal olfactory habituation, but exhibit a striking array of locomotor problems that includes both initiation and motor maintenance defects. Previous studies had suggested that presynaptic release defects at the neuromuscular junction in the multigene deficiency strain were the likely basis of its locomotor phenotype. The locomotor phenotype of the CASK-β mutant, however, cannot be rescued by expression of a CASK-β transgene in motor neurons. Expression in a subset of central neurons that does not include the ellipsoid body, a well-known pre-motor neuropil, provides complete rescue. Full length CASK-β, while widely expressed in the nervous system, appears to have a unique role within central circuits that control motor output (Slawson, 2011).

Previous work has implicated disruption of CASK in a suite of behavioral deficits. These studies, however, all suffered from the same limitation, as the null animals used in these experiments had lost both of the proteins encoded at the CASK locus, and also had disruptions of other third chromosome genes. To address this, a new set of isoform-specific mutants was generated, so as to better dissect the behavioral contribution of the CASK homolog in the fly. While these mutants shared similarities with the 307/313 flies used in previous studies, they were strikingly different in other ways (Slawson, 2011).

The CASK locus encodes two distinct MAGUKs: CASK proteins have been defined as a subfamily of MAGUK protein with a unique N-terminal CaMK-like domain in addition to the more typical L27, PDZ, SH3 and GUK domains. The CaMK-like domain has a constitutively active structure that grants it low levels of Ca2+/calmodulin-independent activity against complexed substrate. Unlike all other known kinases, this activity is inhibited by Mg2+. This domain also participates in regulation of CaMKII autophosphorylation. CASK-β would therefore be expected to have properties different from other MAGUK proteins, and it represents the true ortholog of vertebrate CASK. The Drosophila genome project annotation of the CASK locus predicts that in addition to canonical CASK proteins (CASK-β), this locus has separately initiated transcripts that encode shorter proteins with a unique N-terminal region that is followed by PDZ, SH3 and GUK domains (CASK-α). These proteins are, in structure, more like the p55/MPP1-type MAGUKs than a true CASK. Phylogenetically, the MPP1 MAGUK group in vertebrates appears to be an offshoot of the CASK branch of the tree which arose from a gene duplication with subsequent loss of the CaMK-like and L27 domains (De Mendoza, 2010). Interestingly, Drosophila has no known MPP1 homolog, and it appears that the niche of this type of MAGUK has been filled by the short CASK gene product. It would therefore not be surprising if CASK-β and CASK-α had quite different roles. Indeed the transcripts encoding these two proteins have different developmental profiles. Elucidation of the functions of the MPP1-like isoforms awaits the generation of CASK-α-specific mutants and antibodies, but it is tempting to speculate that the high expression in ovaries might indicate that loss of CASK-α underlies the sterility phenotype of 307/313 flies (Slawson, 2011).

Loss of the CASK isoform containing the CaMK-like and L27 domains underlies the CASK locomotor deficit: Mutants lacking CASK-β displayed an obvious motor defect, which was further dissected using a high-resolution video tracking system. This analysis revealed a very complex defect, with deficits in four major areas: motor initiation, motor maintenance, speed, and acceleration. Furthermore, this defect is clearly dose-dependent, as the severity of the phenotype appears to change in a correlated fashion with the amount of CASK-β protein present in the animal, with CASK^P18/+ heterozygotes being more normal than CASK P18 homozygotes, and with equivalent locomotor behaviors observed between these homozygous null flies and CASK^P18/Df for three independent deficiency lines. Along with this, expression of CASK-β in a null fly rescues the behavioral deficit, also in a dose-dependent fashion; Gal4 lines with stronger expression can even make animals hyperactive. Taken together, these data indicate that the locomotor defect seen in these flies results from loss of CASK-β in the nervous system, and not from extragenic mutations that arose as a result of the P element excision (Slawson, 2011).

The fact that mRNA encoding CASK-α, a CASK gene product that contains the PDZ, SH3 and GUK domains of CASK-β, is still expressed in the CASK^P18 mutant, suggests that there may be unique functions for the CaMK-like and L27 domains of the CASK-β form. The CaMK-like domain has been shown to have both biochemical activity (see Mukherjee, 2008) and specific binding partners, such as MINT1/Lin10 and CaMKII. The L27 domains also have specific binding partners such as DLG/SAP97 and Veli/Lin7. The inability of residual CASK-α to take over CASK-β function might also reflect a difference in localization of the two proteins, as CASK-α has a conserved palmitylation site at its very N-terminus, whereas CASK-β does not have such a motif. This assumes, however, that both CASK-α and CASK-β are expressed in the same populations of neurons, which can not be known for certain until better visualization tools for these proteins are developed (Slawson, 2011).

CASK-β functions in a pre-motor circuit: Although CASK-β is expressed throughout much of the nervous system, its role in locomotor behavior is restricted to a limited number of cells. The C164-Gal4 driver, which rescues locomotor behavior beyond wild type levels, has strong expression in only a subset of central neurons, including the antennal lobes, mushroom bodies, subesophageal ganglion (SOG), pars intercerebralis, and parts of the central complex (fan shaped body), while the periphery is completely devoid of expression. Interestingly, the ellipsoid body, which is known primarily for its role in locomotion, is not a region where the Gal4 protein is expressed with this driver, suggesting that CASK is not acting in this population of cells to rescue behavior (Slawson, 2011).

Strong CASK-β expression in glutamatergic cells with the OK371-Gal4 driver did not rescue locomotor behavior. This is an important finding because insect motor neurons are primarily glutamatergic, implying that this subpopulation of cells within the central nervous system is also not the site of action for CASK-β in locomotion. This finding is at odds with the conclusions of recent work, which has suggested that alterations in the regulation of neurotransmitter release at the NMJ in 307/313 larvae and adults underlie the defective motor behavior of the null. These experiments suggest that these NMJ defects (if they are indeed even present in the CASK-β-specific mutant) are not the basis of the locomotor problems demonstrated by CASK^P18/+ flies. Instead, the site of action is within a premotor population of neurons in the central nervous system that does not include ellipsoid body cells (Slawson, 2011).

Judging by the expression pattern of C164-Gal4, the groups of neurons relevant for CASK-β action in locomotor behavior could include cells from the pars intercerebralis, mushroom bodies, thoracic ganglion interneurons or central complex structures such as the fan-shaped body or protocerebrum, all of which have been previously implicated in regulating insect motor activity. These cells could also include populations of antennal lobe neurons involved in sensory processing, or smaller groups of neurons, but are difficult to identify based on morphology and location alone. Behavioral rescue experiments using Gal4 lines with more restricted expression patterns will be necessary to elucidate the cells relevant for CASK-β action in locomotion. Along with this, the mechanisms behind proper subcellular localization of CASK-β within these cell populations will be of interest, as this could help determine potential binding partners and signaling cascades that interact with CASK-β (Slawson, 2011).

Loss of CASK-β does not impair olfactory habituation: Mutants lacking CASK-β display a lower courtship index and a longer courtship latency than control flies. This indicates that CASK-β mutants are less adept at finding the target fly, which could be explained by a reduced sensitivity to pheromonal cues. Surprisingly, however, when CASK^P18/+ were tested for courtship habituation, which is a task requiring non-associative memory formation and olfactory processing, these flies performed similarly to control flies. This was seen when male CASK^P18/+ flies were trained with either a decapitated target immature male or direct exposure to immature male pheromone. This finding suggests that both olfactory processing and plasticity remain intact in this assay following the loss of CASK-β. It should be noted that these results are specific to male-male courtship, and that plasticity defects involving other pheromonal cues or sensory modalities remain to be examined. 307/313 has additional chromosomal aberrations that affect behavior and fertility: In all behavioral assays, 307/313 flies perform very differently from CASK-β mutants in addition to being sterile. This is not surprising since 307/313 flies are transheterozygous for two overlapping deficiencies. These deficiencies eliminate CASK-α as well as CASK-β, and also contain mutations in genes besides CASK, which could have an effect on the resulting behavior of the flies. The low level of basal courtship observed in CASK^P18/+ flies, which is likely attributable to locomotor problems, is far less severe than the deficit seen in 307/313 flies. Along with this, unlike the CASK^P18/+ mutants, 307/313 flies display an abnormally high and unusually variable habituation index, consistent with previous work (LU et al. 2003). These additional problems of the 307/313 flies could reflect a reduction in olfactory sensitivity, or a short-term plasticity defect, stemming from the loss of CASK-α or from heterozygosity at other genes (Slawson, 2011).

Alternatively, these differences could also stem from the more severe courtship initiation defect observed in 307/313 flies, as a difficulty initiating any kind of movement could affect the reliability of training and testing. This idea is supported by the finding that 307/313 flies display a qualitatively different locomotor profile compared with CASK^P18/+ flies. Importantly, multivariate analysis demonstrates that the individual parameters contributing to the qualitative difference between CASK^P18/+ and 307/313 are primarily initiation parameters. This suggests that the loss of the MPP1-like CASK-α (or potentially genetic interactions between haploinsufficient loci) in 307/313 flies may confer a unique locomotor deficit. For this reason, 307/313 is not a good model for loss of CASK-β, the CaMK-like/L27-containing MAGUK, as it pertains to behavior (Slawson, 2011).

CASK and motor dysfunction: This work with CASK-β mutants shows that there is a clear motor phenotype resulting from loss of the Drosophila CASK homolog. These flies appear to suffer from problems with motor initiation, motor maintenance, speed, and acceleration. Such a complex deficit stemming from a higher-level region within the central nervous system suggests that CASK-β may work to allow integration of multiple parameters of locomotion together into coordinated movement. Not surprisingly, this strong locomotor phenotype also appears to affect other behavioral tasks involving a motor response, such as courtship and habituation (Slawson, 2011).

Many diseases such as Parkinson's Disease and Huntington’s Disease are characterized by motor dysfunction that disrupts multiple motor parameters. Fly models for both of these movement disorders, as well as many others, have been developed and characterized, and show deficits similar to those of CASK^P18/+ flies. Furthermore, recent work has suggested that molecular scaffolds like MAGUK family proteins, of which CASK is a member, interact directly or indirectly with many proteins thought to be associated with these diseases. Determining the role that scaffolds such as CASK play in such interactions may lead to a deeper understanding of motor disease and potentially provide a basis for development of novel therapeutics (Slawson, 2011).

DlgS97/SAP97, a neuronal isoform of discs large, regulates ethanol tolerance

From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, intolerant (intol), a novel allele of discs large 1 (dlg1) was identified. Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly's brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol's sedative/hypnotic effects. It is proposed that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity (Maiya, 2012).

EVOLUTIONARY HOMOLOGS

Characterizaiton of lin-2/CASK

In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, a single interacting protein called CASK has been identified. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 (Drosophila homolog: Discs large). The C-terminal region also contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in the synaptic plasma membranes of the brain, but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca(2+)-activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins (Hata, 1996).

In Caenorhabditis elegans, vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of let-23 RTK at epithelial junctions is essential for the vulval induction, and requires three genes, including lin-2, -7, and -10. The mammalian homolog of lin-2 has been identified as CASK, a protein interacting with neurexin, a neuronal adhesion molecule. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localizes let-23 RTK at epithelial junctions. Three rat homologs of lin-7 are ubiquitously expressed in various tissues. These homologs accumulate at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologs of lin-7 may be implicated in the formation of cell-cell junctions (Irie, 1999).

C. elegans LIN-2 and EGFR trafficking

By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. EGFR substrate protein-8 (EPS-8) has been identified as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis (Stetak, 2006).

Structure of mLIN-2/CASK

LIN-2, -7 (L27) homology domains are putative protein-protein interaction modules found in several scaffold proteins involved in the assembly of polarized cell-signaling structures. These specific interaction pairs are well conserved across metazoan species, from worms to man. L27 domains were expressed and purified from multiple species and it was found that certain domains from proteins such as Caenorhabditis elegans LIN-2 and LIN-7 can specifically heterodimerize. Biophysical analysis of interacting L27 domains demonstrates that the domains interact with a 1:1 stoichiometry. Circular dichroism studies reveal that the domains appear to function as an obligate heterodimer; individually the domains are largely unfolded, but when associated they show a significant increase in helicity, as well as a cooperative unfolding transition. These novel obligate interacting pairs are likely to play a key role in regulating the organization of signaling proteins at polarized cell structures (Harris, 2002).

LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. The solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7 have been solved. Comparison of this structure with other L27 domain structures has allowed formulation of a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. Two keystone positions were identified that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions (Petrosky, 2005).

Initially identified in Caenorhabditis elegans Lin-2 and Lin-7, L27 domain is a protein-protein interaction domain capable of organizing scaffold proteins into supramolecular assemblies by formation of heteromeric L27 domain complexes. L27 domain-mediated protein assemblies have been shown to play essential roles in cellular processes including asymmetric cell division, establishment and maintenance of cell polarity, and clustering of receptors and ion channels. The structural basis of L27 domain heteromeric complex assembly is controversial. The high-resolution solution structure of the prototype L27 domain complex formed by mLin-2 and mLin-7 was determined as well as the solution structure of the L27 domain complex formed by Patj and Pals1. The structures suggest that a tetrameric structure composed of two units of heterodimer is a general assembly mode for cognate pairs of L27 domains. Structural analysis of the L27 domain complex structures further showed that the central four-helix bundles mediating tetramer assembly are highly distinct between different pairs of L27 domain complexes. Biochemical studies revealed that the C-terminal alpha-helix responsible for the formation of the central helix bundle is a critical specificity determinant for each L27 domain in choosing its binding partner. These results provide a unified picture for L27 domain-mediated protein-protein interactions (Feng, 2005)

CASK Functions as a Mg2+-independent neurexin kinase

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. This study shows, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, these data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, this study suggests that other pseudokinases (10% of the kinome) could also be catalytically active (Mukherjee, 2008).

Mg²⁺ acts as an obligate cofactor for ATP binding and phosphotransfer in all known kinases. This study demonstrates that the CASK CaM-kinase domain catalyzes phosphotransfer from ATP to proteins in the complete absence of Mg²⁺. CASK is the first kinase, indeed the first nucleotidase, known to catalyze phosphotransfers in the absence of Mg²⁺ (Mukherjee, 2008).

The structure of the CASK CaM-kinase domain, and comparison of its structure with those of other kinases, illustrates that the CaM-kinase domain of CASK adopts a constitutively active conformation. Biochemical and enzymatic assays demonstrated that CASK binds ATP and catalyzes autophosphorylation and neurexin-1 phosphorylation in the absence of Mg²⁺. Compared to other kinases, CASK contains noncanonical residues in the nucleotide-binding pocket that may account for its unusual catalytic mechanism. Both of the classical metal-coordinating residues in kinases are substituted in the CASK CaM-kinase domain. Moreover, Glu143 of the catalytic loop directly coordinates the metal ion in DAPK1, while in CASK, this Glu is altered to His (Glu145His). These changes likely contribute to the divalent cation-driven inhibition of the CASK CaM-kinase domain. Since the adenine base of ATP makes the most important contacts for the positioning of ATP in the nucleotide-binding pocket, an altered Mg²⁺-coordinating sequence does not exclude ATP binding and, as shown in this study, does not exclude catalysis. Importantly, similar to the CASK CaM-kinase domain, other pseudokinase domains with noncanonical Mg²⁺-binding motifs may coordinate ATP and phosphorylate physiological substrates as well (Mukherjee, 2008).

CASK also differs from other CaM-kinase family members in that its CaM-kinase domain exhibits a constitutively active conformation. In an archetypal CaM kinase, the catalytic domain is followed by an autoinhibitory domain that inhibits kinase activity and is disinhibited by Ca²⁺/calmodulin binding. The CASK CaM-kinase domain is followed by a sequence that is homologous to the autoinhibitory domain of CaM kinases and that also binds Ca²⁺/calmodulin. However, unlike typical CaM kinases, the autoinhibitory helix (αR1) of CASK does not engage in direct contacts with the ATP-binding cleft. No evidence was discerned for further C-terminal residues interacting with the ATP-binding cleft, as in CaMKI, and no stimulatory effect was detected of Ca²⁺ and/or calmodulin on the CASK kinase activity. Thus, the CASK CaM-kinase domain appears to retain a nonfunctional autoinhibitory domain as an evolutionary vestige of CaM kinases. CASK, therefore, differs from other, evolutionarily closely related CaM-kinase domains not only in its Mg²⁺ independence but also in its inherently “closed” active conformation that constitutively binds nucleotides (Mukherjee, 2008).

An almost essential consequence of the constitutively active conformation of the CASK CaM-kinase domain is that the domain exhibits a very low catalytic rate, as shown in autophosphorylation measurements and in measurements of neurexin-1 phosphorylation by the isolated CASK CaM-kinase domain lacking the neurexin-binding PDZ domain of CASK. Mechanistically, this low rate is likely due to the loss of Mg²⁺ coordination by the domain. The low catalytic rate of the CASK CaM-kinase domain presumably serves to ensure that the kinase does not phosphorylate potential substrates randomly. The phosphorylation rate of neurexin-1 is increased dramatically, however, when full-length CASK forms a complex with neurexin-1 via the CASK PDZ domain. This result suggests a general mechanism for CASK kinase activity, whereby CASK couples an intrinsically slow but constitutively active kinase domain to a PDZ domain that recruits the substrates to the kinase domain, thereby increasing the local substrate concentration by many orders of magnitude. According to the model, CASK unites two separate functions—the recruitment activity of MAGUKs and the kinase activity of the CaM-kinase domain—into a single unit whose objective is phosphorylation of specific interacting proteins (Mukherjee, 2008).

CASK phosphorylates neurexin-1 in vitro and in vivo in a reaction that depends on a catalytically active CaM-kinase domain. Neurexin, described in this study as a substrate of CASK, is a presynaptic cell-adhesion molecule. Its heterotypic binding to postsynaptic neuroligins may be involved in synaptic function and could induce synapse formation even on non-neuronal cocultured cells. The neurexin-neuroligin interaction is a candidate for synaptic specialization and pre-post-synapse communication. Both neurexin and neuroligin mutations have been linked to autism spectrum disorders. Deletion of CASK may be connected to X-linked optic atrophy and mental retardation. The evolutionary conservation of CASK and neurexins, and their central importance for survival and synaptic function in mice, indicate that neurexin phosphorylation by CASK may be crucial to neuronal function (Mukherjee, 2008).

In addition to the control of CASK kinase activity by the PDZ-domain-mediated substrate recruitment, whether it is regulated by synaptic activity-driven rises in Ca²⁺ and Mg²⁺ levels was examined. In neurons, synaptic activity triggers a surge in Mg²⁺ and Ca²⁺ levels that could regulate CASK kinase activity. Indeed, a strong increase was observed in neurexin phosphorylation upon silencing synapses in mature neurons, indicating that contrary to other kinases, CASK kinase is inhibited by neuronal activity. It was envisioned that CASK kinase activity is maximal during neuronal development and synaptogenesis and declines with the onset of synaptic function but is reactivated when neurons are silenced. This developmentally regulated activity is in line with the phenotypic defects in CASK knockout mice as well as the developmental nature of CASK- and neurexin-related pathologies (Mukherjee, 2008).

CASK is expressed ubiquitously at low levels. The non-neuronal functions of CASK are evident from developmental defects in CASK/Lin-2 null animals, such as cleft palate in mice and vulval dysgenesis in C. elegans. In non-neuronal cells, CASK-interacting adhesion molecules of the syndecan or JAM families could be substrates. These molecules share the PDZ-domain-mediated CASK association, and at least in the case of syndecan-2, serine residues in the cytoplasmic tail homologous to those of neurexins are phosphorylated in vivo (Mukherjee, 2008).

Finally, of the 518 known kinases in the human genome, 48 are predicted to be pseudokinases. In each of these pseudokinases, one or more of the invariant motifs are altered. Nine of the presumed pseudokinases, including CASK, lack a canonical DFG motif. Furthermore, this motif is altered along with other canonical motifs (HRD and/or VAIK) in 22 additional pseudokinases. These data on CASK suggest that other pseudokinases, especially those with atypical DFG motifs, could be active in physiologically relevant environments, indicating that the catalytically active kinome may be more diverse than originally envisioned (Mukherjee, 2008).

Mint interacts with CASK

Mint1 (X11/human Lin-10) and Mint2 are neuronal adaptor proteins that bind to Munc18-1 (n/rb-sec1), a protein essential for synaptic vesicle exocytosis. Mint1 has previously been characterized in a complex with CASK, another adaptor protein which in turn interacts with neurexins. Neurexins are neuron-specific cell surface proteins that act as receptors for the excitatory neurotoxin alpha-latrotoxin. Hence, one possible function for Mint1 is to mediate the recruitment of Munc18 to neurexins. In agreement with this hypothesis, it has been shown that the cytoplasmic tail of neurexins captures Munc18 via a multiprotein complex that involves Mint1. Furthermore, both Mint1 and Mint2 can directly bind to neurexins in a PDZ domain-mediated interaction. Various Mint and/or CASK-containing complexes can be assembled on neurexins, and Mint1 can bind to Munc18 and CASK simultaneously. These data support a model whereby one of the functions of Mints is to localize the vesicle fusion protein Munc18 to those sites at the plasma membrane that are defined by neurexins, presumably in the vicinity of points of exocytosis (Biederer, 2000).

Interaction of CASK and human Dlg

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). One MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding (Nix, 2000).

Neph1 and Neph2 interact with the PDZ domain of CASK

Formation, differentiation, and plasticity of synapses require interactions between pre- and postsynaptic partners. Recently, it was shown that the transmembrane immunoglobulin superfamily protein SYG-1 is required for providing synaptic specificity in C. elegans. However, it is unclear whether the mammalian orthologs of SYG-1 are also involved in local cell interactions to determine specificity during synapse formation. In situ hybridization, immunohistochemistry, and immunogold electron microscopy were used to study the temporal and spatial expression of Neph1 and Neph2 in the developing and adult mouse brain. Both proteins show similar patterns with neuronal expression starting around embryonic days 12 and 11, respectively. Expression is strongest in areas of high migratory activity. In the adult brain, Neph1 and Neph2 are predominantly seen in the olfactory nerve layer and the glomerular layer of the olfactory bulb, in the hippocampus, and in Purkinje cells of the cerebellum. At the ultrastructural level, Neph1 and Neph2 are detectable within the dendritic shafts of pyramidal neurons. To a lesser extent, there is also synaptic localization of Neph1 within the stratum pyramidale of the hippocampal CA1 and CA3 region on both pre- and postsynaptic sites. Here it colocalizes with the synaptic scaffolder calmodulin-associated serine/threonine kinase (CASK), and both Neph1 and Neph2 interact with the PDZ domain of CASK via their cytoplasmic tail. These results show that Neph proteins are expressed in the developing nervous system of mammals and suggest that these proteins may have a conserved function in synapse formation or neurogenesis (Gerke, 2006).

CASK protein complexes

CASK, an adaptor protein of the plasma membrane, is composed of an N-terminal calcium/calmodulin-dependent protein (CaM) kinase domain, central PSD-95, Dlg, and ZO-1/2 domain (PDZ) and Src homology 3 (SH3) domains, and a C-terminal guanylate kinase sequence. The CaM kinase domain of CASK binds to Mint 1, and the region between the CaM kinase and PDZ domains interacts with Velis, resulting in a tight tripartite complex. CASK, Velis, and Mint 1 are evolutionarily conserved in Caenorhabditis elegans, in which homologous genes (called lin-2, lin-7, and lin-10) are required for vulva development. The N-terminal CaM kinase domain of CASK binds to a novel brain-specific adaptor protein called Caskin 1. Caskin 1 and a closely related isoform, Caskin 2, are multidomain proteins containing six N-terminal ankyrin repeats, a single SH3 domain, and two sterile alpha motif domains followed by a long proline-rich sequence and a short conserved C-terminal domain. Unlike CASK and Mint 1, no Caskin homolog was detected in C. elegans. Immunoprecipitations showed that Caskin 1, like Mint 1, is stably bound to CASK in the brain. Affinity chromatography experiments demonstrated that Caskin 1 coassembles with CASK on the immobilized cytoplasmic tail of neurexin 1, suggesting that CASK and Caskin 1 coat the cytoplasmic tails of neurexins and other cell-surface proteins. Detailed mapping studies revealed that Caskin 1 and Mint 1 bind to the same site on the N-terminal CaM kinase domain of CASK and compete with each other for CASK binding. These data suggest that in the vertebrate brain, CASK and Velis form alternative tripartite complexes with either Mint 1 or Caskin 1 that may couple CASK to distinct downstream effectors (Tabuchi, 2002).

Nephrin is a cell surface receptor of the Ig superfamily that localizes to slit diaphragms, the specialized junctions between the interdigitating foot processes of the glomerular epithelium (podocytes) in the kidney. Mutations in the NPHS1 gene encoding nephrin lead to proteinuria and congenital nephrotic syndrome, indicating that nephrin is essential for normal glomerular development and function. To identify nephrin-binding proteins, mass spectrometry was performed on proteins obtained from pull-down assays with GST-nephrin cytoplasmic domain. Nephrin specifically pulled down six proteins from glomerular lysates, MAGI-2/S-SCAM (membrane-associated guanylate kinase inverted 2/synaptic scaffolding molecule), IQGAP1 (IQ motif-containingGTPase-activatingprotein1), CASK (calcium/calmodulin-dependent serine protein kinase), alpha-actinin, alphaII spectrin, and betaII spectrin. All of these scaffolding proteins are often associated with cell junctions. By immunofluorescence these proteins are expressed in glomerular epithelial cells, where they colocalize with nephrin in the foot processes. During glomerular development, IQGAP1 is expressed in the junctional complexes between the earliest identifiable podocytes, MAGI-2/S-SCAM is first detected in junctional complexes in podocytes after their migration to the base of the cells. Thus, the nephrin-slit diaphragm protein complex contains a group of scaffolding proteins that function to connect junctional membrane proteins to the actin cytoskeleton and signaling cascades. Despite their special morphology and function, there is considerable compositional similarity between the podocyte slit diaphragm and typical junctional complexes of other epithelial cells (Lehtonen, 2005).

Hearing in mammals depends upon the proper development of actin-filled stereocilia at the hair cell surface in the inner ear. Whirlin, a PDZ domain-containing protein, is expressed at stereocilia tips and, by virtue of mutations in the whirlin gene, is known to play a key role in stereocilia development. Whirlin interacts with the membrane-associated guanylate kinase (MAGUK) protein, erythrocyte protein p55 (p55). p55 is expressed in outer hair cells in long stereocilia that make up the stereocilia bundle as well as surrounding shorter stereocilia structures. p55 interacts with protein 4.1R in erythrocytes, and 4.1R is also expressed in stereocilia structures with an identical pattern to p55. Mutations in the whirlin gene (whirler) and in the myosin XVa gene (shaker2) affect stereocilia development and lead to early ablation of p55 and 4.1R labeling of stereocilia. The related MAGUK protein Ca2+-calmodulin serine kinase (CASK) is also expressed in stereocilia in both outer and inner hair cells, where it is confined to the stereocilia bundle. CASK interacts with protein 4.1N in neuronal tissue, and 4.1N is expressed in stereocilia with an identical pattern to CASK. Unlike p55, CASK labeling shows little diminution of labeling in the whirler mutant and is unaffected in the shaker2 mutant. Similarly, expression of 4.1N in stereocilia is unaltered in whirler and shaker2 mutants. p55 and protein 4.1R form complexes critical for actin cytoskeletal assembly in erythrocytes, and the interaction of whirlin with p55 indicates it plays a similar role in hair cell stereocilia (Mburu, 2005; full text of article).

CASK has been implicated in synaptic protein targeting, synaptic organization, and transcriptional regulation. Three more CASK associated proteins, GRIP1, PKCepsilon, and RGS4, were initially identified by immunoprecipitation and mass analysis, and confirmed by immunoprecipitation-immunoblotting assay using rat brain extract. Via the interaction with GRIP1, GluR2/3 was also co-immunoprecipitated by CASK antibody from rat brain. The PDZ and SH3-GK domains of CASK were demonstrated as the associated domains for GRIP1 and PKCepsilon, respectively. The associations between CASK, PKCepsilon, and RGS4 were up-regulated in the adult brain compared with postnatal day 11 rat brain. In contrast, the associations of CASK with Mint1, GRIP1, and GluR2/3 were down-regulated in the adult brain. These results suggest that CASK protein complex is developmentally regulated by unknown signals. In conclusion, this study suggests that the CASK protein complex not only functions as a scaffold but also recruits signaling molecules and may contribute to signal transduction (Hong, 2006).

Transcriptional modification by a CASK-interacting nucleosome assembly protein

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. This study reports that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene (Wang, 2004).

Murine CASK is disrupted in a sex-linked cleft palate mouse mutant

A transgenic mouse insertional mutant displayed the phenotype of altered cranial morphology with sex-linked cleft palate. The disrupted genomic X-linked locus was cloned and identified as the mCASK gene. The gene is transcribed to produce two messages of 4.5 and 9.5 kb expressed during development and in adult tissues, particularly the brain. Two differentially spliced mouse cDNAs were cloned from the locus (mCASK-A and mCASK-B). The mCASK-B cDNA probably represents the full-length product of the 4.5-kb transcript. The identical N-termini of the predicted encoded proteins (mCASK-A and -B) are highly homologous to Ca2+/calmodulin-dependent protein kinase II, while the deduced C-terminus of mCASK-B is highly homologous to a family of multidomain proteins containing a guanylate kinase motif, the MAGUK proteins. mCASK-B is a new member of an emerging family of genes in which the encoded proteins combine these domains, termed here, the CAMGUKs, including rat CASK, Caenorhabditis elegans lin-2, and Drosophila caki/camguk. The CAMGUKs are likely to be effectors in signal transduction as regulatory partners of transmembrane molecules, modulated by calcium and nucleotides. The transgene in this mutant mouse line integrated into an intron that bisects the encoded calmodulin-binding domain, a potentially important regulatory domain of the predicted protein, generating hybrid transcripts (Laverty, 1998).

Deletion of CASK in mice is lethal and impairs synaptic function

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca(2+)/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, CASK 'knockdown' mice in which CASK expression was suppressed by approximately 70%, and CASK knockout (KO) mice, in which CASK expression was abolished, were analyzed. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. These data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca(2+)/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca(2+)-triggered presynaptic release, or postsynaptic receptor functions (Atasoy, 2007).

Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK

Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins was investigated in a mammalian system. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. It was found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis (Samuels, 2007).

Homologs of liprin-α proteins are essential for presynaptic terminal formation in C. elegans and Drosophila . Mutations in C. elegans syd-2 result in a diffuse localization of several presynaptic proteins and abnormally sized active zones, and loss- and gain-of-function experiments demonstrate that presynaptic organization is dependent on syd-2. Likewise, Dliprin-α is required for normal synaptic morphology including the size and shape of the presynaptic active zone in Drosophila . Cdk5-dependent phosphorylation of CASK occurs in both the CaMK and L27 domains, and only mutation of both sites yields a localization phenotype. Since liprin-α proteins require the presence of both domains to interact with CASK, the phosphorylation sites are in a prime spot to mediate the interaction. According to the model described in this study, liprin-α is required for initial CASK localization to presynaptic terminals. Since, liprin-α binds directly to the kinesin motor KIF1A and in Drosophila liprin-α mutant axons there is decreased anterograde processivity resulting in reduced levels of presynaptic markers at terminals, it is feasible that liprin-α acts as a cargo receptor that delivers CASK, as well as other components, to and within the developing synapse. Cdk5-dependent phosphorylation could then act to coordinate distinct pools of CASK that are bound to liprin-α or are bound to other components of the presynaptic machinery. Importantly, it is not believed that Cdk5 loss of function generally affects liprin-α-mediated transport since synaptophysin, a marker of synaptic vesicles, is still properly localized within synaptosomes. In this model, there would be advantages of having locally enhanced Cdk5 activity within the presynaptic terminal relative to some other cellular compartments. Supporting this idea, phospho-CASK is particularly enriched at synaptic membranes, and Cdk5 has been shown to phosphorylate and regulate several proteins, including Munc-18, Dynamin-1, Amphiphysin-1, and Synaptojanin-1, that function to control multiple rounds of the synaptic vesicle cycle. Synapsin-1 is also a Cdk5 substrate. With regard to the role of liprin-α, it will ultimately be essential to assay synapse formation and CASK localization in mammalian liprin-α loss-of-function models (Samuels, 2007).

The netrin receptor UNC-40/DCC assembles a postsynaptic scaffold and sets the synaptic content of GABAA receptors

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC (see Drosophila Frazzled) controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs; see Drosophila Rdl) at inhibitory neuromuscular junctions. This study show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK (see Drosophila Cask), that binds the synaptic adhesion molecule NLG-1/Neuroligin (see Drosophila Neuroligin) and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4 (a member of the ADAMTS family of proteases), which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain (Zhou, 2020).

REFERENCES

Search PubMed for articles about Drosophila CASK ortholog

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date revised: 25 April 2024

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