Interactive Fly, Drosophila

Sub-cellular distribution of PKA

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. A 78 kDa AKAP that is enriched in gastric parietal cells has been purified to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP is recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produces in either Sf9 cells or bacteria also binds RII. Recombinant radixin and moesin, ezrin-related proteins, also binds RII in blot overlay. Analysis of recombinant truncations of ezrin map the RII binding site to a region between amino acids 373 and 439. This region contains a 14-amino-acid amphipathic [alpha]-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocks RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 fails to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributes from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus (Dransfield, 1997).

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Another novel cDNA isolated from that screen has been cloned and characterized. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids, including the R binding fragment (residues 333-372) at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein, thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase (Huang, 1997).

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. In transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP result in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation are facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79, one that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity are not observed. The association of an AKAP with PKA is required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit results in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928 (Gao, 1997).

Stimulation of beta-adrenergic receptors activates type I and II cyclic AMP-dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase-anchoring proteins (AKAPs) regulates cyclic AMP-dependent signaling in the cell. The expression and localization of AKAP100 in adult hearts was investigated. AKAP100 was identified in adult rat and human hearts: type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. In rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, AKAP100 was localized to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus alpha-actinin-specific antibodies or AKAP100 plus ryanodine receptor-specific antibodies, confocal images shows AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, colocalizes with AKAP100 in the rat heart. These studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart (Yang 1998).

The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). Capacitation of cauda epididymal mouse sperm in vitro is accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity does not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, are found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation is associated with enzyme activity found in the soluble fraction. The regulatory and catalytic subunits of PK-A are present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum, which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. The RII subunit-binding domain of this protein maps to a 57-amino-acid residue region at its N-terminus. Computer analysis reveals a 14-amino-acid region that resembled the RII-binding domains of other A kinase anchor proteins. A synthetic peptide corresponding to this domain inhibits AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function (Visconti, 1997).

The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A kinase anchor protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy has demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 [M(r) 97,000]. In immunoblotting experiments, the antibody detects pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M(r) 109,000 (p109) and M(r) 26,000 (p26, representing the "pro" domain of the precursor) are present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrates that p109 is a phosphorylated form of pro-AKAP82 that remains in sperm. By immunofluorescence, pro-AKAP82 is found to be localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm, p109 and p26 are present only in the proximal portion of the principal piece. Pro-AKAP82 is solubilized when germ cells are extracted with Triton X-100. However, in sperm, both AKAP82 and p109 are almost totally resistant to these extraction conditions and remain in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A is present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also becomes particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82 (Johnson, 1997).

Impaired insulin secretion is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). One possible therapeutic agent for NIDDM is the insulinotropic hormone glucagon-like peptide 1 (GLP-1). GLP-1 stimulates insulin secretion by means of several mechanisms, including activation of protein kinase A (PKA). The subcellular targeting of PKA through association with A-kinase-anchoring proteins (AKAPs) facilitates GLP-1-mediated insulin secretion. Disruption of PKA anchoring by the introduction of anchoring inhibitor peptides or expression of soluble AKAP fragments blocks GLP-1 action in primary islets and cAMP-responsive insulin secretion in clonal beta cells (RINm5F). Displacement of PKA also prevents cAMP-mediated elevation of intracellular calcium, suggesting that localized PKA phosphorylation events augment calcium flux (Lester, 1997).

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. All A-kinse anchoring proteins (AKAPs) contain a common structural motif that tethers PKA through interaction with the regulatory subunit (R) dimer of the kinase. While all AKAPs contain a conserved RII binding domain, a second motif unique to each anchoring protein allows it to sequester the kinase to specific intracellular locations. For example, subcellular fractionation and immunohistochemical analyses have detected AKAPs at specific subcellular sites, such as the cytoskeleton, endoplasmic reticulum, filopodia, golgi, microtubules, plasma membrane, postsynaptic density and secretory granules. Therefore, the role of AKAP targeting is to provide specificity in cAMP-responsive events by placing the anchored kinase close to specific substrates. A low-molecular weight A-kinase anchoring protein, called AKAP18, has been cloned; it targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5, which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promotes a marked increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 has no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates insulinotropic hormone glucagon-like peptide 1 (GLP-1) mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation (Fraser, 1998).

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). AKAP15 is a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes (Gray, 1998).

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 has been identified and sequenced, and reveals a 692-amino acid open reading frame that is 89% homologous to the rat AKAP95. The gene encoding AKAP95 maps to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 is expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolishes RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts show a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining is markedly changed and appears to be excluded from the condensed chromatin and localized outside the metaphase plate. The subcellular localizations of AKAP95 and RIIalpha overlap in metaphase but start to segregate in anaphase and are again separated as AKAP95 reenters the nucleus in telophase. RIIalpha can be coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent (Eide, 1998).

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c). The PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/-4 nM in vitro. Immunoprecipitation of PP1 from cell extracts results in a 10.4 +/-3.8-fold enrichment of PKA activity. AKAP220 co-purifies with PP1c by affinity chromatography on microcystin sepharose. Immunocytochemical analysis demonstrates that the kinase, the phosphatase and the anchoring protein have distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme (Schillace, 1999).

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encodes a 3908 amino acid protein of 453 kDa that has been designated AKAP450. Sequence comparison demonstrates that the open reading frame contains a previously characterized cDNA encoding Yotiao, as well as the human homolog of AKAP120. Numerous coiled-coil structures are predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins is observed. A putative RII-binding site has been identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay, and an amphipatic helix is predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrates co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it has been demonstrated that AKAP450 localizes to centrosomes. Furthermore, AKAP450 co-purifies in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene (Witczak, 1999).

Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. A complete sequence of human gastric AKAP350 has now been obtained, as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants have also been identified. Both of the major splice variants show tissue- and cell-specific expression patterns. Immunofluorescence microscopy demonstrates that AKAP350 is associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localizes asymmetrically to one pole of the centrosome, and nocodazole does not alter its localization. During the cell cycle, AKAP350 is associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrate noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types (Schmidt, 1999).

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA). Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs). AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. Both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo (Diviani, 2000).

The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein-protein interactions. A prototypic example is the compartmentalization of the cAMP-dependent protein kinase (PKA) through its association with A-kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. The structures of two D/D-AKAP peptide complexes obtained by solution NMR methods are reported in this study, one with Ht31(493-515) and the other with AKAP79(392-413). The first direct structural data demonstrating the helical nature of the peptides is reported. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are both responsible for mediating the high affinity association in the complexes. In a departure from the dimer-dimer interactions seen in other X-type four-helix bundle dimeric proteins, these structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIalpha D/D (Newlon, 2001).

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain have been identified by means of a yeast two-hybrid screen. Full-length human D-AKAP2 (662 residues) with an additional putative RGS domain has now been cloned, as well as the corresponding mouse protein, less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The physiological relevance of interactions between PKA and AKAPs with mitochondria is not fully understood. Several PKA substrates involved in mitochondrial respiration have been identified recently. In addition to their central role in aerobic energy production, mitochondria are also involved in triggering the apoptotic pathway. PKA exhibits either pro-apoptotic or anti-apoptotic roles in different cell lines, or the same cell line under different conditions. BAD, a pro-apoptotic member, is phosphorylated and inactivated by mitochondria-anchored PKA. Furthermore, this process is inhibited if the association of PKA and AKAP is disrupted by the universal anchoring motif peptide mimetic, Ht31. Future experiments will elucidate whether D-AKAP2 plays a direct role in apoptosis by mediating the localization of PKA to the mitochondria (Wang, 2001).

The second messenger cyclic adenosine 5'monophosphate (cAMP) has been implicated in controlling meiotic maturation. To date, there have been no direct measurements of cAMP in living mammalian oocytes. The fluorescently labelled cAMP-dependent protein kinase A (PKA), FlCRhR, has been used to monitor cAMP in mouse oocytes. In cumulus-enclosed oocytes, follicle-stimulating hormone (FSH) stimulates an increase in the oocyte [cAMP] that is prevented by using the gap junction inhibitor, carbenoxolone. The FSH-induced increase in oocyte [cAMP] is suppressed in a time-dependent manner by prior exposure to ATP. Using confocal microscopy, it has been shown that the regulatory and catalytic subunits of the microinjected PKA are distributed in a punctate manner with a stronger accumulation in the perinuclear region. On an increase in [cAMP], in response to phosphodiesterase inhibition or FSH, the catalytic subunit diffuses throughout the cytoplasm and germinal vesicle, while the regulatory subunit remains anchored. These experiments show that increases in cAMP in ovarian somatic cells are communicated via gap junctions to the oocyte, where the cAMP can lead to a redistribution of the catalytic subunit of PKA. These results suggest that the cAMP increase in the oocyte is a result of cAMP diffusing from the cumulus cells to the oocyte. Alternative explanations cannot be completely discounted. It is feasible that, in addition to cAMP, FSH stimulates the production of an additional small messenger molecule that is able to modulate oocyte cAMP levels by inhibiting the PDE or stimulating adenylyl cyclase. However, there is no evidence for this form of signaling in other cell types, while evidence exists for cAMP diffusing through gap junctions (Webb, 2002).

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Evidence is provided for interactions between AKAP79/150 and AC isoenzymes ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production (Bauman, 2006).

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP in human embryonic kidney cell cultures using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. It is proposed that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities (Willoughby, 2005).

NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, it has been demonstrated that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP-PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150-PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, these observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components (Smith, 2006).

PKA and the cell cycle

The activity of S. cerevisiae cAPK is regulated by a complex signaling pathway that includes two yeast homologs of mammalian ras proteins. The yeast RAS1 and RAS2 gene products (Ras) are small GTP-binding proteins that are activated by a GTP-exchange factor (GEF), encoded by CDC25, and inactivated by stimulation of the intrinsic GTPase-activity via GTPase-activating proteins (GAP), encoded by IRA1 and IRA2. Activated GTP-bound Ras stimulates adenylate cyclase (encoded by CYR1/CDC35) to yield increased levels of cAMP that can be degraded by the low- and high-affinity phosphodiesterases encoded by the PDE1 and PDE2 genes, respectively. Binding of cAMP to the regulatory subunits of cAPK (encoded by BCY1) has two results: (1) the dissociation if the cAPK subunits from the catalytic subunits, which are encoded by three functionally redundant genes, TPK1, TPK2, and TPK3, and (2) the stimulation of cAPK activity. While the components of the Ras/cAMP pathway required for activation of cAPK are well established, little is known about the mechanisms of activation of the pathway by biological signals (i.e., nutrients) or about the potential biochemical targets of cAPK (Reinders, 1998 and references).

The S. cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation; in transcriptional derepression of HSP12, HSP26, and SSA3; in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase (Reinders, 1998).

Cell cycle progression in cycling Xenopus egg extracts is accompanied by fluctuations in the concentration of adenosine 3',5'-monophosphate (cAMP) and in the activity of the cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase. Blocking the activation of PKA at metaphase prevents the transition into interphase; the activity of M phase-promoting factor (MPF; the cyclin B-p34cdc2 complex) (see Drosophila cdc2) remains high, and mitotic cyclins are not degraded. The arrest in mitosis is reversed by the reactivation of PKA. The inhibition of protein synthesis prevents the accumulation of cyclin and the oscillations of MPF, PKA, and cAMP. Addition of recombinant nondegradable cyclin B activates p34cdc2 and PKA and induces the degradation of full-length cyclin B. Addition of cyclin A activates p34cdc2 but not PKA, nor does it induce the degradation of full-length cyclin B. This suggests that cyclin degradation and exit from mitosis require MPF-dependent activation of the cAMP-PKA pathway (Grieco, 1996).

The 20S cyclosome complex (also known as the anaphase-promoting complex) has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation. The formation and activation of the 20S cyclosome complex is regulated by an unknown mechanism. Cut4 is an essential component of the cyclosome in fission yeast. Cut4 shares sequence similarity with BimE, a protein that regulates mitosis in Aspergillus nidulans. Mutations in cut4 result in hypersensitivity to cyclic AMP and to stress-inducing heavy metals, inhibition of the onset of anaphase, disruption of the 20S complex, and inhibition of mitotic cyclin ubiquitination. These phenotypes are fully suppressed by cAMP phosphodiesterase and the protein kinase A (PKA) regulatory subunit and weakly suppressed by Sti1 (an activator of the Hsp70 and Hsp90 chaperones). Suppression correlates with the amount of 20S complex, indicating that cyclosome formation and activation is inhibited by the cAMP/PKA pathway (Yamashita, 1996).

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome. As a consequence, this proteolysis regulates metaphase-anaphase transition and exit from mitosis. Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC; at metaphase, Plk activity peaks whereas PKA activity is falling. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity (Kotani. 1998).

The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. A polyclonal antibody raised to purified recombinant PKI alpha was prepared. Using this antibody, the intracellular distribution of endogenous PKI alpha was assessed by immunostaining. The PKI alpha expression and intracellular distribution varies as a function of cell cycle progression. PKI alpha expression appears low in serum-starved cells and in cells in G1 and increases as cells progress through S phase. Its distribution becomes increasingly nuclear as cells enter G2/M. Nuclear levels of PKI alpha remain high through cell division and decrease again as cells reenter G1. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G2/M phases of the cell cycle. Consistent with this, microinjection of PKI alpha antibody into serum-starved cells prevents their subsequent cell cycle progression. Similarly, overexpression of the C subunit in cells arrested at the G1/S boundary prevents their subsequent division. Together these results support the idea that PKI alpha plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis (Wen, 1995).

Mitosis requires activity of the cyclin B cyclin-dependent kinase 1 (cdc2) heterodimer. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B. Cdk2 is also active during mitosis. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2. Cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. These findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit (D'Angiolella, 2001).

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, a cell-free oocyte extract was used that carries out the signaling events of oocyte maturation after addition of PKI, the heat-stable inhibitor of PKA. PKI stimulates the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos does not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delays, but does not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide has a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibits activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 has no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity. It is evident that Plx1 is an essential trigger kinase for Cdc25C activation at the G2/M transition. No other kinase appears to be able to substitute for this function of Plx1 in G2, although, once activated, cyclin B-Cdc2 is capable of activating Cdc25C in a positive feedback loop (Qian, 2001).

Cyclic adenosine monophosphate (cAMP) has been implicated as an important regulator of meiotic maturation in mammalian oocytes. A decrease in cAMP, brought about by the action of cAMP phosphodiesterase (PDE), is thought to initiate germinal vesicle breakdown (GVB) by the inactivation of cAMP-dependent protein kinase. However, the product of PDE activity, 5'-AMP, is a potent activator of an important regulatory enzyme, AMP-activated protein kinase (AMPK). The aim of this study was to evaluate a possible role for AMPK in meiotic induction, using oocytes obtained from eCG-primed, immature mice. Alpha-1 and -2 isoforms of the catalytic subunit of AMPK were detected in both oocytes and cumulus cells. When 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICA riboside), an activator of AMPK, was tested on denuded oocytes (DO) and cumulus cell-enclosed oocytes (CEO) maintained in meiotic arrest by dbcAMP or hypoxanthine, GVB was dose-dependently induced. Meiotic induction by AICA riboside in dbcAMP-supplemented medium is initiated within 3 h in DO and 4 h in CEO and is accompanied by increased AMPK activity in the oocyte. AICA riboside also triggers GVB when meiotic arrest is maintained with hypoxanthine, 8-AHA-cAMP, guanosine, or milrinone, but is ineffective in olomoucine- or roscovitine-arrested oocytes, indicating that it acts upstream of maturation-promoting factor. AMP dose-dependently stimulates GVB in DO when meiotic arrest is maintained with dbcAMP or hypoxanthine. This effect is not mimicked by other monophosphate or adenosine nucleotides and is not affected by inhibitors of ectophosphatases. Combined treatment with adenosine and deoxycoformycin, an adenosine deaminase inhibitor, stimulates GVB in dbcAMP-arrested CEO, suggesting AMPK activation due to AMP accumulation. It is concluded that phosphodiesterase-generated AMP may serve as a transducer of the meiotic induction process through activation of AMPK (Downs, 2002).

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye (Masai, 2005).

In most species, the meiotic cell cycle is arrested at the transition between prophase and metaphase through unclear somatic signals. Activation of the Cdc2-kinase component of maturation promoting factor (MPF) triggers germinal vesicle breakdown after the luteinizing hormone (LH) surge and reentry into the meiotic cell cycle. Although high levels of cAMP and activation of protein kinase A (PKA) play a critical role in maintaining an inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. Using a small-pool expression-screening strategy, several putative PKA substrates have been identified from a mouse oocyte cDNA library. One of these clones encodes a Wee1-like kinase that prevents progesterone-induced oocyte maturation when expressed in Xenopus oocytes. Unlike the widely expressed Wee1 and Myt1, mWee1B mRNA and its protein are expressed only in oocytes, and mRNA downregulation by RNAi injection in vitro or transgenic overexpression of RNAi in vivo causes a leaky meiotic arrest. Ser15 residue of mWee1B is the major PKA phosphorylation site in vitro, and the inhibitory effects of the kinase are enhanced when this residue is phosphorylated. Thus, mWee1B is a key MPF inhibitory kinase in mouse oocytes, functions downstream of PKA, and is required for maintaining meiotic arrest (Han, 2005).

PKA and circadian rhythms

Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. In contrast, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. By using whole-cell isotope labeling and quantitative mass spectrometry methods, this study shows that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. The cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop (Huang, 2007).

Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. This prediction is tested directly in this study. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. Twenty-six genes required for Drosophila melanogaster sleep were identified. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, a conserved genetic pathway was delineated by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented in this study suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry (Singh, 2014).

PKA and oocyte maturation

In the final stages of ovarian follicular development, the mouse oocyte remains arrested in the first meiotic prophase, and cAMP-stimulated PKA plays an essential role in this arrest. After the LH surge, a decrease in cAMP and PKA activity in the oocyte initiates an irreversible maturation process that culminates in a second arrest at metaphase II prior to fertilization. A-kinase anchoring proteins (AKAPs) mediate the intracellular localization of PKA and control the specificity and kinetics of substrate phosphorylation. Several AKAPs have been identified in oocytes including one at 140 kDa that is now identified as a product of the Akap1 gene. PKA interaction with AKAPs is essential for two sequential steps in the maturation process: the initial maintenance of meiotic arrest and the subsequent irreversible progression to the polar body extruded stage. A peptide inhibitor (HT31) that disrupts AKAP/PKA interactions stimulates oocyte maturation in the continued presence of high cAMP. However, during the early minutes of maturation, type II PKA moves from cytoplasmic sites to the mitochondria, where it associates with AKAP1, and this is shown to be essential for maturation to continue irreversibly (Newhall, 2006).

This work reveals that the localization of PKA is an essential element of the decision of oocytes to commit to the maturation program. In germinal vesicle-stage oocytes, the RIIα PKA holoenzyme localizes in a widely distributed punctate pattern throughout the cytoplasm. It is speculated that this holoenzyme is binding to an as yet unidentified AKAP that positions PKA close to its targets (e.g., Cdc25b phosphatase and the recently identified Wee1B kinase), maintaining the inactive state of Cdc25b and the active state of Wee1B by phosphorylation. In support of this, it was found that germinal vesicle-stage oocytes injected with the PKA anchoring disruptor HT31 initiate maturation even when the oocytes are maintained in the phosphodiesterase inhibitor IBMX to keep cAMP and PKA activity high. These experiments with analogs demonstrate greater synergistic inhibition of GVBD (meiotic resumption) with RII analogs compared with RI analogs, suggesting that the type II kinase is more closely linked to the arrest of maturation in wild-type oocytes (Newhall, 2006).

When cAMP levels in the oocyte decline, PKA activity declines and no longer maintains meiotic arrest. CDK1 is dephosphorylated and becomes active, and maturation is initiated. It is suggested that activation of CDK1 sets in motion the relocalization of RIIα-PKA to the mitochondria, which occurs prior to GVBD. The molecular mechanisms that regulate this relocalization are unknown but may involve posttranslational modifications of either RIIα or AKAP1 that increase their interaction with each other. In the AKAP1 knockout, RIIα does not associate with mitochondria, and this loss of RIIα localization prevents the 'meiotic switch' from being thrown irreversibly. AKAPs are therefore playing a dual role in oocyte maturation: they are required to maintain the PKA-dependent arrest of oocytes at meiosis I prophase prior to the LH surge, and, after the initiation of maturation, AKAP1 plays an alternative and equally important role in sequestering PKA away from its targets in order for oocyte maturation to proceed normally and irreversibly. Similarly, a sequestration role has been proposed for Drosophila AKAP200 similar to that proposed in this study for AKAP1 based on studies of egg chamber and actin-enriched ring canal development (Newhall, 2006).

The increase in IVF procedures in the human population has focused attention on oocyte maturation as a determinant of successful fertilization, and several reports have documented oocyte maturation defects as a cause of IVF failure. The current results demonstrate the importance of temporal and spatial control of PKA signaling during mammalian oocyte maturation (Newhall, 2006).

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