cAMP-dependent protein kinase 1


Table of contents

PKA interaction with its regulatory subunits

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes, any or all of which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase; the other is homologous to response regulators (RRs) of two-component signal transduction systems. In the simplest configuration of a two-component system, phosphates are transferred directly from a histidine kinase to the Asp of the RR. However, in more complex systems such as those controlling initiation of sporulation in Bacillus subtilis, and in the yeast osmo-regulatory pathway, phosphates flow through a four-step phosphorelay. In the yeast system, phosphates are relayed from His to Asp on the kinase (Sln1p) and then to a His on an intermediate phosphotransfer protein, Ypd1p, before reaching the RR (Ssk1p). Dictyostelium RegA can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. This dominant-negative effect is interpreted as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. It is proposed that cAMP breakdown is controlled by a phosphorelay system that activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited (Thomason, 1998).

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. The neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, has been identified as a binding partner for RIbeta. Merlin and RIbeta demonstrate a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins coprecipitate from brain lysates by cAMP-agarose and coimmunoprecipite from cellular lysates with specific antibodies. In vitro binding studies have verified that the interaction is direct. The interaction appears to be under conformational regulation and is mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling (Gronholm, 2003).

Protein kinase A (PKA) holoenzyme is one of the major receptors for cyclic adenosine monophosphate (cAMP), where an extracellular stimulus is translated into a signaling response. This paper reports the structure of a complex between the PKA catalytic subunit and a mutant RI regulatory subunit, RIα(91-379:R333K), containing both cAMP-binding domains. Upon binding to the catalytic subunit, RI undergoes a dramatic conformational change in which the two cAMP-binding domains uncouple and wrap around the large lobe of the catalytic subunit. This large conformational reorganization reveals the concerted mechanism required to bind and inhibit the catalytic subunit. The structure also reveals a holoenzyme-specific salt bridge between two conserved residues, Glu261 and Arg366, that tethers the two adenine capping residues far from their cAMP-binding sites. Mutagenesis of these residues demonstrates their importance for PKA activation. These structural insights, combined with the mutagenesis results, provide a molecular mechanism for the ordered and cooperative activation of PKA by cAMP (Kim, 2007).

Other PKA interactions and targets

Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization is rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p correlates inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to an SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolish cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. It is proposed that stress and cAMP-regulated intracellular localization of Msn2p are key steps in STRE-dependent transcription and in the general stress response (Görner, 1998).

An investigation was carried out of the role of cAMP-dependent protein kinase A (PKA) in the induction of the early mesodermal marker genes goosecoid and no tail by activin in zebrafish embryos. Upon treatment with activin, zebrafish blastula cells exhibit a rapid and transient increase in PKA activity. In these cells, activin rapidly induces the expression of the immediate early response genes goosecoid and no tail. Stimulation and inhibition of PKA by activin, respectively, enhances and reduces the induction of goosecoid and no tail mRNA expression. Similar effects of PKA stimulation and inhibition on the induction by activin of a 1.8 kb zebrafish goosecoid promoter construct are observed. The induction by activin of a fragment of the zebrafish goosecoid promoter that mediates an immediate early response to activin is blocked by inhibition of PKA. Activation of PKA alone has no effect in these experiments. Finally, inhibition of PKA in whole embryos by overexpression of a dominant negative regulatory subunit of PKA reduces the expression of no tail and goosecoid, whereas the expression of even-skipped1 remains unaltered. Overexpression of the catalytic subunit of PKA in embryos does not affect expression of goosecoid, no tail or even-skipped1. These data show that in dissociated blastulae, PKA is required, but not sufficient for activin signalling towards induction of goosecoid and no tail. In intact zebrafish embryos, PKA contributes to induction of goosecoid and no tail, although it is neither required nor sufficient (Joore, 1998).

Stimulation of cells with inducers of NF-kappaB (a Rel protein related to Drosophila Dorsal) such as LPS and IL-1 leads to the degradation of IkappaB-alpha and IkappaB-ß proteins (homologs of Drosophila Cactus) and translocation of NF-kappaB to the nucleus. Besides the physical partitioning of inactive NF-kappaB to the cytosol, the transcriptional activity of NF-kappaB is regulated through phosphorylation of NF-kappaB p65 by protein kinase A (PKA). The catalytic subunit of PKA (PKAc) is maintained in an inactive state through association with IkappaB-alpha or IkappaB-ß in an NFkappa-B-IkappaB-PKAc complex. Signals that cause the degradation of IkappaB result in activation of PKAc in a cAMP-independent manner and the subsequent phosphorylation of p65. Therefore, this pathway represents a novel mechanism for the cAMP-independent activation of PKA and the regulation of NF-kappa B activity (Zhong, 1997).

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in the olfaction of various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) which can function in turn to negatively regulate guanylyl cyclase, thus limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events (Moon, 1998).

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. It was hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increase to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity is observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle is also unchanged, as compared with wild type, although it is more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalizes with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retain this localization in knockout muscle. The RIalpha subunit binds AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions (Burton, 1997).

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encodes a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa. It has been named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence reveals high similarity to rat AKAP220 in the 1167 C-terminal residues, but contains 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA is expressed at high levels in testis and in isolated pachytene spermatocytes and round spermatids. The hAKAP220 protein is present in male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicates that hAKAP220 is localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization is found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I is resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, is extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitates both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s) (Reinton, 2000).

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. Two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, were used to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35 (a mutant that binds Raf) or RasG37 (a mutant that binds RalGDS) exhibit TSH-independent proliferation. RasS35 stimulates morphological transformation and anchorage-independent growth. RasG37 stimulates proliferation but not transformation as measured by these indices. TSH exerts markedly different effects on the Ras mutants and transiently represses MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulates MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exert differential effects on TSH signaling. RasS35 abolishes TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 has no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways (Miller, 1998).

The Wilms' tumor suppressor gene, WT1, encodes a transcription factor in the zinc finger family, which binds to GC-rich sequences and functions both as a transcriptional activator or repressor. The WT1 protein plays a crucial role in urogenital development in mammals; its function is thought to be conserved during vertebrate evolution. Although accumulating evidence suggests that WT1 regulates a subset of genes including growth factor and growth factor receptor genes, little is known about regulators or signal cascades that could modulate the function of WT1. The WT1 protein expressed exogenously in fibroblasts is phosphorylated in vivo; treatment with forskolin, which activates the cAMP-dependent protein kinase (PKA) in vivo, induces phosphorylation of additional sites in WT1. Forskolin-induced phosphorylation sites were identified as Ser-365 and Ser-393, which lie in the zinc finger domain in zinc fingers 2 and 3, respectively. PKA phosphorylates WT1 at Ser-365 and Ser-393 in vitro, as well as at additional sites. This phosphorylation abolishes the DNA-binding activity of WT1 in vitro. Using WT1 mutants in which Ser-365 and Ser-393 are mutated to Ala individually and in combination, it has been shown that phosphorylation of these sites is critical for inhibition of DNA binding in vivo. Thus, coexpression of the PKA catalytic subunit with wild type WT1 reduces the level of WT1 DNA-binding activity detected in nuclear extracts, and decreases transcriptional repression activity in vivo. In contrast to wild type WT1, all of the phosphorylation site mutants retain significant DNA-binding activity and repression activity in the presence of PKA. Analysis of the mutants shows that phosphorylation of Ser-365 and Ser-395 has additive inhibitory effects on WT1 DNA-binding in vivo and that phosphorylation at both sites is required for neutralization of repression activity. Therefore, it is concluded that PKA modulates the activity of WT1 in vivo through phosphorylation of Ser-365 and Ser-393, which inhibits DNA binding. This in turn results in a decrease in WT1 transcriptional repression. These findings provide the first evidence that the function of WT1 can be modulated by its phosphorylation in vivo (Sakamoto, 1997).

Zebrafish neurogenin1 (Drosophila homolog Atonal) encodes a basic helix-loop-helix protein that shares structural and functional characteristics with proneural genes in Drosophila melanogaster. neurogenin1 is expressed in the early neural plate in domains comprising more cells than the primary neurons known to develop from these regions; its expression is modulated by Delta/Notch signaling, suggesting that it is a target of lateral inhibition. Misexpression of neurogenin1 in the embryo results in development of ectopic neurons. Markers for different neuronal subtypes are not ectopically expressed in the same patterns in neurogenin1-injected embryos suggesting that the final identity of the ectopically induced neurons is modulated by local cues. Induction of ectopic motor neurons by neurogenin1 requires coexpression of a dominant negative regulatory subunit of protein kinase A, an intracellular transducer of Hedgehog signals. Inhibition of ngn1 expression in the lateral plate in embryos injected with constitutively active PKA suggests that PKA may act as a dominant repressor of ngn1 expression. The pattern of endogenous neurogenin1 expression in the neural plate is expanded in response to elevated levels of Hedgehog (Hh) signaling or abolished as a result of inhibition of Hh signaling. Other factors induced by Hedgehogs must be required in addition to Neurogenin1 for development of motor neurons. It is possible that ngn1 expression in the lateral neural plate is controlled by BMP4/7 and that the interplay of the two signaling centers causes the striped pattern of Ngn1 in the posterior neural plate. Together these data suggest that Hh signals regulate neurogenin1 expression and subsequently modulate the type of neurons produced by Neurogenin1 activity (Blader, 1997).

The mammalian Pbx homeodomain proteins (homologs of Drosophila Extradenticle) provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1 (Drosophila homolog: Homothorax), is now also demonstrated to be a CRS1-binding protein when purified using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs Extradenticle from Drosophila and ceh-20 from C. elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permits these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence (Bischof, 1998).

The basis for this cAMP response is not yet well understood. Pbx1 enhances the cAMP activated transcriptional response, mediated by protein kinase A, of CRS1's regulation of a reporter gene. There are several possible mechanisms by which protein kinase A could regulate the activity of CRS1: (1) the levels of one or more of the CRS1-binding proteins could be regulated, for example, at the level of gene expression or translation. (2) Alternatively, Pbx1 or Meis1 could be posttranslationally modified by phosphorylation, which could affect DNA binding, dimerization ability, or transactivation function. For example, protein kinase A phosphorylation of the homeodomain protein thyroid transcription factor 1 is involved in the expression of the surfactant protein B gene promoter by this homeodomain protein. (3) An additional possibility is that protein kinase A modifies a non-DNA-bound protein, which may interact with the Pbx-Meis1 complex. (4) Concerning CRS1 activity, there may be other components of the CRS1 binding complex, such as the 60-kDa protein that was copurified. Preliminary data indicate that this protein may be the homeodomain protein, Pknox1, which is related to Meis1. The identification of this protein, as well as how protein kinase A may regulate transcription through this element, is being investigated (Bischof, 1998).

ROMK inward-rectifier K+ channels control renal K+ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Since direct interaction with membrane phosphatidylinositol 4, 5-bisphosphate (PIP2) is essential for channel activation, the role of PIP2 in regulation of ROMK1 by PKA was investigated. By using adenosine-5'-[gamma-thio]triphosphate) (ATP[gammaS]) as the substrate, PKA was found not to directly activate ROMK1 channels in membranes that are devoid of PIP2. Rather, phosphorylation by PKA + ATP[gammaS] lowers the concentration of PIP2 necessary for activation of the channels. In solution-binding assays, anti-PIP2 antibodies bind PIP2 and prevent PIP2-channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 to inhibition by the antibodies, indicating an enhanced interaction between PIP2 and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP2 interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP2-channel interaction (Liou, 1999).

Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5' deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, it has been demonstrated that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5' untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE represses basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain reveals a protein complex of approximately 110 kDa that specifically binds to the DRE. Forskolin treatment reduces binding to the DRE, and the time course parallels that for an increase in prodynorphin gene expression. These results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. A model is proposed for human prodynorphin activation through PKA-dependent derepression at the DRE site. Two independent experimental techniques, UV cross-linking and Southwestern analysis, have allowed the initial characterization of the DRE binding activity, a 110-kDa nuclear complex that specifically binds to the DRE. In addition, a weaker 55-kDa band observed after UV cross-linking was competed with unlabeled DRE. It is tempting to speculate that the 110-kDa complex represents a dimer of the 55-kDa protein and that these two forms generate the two DRE-specific retarded bands. The DRE binding activity has been detected reproducibly in the prodynorphin-expressing cell lines tested, as well as in caudate samples from human brain. The latter finding further strengthens the functional significance of the DRE binding activity, since it is associated with the high level of expression of the prodynorphin gene in the caudate in vivo. However, the 110-kDa nuclear activity is observed also in human cerebellum. Since prodynorphin is expressed at very low levels in rat cerebellum, this suggests that the DRE binding protein complex may be involved in the regulation of other genes having DRE or DRE-like elements in their promoters (Carrion, 1998).

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin is shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolishes the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevents cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism (Dong, 1998b).

WAVE proteins (see Drosophila Scar/Wave)are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. WAVE-1 has been identified in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin because the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, a previously unrecognized function is proposed for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl (Westphal, 2000).

A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. This study tests the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores (Rawe, 2004).

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Snapin, a protein of relative molecular mass 15,000 has been identified that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolyzable cAMP analog induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. These results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway (Chheda, 2001).

The results presented here indicate that Snapin can serve as a target for PKA at the synapse to modulate transmitter release and neuronal plasticity through a second-messenger pathway. Phosphorylation of Snapin at serine 50, both in vitro and in vivo, leads to an increase in binding of Snapin to SNAP-25. Furthermore, this phosphorylation apparently strengthens the association of synaptotagmin, a proposed Ca2+ sensor for exocytosis, with the assembled SNARE complex. In physiological experiments, overexpression of constitutively phosphorylated Snapin (S50D Snapin) leads to an increase in the number of release-ready vesicles in adrenal chromaffin cells, whereas overexpression of the unphosphorylated form (S50A Snapin) reduces this number. In the context of the current model of exocytosis in chromaffin cells, these findings lead to the following conclusions: (1) both the S50D and S50A mutations increase the magnitude of the sustained component of exocytosis, indicating that Snapin acts as a positive modulator of the priming step by increasing the forward rate constant of priming; (2) S50D Snapin increases the size of the exocytotic burst, wheresa S50A Snapin reduces it. Thus, phosphorylation of Snapin leads to stabilization of release-ready vesicles, most probably by reducing the backward rate constant for the priming-unpriming reaction (Chheda, 2001).

Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signaling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signaling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction (Howe, 2000).

The PBC subfamily of TALE (Three-Amino-acid-Loop-Extension) homeodomain proteins includes the products of the vertebrate Pbx1, Pbx2, Pbx3 and Pbx4 genes, and the Drosophila extradenticle (exd) gene. PBC proteins form stable heterodimers with MEINOX proteins, which belong to a different subfamily of TALE homeodomain proteins and include the products of the vertebrate Meis and Prep genes and the Drosophila homothorax (hth) gene. The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. The processes controlling PBX1 nuclear export was investigated. In the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. These results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain (Kilstrup-Nielsen, 2003).

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from lysine residues of histone and nonhistone proteins. Recent studies suggest that they are key regulators of many cellular events, including cell proliferation and cancer development. Human class I HDACs possess homology to the yeast RPD3 protein and include HDAC1, HDAC2, HDAC3, and HDAC8. While HDAC1, HDAC2, and HDAC3 have been characterized extensively, almost nothing is known about HDAC8. HDAC8 is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro and in vivo. The PKA phosphoacceptor site of HDAC8 is Ser(39), a nonconserved residue among class I HDACs. Mutation of Ser(39) to Ala enhances the deacetylase activity of HDAC8. In contrast, mutation of Ser(39) to Glu or induction of HDAC8 phosphorylation by forskolin, a potent activator of adenyl cyclase, decreases HDAC8's enzymatic activity. Remarkably, inhibition of HDAC8 activity by hyperphosphorylation leads to hyperacetylation of histones H3 and H4, suggesting that PKA-mediated phosphorylation of HDAC8 plays a central role in the overall acetylation status of histones (Lee, 2004).

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

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I: Drosophila homolog Hephaestus) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. The nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB (Xie, 2003).

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. CaM-binding protein, designated regulator of calmodulin signaling (RCS), has been identified. G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acts as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocks GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increases this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B (Rakhilin, 2004).

Table of contents

cAMP-dependent protein kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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