xf Interactive Fly, Drosophila

cAMP-dependent protein kinase 1


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PKA and Hedgehog signaling

Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as a transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. Processing of Drosophila Ci155 protein depends upon activity of the cyclic AMP-dependent protein kinase (PKA). In addition, responses to Shh signaling in vertebrates are blocked either by pharmacological manipulations that increase cAMP levels or by coexpression with the constitutively active form of the PKA catalytic subunit. These findings are consistent with a possible role for PKA phosphorylation activity in the formation of a Gli repressor. Transfected cells were treated either with forskolin (FSK), a membrane-permeable activator of adenylate cyclase, and its inactive analog dideoxy forskolin (ddFSK) as a control, or 3-isobutyl-1-methyl-xanthine (IBMX), a membrane-permeable inhibitor of cAMP phosphodiesterase. Treatment of both COS1 cells and primary limb bud cultures with FSK or IBMX but not with ddFSK or the DMSO vehicle causes the appearance of a novel species detectable by antiGli3 antiserum. This novel species has been designated Gli3-83 based upon its electrophoretic mobility. If Gli3-83 is formed by a single cleavage of the Gli3-190 precursor, as appears to occur in the processing of Ci155, this cleavage occurs between residues 700 and 740 within the Gli3-190 precursor. The Gli3-83 protein would thus contain amino-terminal sequences extending just C-terminal to the zinc finger region, corresponding to the Drosophila Ci75 repressor in its structure. The cAMP-stimulated appearance of Gli3-83 in COS1 cells and in primary limb bud cultures suggests that Gli3 processing may be dependent on PKA activity (Wang, 2000).

PKA-dependent processing of vertebrate Gli3 in the developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation (Wang, 2000).

The large proportion of total Gli3 protein that exists as the Gli3-83 species suggests that Gli3-190 is readily processed. The extent, if any, of endogenous Gli1 and Gli2 processing cannot be examined until high-affinity antibodies are available. It is interesting to note, however, that neither Gli1 nor Gli2 appear to be processed in transfection experiments with established cell lines, even though Gli2 is phosphorylated upon stimulation of PKA. Gli proteins display biologically distinct properties upon high-level expression in Xenopus embryos, such that ectopic development of Shh-inducible cell fates appears to be promoted by Gli1, by Gli2 to a lesser extent, and not at all by Gli3, which actually seems to suppress the positive activities of Gli1 and Gli2. Differences in propensity to be processed may help account for these differences, with Gli3 representing a potent repressing activity for Shh-inducible fates simply because it is readily processed to form Gli3-83 repressor, particularly at sites distant from endogenous Shh activity (Wang, 2000).

The pattern of Gli-dependent transcription of a developing structure would depend upon the combined activities of all Gli protein species present. In the case of Gli3, Shh signaling appears to reduce transcription, and the protein has a high propensity for repressor formation; these properties of the Gli3 gene and of the Gli3 protein help insure that highest repressor levels are found in cells experiencing the lowest levels of Shh pathway activation. Conversely, transcription of Gli1 and Gli2 is respectively stimulated or unaffected by Shh pathway activation, at least outside the immediate zone of Shh gene expression: this neutral or positive effect is coupled to the more positive effects of Gli1 and Gli2 proteins on Gli-dependent transcription of target genes. These more positive effects of Gli1 and Gli2 proteins may be in part due to a reduced propensity for processing and repressor formation. As compared to Drosophila, the vertebrate innovation of distinctly regulated Gli genes encoding proteins with distinctly regulated propensities for processing may provide the opportunity to establish a more robust and finer gradation of Gli-dependent transcription within a developing field of cells (Wang, 2000).

The hedgehog (Hh) pathway plays a critical role during embryo development and in cancer. Although the molecular basis by which protein kinase A (PKA) regulates the stability of Hedgehog's downstream transcription factor Cubitus interruptus (the Drosophila homologue of vertebrate Gli molecules) is well documented, the mechanism by which PKA inhibits the functions of Gli molecules in vertebrates remains elusive. This study reports that activation of PKA retains Gli1 in the cytoplasm. Conversely, inhibition of PKA activity promotes nuclear accumulation of Gli1. Mutation analysis identifies Thr374 as a major PKA site determining Gli1 protein localization. In the three-dimensional structure, Thr374 resides adjacent to the basic residue cluster of the nuclear localization signal (NLS). Phosphorylation of this Thr residue is predicted to alter the local charge and consequently the NLS function. Indeed, mutation of this residue to Asp (Gli1/T374D) results in more cytoplasmic Gli1 whereas a mutation to Lys (Gli1/T374K) leads to more nuclear Gli1. Disruption of the NLS causes Gli1/T374K to be more cytoplasmic. The change of Gli1 localization is correlated with the change of its transcriptional activity. These data provide evidence to support a model that PKA regulates Gli1 localization and its transcriptional activity, in part, through modulating the NLS function (Sheng, 2006).

Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube

Protein kinase A (PKA) is an evolutionarily conserved negative regulator of the hedgehog (Hh) signal transduction pathway. PKA is known to be required for the proteolytic processing event that generates the repressor forms of the Ci and Gli transcription factors that keep target genes off in the absence of Hh. This study shows that complete loss of PKA activity in the mouse leads to midgestation lethality and a completely ventralized neural tube, demonstrating that PKA is as strong a negative regulator of the sonic hedgehog (Shh) pathway as patched 1 (Ptch1) or suppressor of fused (Sufu). Genetic analysis shows that although PKA is important for production of the repressor form of Gli3, the principal function of PKA in the Shh pathway in neural development is to restrain activation of Gli2. Activation of the Hh pathway in PKA mutants depends on cilia, and the catalytic and regulatory subunits of PKA are localized to a compartment at the base of the primary cilia, just proximal to the basal body. The data show that PKA does not affect cilia length or trafficking of smoothened (Smo) in the cilium. Instead, there is a significant increase in the lev el of Gli2 at the tips of cilia of PKA-null cells. The data suggest a model in which PKA acts at the base of the cilium after Gli proteins have transited the primary cilium; in this model the sequential movement of Gli proteins between compartments in the cilium and at its base controls accessibility of Gli proteins to PKA, which determines the fates of Gli proteins and the activity of the Shh pathway (Tuson, 2011).

PKA and other signaling pathways

The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. Activated CaM-KIV can activate the mitogen-activated protein kinases. The present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gives rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which is due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains are deleted, is still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site is identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators give 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway (Wayman, 1997).

Hepatocyte nuclear factor 4 (See Drosophila HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for liver development and liver-specific gene expression. Its DNA-binding activity is modulated posttranslationally by phosphorylation. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids are able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly represses the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activate transcription more efficiently than do those from the wild-type protein, when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA that might be involved in the transcriptional inhibition of liver genes by cAMP inducers (Viollet, 1997).

The cAMP signaling cascade has been implicated in several stages of memory formation. Activation of this cascade by serotonin (5-HT) in the sensory neurons of Aplysia has been examined. Different patterns of 5-HT exposure induce three distinct modes of PKA activation: (1) a single 5 min pulse induces transient (5 min) PKA activation that requires neither transcription nor translation; (2) 4-5 pulses induce intermediate-term persistent activation (3 hr duration) that requires translation but not transcription; (3) 5 pulses of 5-HT, as well as continuous (90 min) exposure, induce long-term persistent activation 20 hr later, which requires both transcription and translation. Thus, in the sensory neurons, different patterns of 5-HT give rise to three independent phases of PKA activation that differ in their induction requirements, their temporal profiles, and their molecular mechanisms (Muller, 1998).

The regulation of ribosome biogenesis in response to environmental conditions is a key aspect of cell growth control. Ribosomal protein (RP) genes are regulated by the nutrient-sensitive, conserved target of rapamycin (TOR) signaling pathway. TOR controls the subcellular localization of protein kinase A (PKA) and the PKA-regulated kinase YAK1. However, the target transcription factor(s) of the TOR-PKA pathway are unknown. Regulation of RP gene transcription via TOR and PKA in yeast involves the Forkhead-like transcription factor FHL1 and the two cofactors IFH1 (a coactivator) and CRF1 (a corepressor). TOR, via PKA, negatively regulates YAK1 and maintains CRF1 in the cytoplasm. Upon TOR inactivation, activated YAK1 phosphorylates and activates CRF1. Phosphorylated CRF1 accumulates in the nucleus and competes with IFH1 for binding to FHL1 at RP gene promoters, and thereby inhibits transcription of RP genes. Thus, a signaling mechanism is described linking an environmental sensor to ribosome biogenesis (Martin, 2004).

In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. Mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Delta background. The slow-growth phenotype of mob2Delta ras2Delta cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described. In addition, mob2Delta strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2. Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, it is concluded that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression (Schnaper, 2004).

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. This study shows that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocks wnt-induced alterations in HSC formation. PGE2 modifies the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulates murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt is also conserved during regeneration of other organ systems. This work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery (Goessling, 2009).

PKA and development

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. The discoidins are among the first genes to be induced upon exhaustion of food and are used as a molecular marker for the initiation of development. Overexpression of PKA leads to overexpression of the discoidinI gammma promoter, while expression of the discoidinI gamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. Signaling molecules that are active at the transition from growth to development are the glycosylated proteins PSF (prestarvation factor) and CMF (conditioned medium factor). PKA-C null cells are able to respond to media conditioned by PSF and CMF. It is concluded that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes (Primpke, 2000).

Constitutive inhibition of cAMP-dependent protein kinase (PKA) in Dictyostelium cells blocks cell aggregation and development. Overexpressing dominant-negative PKA regulatory subunits (PKA-RM) under an actin 15 promoter results in mutants that can not relay pulses of the chemoattractant cAMP, due to a defect in expression of the aggregative adenylyl cyclase (ACA) gene. Also strongly reduced in the mutants are unstimulated and cAMP pulse-induced expression of other aggregative genes encoding the cAMP receptor cAR1, adhesive contact sites A, and cAMP-phosphodiesterase. Additionally, the expression of the discoidin I gene, expressed early in development in response to cell density sensing factors, is almost completely absent. These data contrast with observations that cAMP relay and aggregative gene expression are normal in null mutants for the PKA catalytic (C) subunit, and suggest the presence of multiple C subunit genes in Dictyostelium and an almost universal requirement for PKA activity in developmental gene expression (Schulkes, 1995).

The pseudoplasmodium or migrating slug of Dictyostelium is composed of non-terminally differentiated cells, organized along an anteroposterior axis. Cells in the anterior region of the slug define the prestalk compartment, whereas most of the posterior zone consists of prespore cells. Evidence is presented that the cAMP-dependent protein kinase (PKA) and the RING domain/leucine zipper protein rZIP interact genetically to mediate a transcriptional activation gradient that regulates the differentiation of prespore cells within the posterior compartment of the slug. PKA is absolutely required for prespore differentiation. In contrast, rZIP negatively regulates prespore patterning; rzpA- cells, which lack rZIP, have reduced prestalk differentiation and a corresponding increase in prespore-specific gene expression. Using cell-specific markers and chimaeras of wild-type and rzpA- cells, it has been shown that rZIP functions non-autonomously to establish a graded, prespore gene activation signal, but autonomously to localize prespore expression. Overexpression of either the catalytic subunit or a dominant-negative regulatory subunit of PKA further demonstrates that PKA lies within the intracellular pathway that mediates the extracellular signal and regulates prespore patterning. A 5'-distal segment within a prespore promoter that is responsive to a graded signal is also sensitive to PKA and rZIP, indicating that the signal acts directly at the level of prespore-specific gene transcription for regulation (Balint-Kurti, 1998).

Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. Thus CRAC acts upstream of ACA. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains form mounds, they do not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescues sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation is observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. The development of CRAC-null ACA-null double mutants can be rescued by the activation of PKA, together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway. How CRAC executes its function in prespore cell differentiation and morphogenesis during postaggregative development remains unknown. Possibilities include the participation of CRAC in a PKA-independent pathway required for sporulation, or the additional utilization of CRAC in the PKA pathway 'downstream' of PKA-C (B. Wang, 1999).

The S. pombe pcr1 gene encodes a bZIP protein that apparently binds to and activates promoters containing cyclic AMP response elements (CRE). PCR1 consists of 171 amino acid residues and is most similar to the mammalian CRE-BP1. Disruption of the pcr1 gene is not lethal, but mutants show cold-sensitive growth on rich medium. Mutants are also inefficient in mating and sporulation, though they are not completely sterile. Expression of the ste11 gene, which encodes a key transcription factor for sexual development, is greatly reduced, and overexpression of ste11+ suppresses the deficiency of the pcr1mutants in sexual development. Expression of ste11 is negatively regulated by cyclic AMP-dependent protein kinase (PKA). The loss of PKA activity results in ectopic sexual development. Disruption of pcr1 blocks ectopic sexual development. Furthermore, disruption of pcr1 reduces expression of fbp1, a glucose-repressible gene negatively regulated by PKA. Pcr1 appears to be a transcriptional regulator whose activity may be controlled by PKA. Alternatively, its activity may be independent of PKA, and full induction of ste11 and fbp1 expression requires the function of Pcr1 in addition to elimination of the repression by PKA (Watanabe, 1996).

Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through the utilization of several different classes of G proteins. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. The mechanism that controls the specificity of this coupling has been examined. In HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. These results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs, and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' the coupling of this receptor from Gs to Gi and initiate a new set of signaling events (Daaka, 1997).

Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. Sonic hedgehog (Shh), which is made by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum (Wechsler-Reya, 1999).

Expression of the Nodal gene, which encodes a member of the TGFß superfamily of secreted factors, localizes to the left side of the developing embryo in all vertebrates examined so far. This asymmetric pattern correlates with normal development of the left-right axis. The Wnt and PKA signaling pathways control left-right determination in the chick embryo through Nodal. A Wnt/ß-catenin pathway controls Nodal expression in and around Hensen's node, without affecting the upstream regulators Sonic hedgehog, Car and Fibroblast Growth Factor 8. Transcription of Nodal is also positively regulated by a protein kinase A-dependent pathway. Both the adhesion protein N-cadherin and PKI (an endogenous protein kinase A inhibitor) are localized to the right side of the node and may contribute to restrict Nodal activation by Wnt signaling; PKA is localized to the left side of the node (Rodriguez-Esteban, 2001).

A model is presented for the role of Wnts and PKA in LR determination in the chick embryo. At the time at which Nodal becomes restricted to the left side of Hensen's node (HH stage 5+), Wnt/ß-catenin and PKA act as positive regulators of Nodal transcription through Shh-independent mechanisms. Activation (or maintenance) of Nodal on the right side of the node might be prevented by at least two mechanisms: (1) the presence of N-cadherin on the right side, which could inhibit activation of Nodal transcription by ß-catenin; (2) the presence of high levels of PKI, which could interfere with the activation of Nodal by PKA. Expression of N-cadherin and PKI is biased towards the right side of the node at this stage; expression of Shh, Nodal and lefty-1 is left-specific. At this stage, several Wnts that are known to signal through ß-catenin are expressed in or around the node, and thus in this model a sum of Wnt activities is considered to be mediated by ß-catenin. In the mouse, Wnt-8c is expressed in a pattern very similar to that of its chick counterpart, but its role in LR development has not been described yet. Ectopic expression of Wnt-8c in a transgenic line induces an ectopic embryonic axis and causes a truncation of the anterior neuroectoderm. Also, mice deficient in ß-catenin have been shown to display severe defects of the anterior-posterior axis, which prevents an analysis of possible defects in LR determination (Rodriguez-Esteban, 2001).

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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