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

Gene name - cAMP-dependent protein kinase 1

Synonyms - DC0, PKA

Cytological map position - 30C1--2

Function - Signal transduction

Keywords - Learning pathway, oogenesis, segment polarity

Symbol - Pka-C1

FlyBase ID:FBgn0000273

Genetic map position - 2-[34]

Classification - cAMP-dependent protein kinase A, serine/threonine kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Drosophila research is currently uncovering the role G-protein coupled receptors play in development. G-protein coupled receptors have been extensively characterized for their involvement in adult physiology (see The Learning Pathway). In mammals for example, G-protein coupled receptors are commonly found for hormones, neuropeptides and neurotransmitters. However, the involvement of G-protein coupled receptors in developmental pathways is a fairly new area of inquiry.

Cyclic AMP is the classic second messenger (see Cyclic AMP Second Messenger System for more information). It is used in Drosophila to transduce external signals sensed by G protein coupled receptors (e.g., Smoothened) or as a signal transducer of cytoplasmic events such as a change in Ca++ levels, for example, sensed by Calmodulin, which then interacts with Rutabaga).

Mammalian cyclic AMP-dependent protein kinase A (PKA) may serve here as a model for its Drosophila protein counterpart. The mammalian protein is a tetramer composed of two regulatory subunits and two catalytic subunits. In the holoenzyme, the enzymatic activity of the catalytic subunit, coded for by the gene described here, PKA-C1) is latent because of the inhibition conferred by the inhibitory domain of the regulatory subunit. When two mammalian cAMP molecules bind per regulatory subunit, the affinity of the regulatory subunit for the catalytic subunit decreases 10,000-100,000 fold, the tetramer dissociates into dimeric regulatory subunits, two monomers of the catalytic subunit are released, and inhibition is relieved. The catalytic subunit is subject to autophosphosphorylation that may stabilize the catalytic site and foster optimal alignment of peptide substrate within the site for phosphotransfer. Likewise, the catalytic subunit phosphorylates the regulatory subunit in an autoinhibitory domain (Francis, 1994).

Mutation in the catalytic subunit of PKA mimics the phenotype generated by ectopic expression of hedgehog in the anterior compartment of wing and leg discs and ahead of the morphogenetic furrow in the developing eye. That is, either PKA mutation or hedgehog ectopic expression induce ectopic expression of decapentaplegic, wingless and patched. Based on this evidence, it has been suggested that PKA is a component of a signaling pathway that represses dpp, wg and ptc expression and that hh antagonizes this pathway to maintain target gene expression at the anterior-posterior compartment border of appendages and in the morphogenetic furrow (Pan, 1995 and Li, 1995).

With the recent purification of Smoothened, the HH receptor, these findings can now be placed in a broader context. Smoothened is a 7 pass transmembrane receptor, characteristic of G-protein coupled receptors. As mentioned above, these receptors have well characterized roles as both hormone and neurotransmitter signal transducers, but Smoothened has a developmental role as HH receptor (Alcedo, 1996). Like other members of the serpentine receptor family, Smoothened is also coupled to G proteins. The presumed target of SMO is Adenylate cyclase (See Rutabaga), an enzyme that converts ATP to cyclic AMP. In turn, cAMP regulates Protein kinase A.

The target of PKA in segmentation is not yet known, although recent work suggests that this target is likely to be Cubitus interruptus (see below). In the learning pathway the target is CREB, the cyclic AMP response element binding protein, a transcription factor activated by PKA phosphorylation. Likewise, PKA targeted CREB is implicated in the regulation of proliferation of anterior pituitary somatotropic cells. Blocking CREB function in transgenic mice prevents proliferation of somatotropic cells, resulting in pituitary atrophy and dwarfism (Struthers, 1991).

PKA has a another documented role in Drosophila, in addition to signal transduction in segmentation and learning. PKA is associated with germ cell membranes and involved in establishment of oocyte polarity, acting to transduce a signal for microtubule reorganization involved in oocyte mRNA localization (Lane, 1994 and 1995).

PKA also controls quantal size in the Drosophila neuromuscular junction (NMJ). The NMJ is a glutamatergic synapse (the neurotransmitter is glutamate) with ultrastructural similarities to glutamatergic central synapses of vertebrates. Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. The gene encoding a second muscle-specific glutamate receptor, DGluRIIB, has been cloned and is closely related to the previously identified DGluRIIA, which is located adjacent to it in the genome. Both are non-NMDA type but cannot be classified as either AMPA or kainate type receptors. Both genes are expressed in muscle from stage 12, and neither gene is expressed in the nervous system. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal, because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function (Petersen, 1997).

Two distinct mechanisms regulate synaptic efficacy at the Drosophila neuromuscular junction: a PKA-dependent modulation of quantal size and a retrograde regulation of presynaptic release. Postsynaptic expression of a constitutively active PKA catalytic subunit decreases quantal size, whereas overexpression of a mutant PKA regulatory subunit (inhibiting PKA activity) increases quantal size. Increased PKA activity also decreases the response to direct iontophoresis of glutamate onto postsynaptic receptors. The PKA-dependent modulation of quantal size requires the presence of the muscle-specific glutamate receptor DGluRIIA, since PKA-dependent modulation of quantal size is lost in viable homozygous DGluRIIA- mutants. The DGluRIIA sequence contains an optimal PKA consensus phosphorylation site on the C-terminal tail (RRXS), believed to be located in the cytoplasmic portion of the protein. Elevated postsynaptic PKA reduces the quantal amplitude and the time constant of miniature excitatory junctional potential (mEJP) decay to values that are nearly identical to those observed in DGluRIIA mutants. PKA modulation of quantal size is sensitive to the copy number of DGluRIIA. Larvae heterozygous for a deletion of DGluRIIA show significantly less modulation by PKA than wild-type controls. This suggests that PKA-dependent modulation of receptor function may be influenced by the subunit composition of postsynaptic receptors. PKA activity appears to constitutively regulate synaptic function at the wild-type synapse. The demonstration that inhibition of PKA leads to a large increase in quantal size suggests that there is a high basal phosphorylation of DGluRIIA at the wild-type synapse. The PKA-dependent reduction in quantal size is accompanied developmentally by an increase in presynaptic quantal content, indicating the presence of a retrograde signal that regulates presynaptic release. A retrograde regulation of presynaptic transmitter release may serve to maintain postsynaptic excitation during the developmental growth of this synapse (Davis, 1998).

Analysis of protein kinase A (PKA) has been initiated in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. GAL4/UAS-regulated transgenes were constructed in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. Evidence is provided that the wild-type transgenes are active, and a summary is given of the phenotypes produced by a number of GAL4 enhancer-detector strains. The effects of transgenes encoding PKI(1-31) are compared with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits (Kiger, 1999).

Phenotypes produced by PKI(1-31) and PKAr* are surprisingly different. The phenotypic effects of PKI(1-31) appear to represent a subset of those of PKAr*. Both retard or otherwise block larval growth. PKAr* alone affects patterning in embryos and imaginal discs by activating Hedgehog signaling, and it alone causes abnormal differentiation in imaginal discs (which may reflect minor aberrations in patterning). The origin of this difference might reside in some fundamental difference in the biological properties of PKI(1-31) and PKAr* or perhaps in their relative stabilities in different cell types. However, PKI(1-31) is demonstrably active in wing imaginal discs and in other tissues since it is capable of inhibiting ectopic PKAcF (epitope tagged PKA catalytic subunit). Regardless of the origin of the difference, it would appear that PKI(1-31) specifically targets larval growth (Kiger, 1999).

Newly hatched larvae consist of two cell types: (1) mitotic cells composing the imaginal discs, gonad, and some neuroblasts, and (2) endoreplicating cells making up the exclusively larval tissues. These latter cells do not divide after hatching, but they increase in size as the larva grows, being maintained by cycles of DNA replication without nuclear division. These two cell types are regulated in fundamentally different ways, as demonstrated by their responses to nutritional deprivation. It would appear that mitotic cells are not sensitive to expression of PKI(1-31), but only to expression of PKAr*, whereas endoreplicating cells are sensitive to both. Both proteins are effective inhibitors of the catalytic site of PKAc, possessing a pseudosubstrate binding site with a pair of adjacent Arg residues that interact with the catalytic site. PKAr, which is larger than PKI(1-31) or full-length PKI(1-77), makes additional contacts with PKAc that make the PKAr:PKAc complex more stable than the PKI(1-77):PKAc complex. PKI(5-24) and PKI(1-77) bind to PKAc with the same affinity, and PKI(1-31) is probably no different. The C terminus of PKI(5-24) is not involved in binding to PKAc, so the FLAG epitope at the C terminus of PKI(1-31) should not interfere with its inhibitory function. Free PKAc and PKI(1-77) are small enough to enter the nucleus by diffusion. PKI(1-77) possesses a nuclear export signal (residues 35-49) that is hidden until PKAc is bound, whereupon the PKI(1-77):PKAc complex is extruded from the nucleus. Moreover, the expression and intracellular distribution of PKI(1-77) is regulated during the cell cycle and is necessary for cell cycle progression. PKI(1-31) would be expected to inhibit both nuclear and cytoplasmic PKAc. In addition, PKI(1-31) could compete with a Drosophila homolog of PKI(1-77) for nuclear PKAc and block PKAc export. In contrast, PKAr is cytoplasmic whether or not it is complexed with PKAc because it is too large to enter the nucleus. In addition, anchoring proteins have been identified that bind PKAr to the membrane or cytoskeleton. PKAr* would be expected to inhibit cytoplasmic PKAc and to deplete nuclear PKAc by forming a cytoplasmic sink for PKAc that diffuses from the nucleus (Kiger, 1999 and references).

With regard to Hedgehog signaling, a possible target of PKAr* and PKI(1-31) in the cytoplasm would be the complex responsible for the proteolysis of the transcription factor Ci, where PKAr* and PKI(1-31) would inhibit phosphorylation of the PKA sites necessary for proteolysis of Ci155 to the repressor form Ci75. The ability of PKAr* to interact with anchoring or other proteins might give it greater access to this complex than PKI(1-31), accounting for the failure of the latter to activate Hedgehog target genes (Kiger, 1999).

Another possible explanation for the different actions of PKAr* and PKIF(1-31) is that free PKAr* (and by implication free PKAr) has a target other than PKAc through which it activates Hedgehog signaling. Precedent for such a role exists. In Dictyostelium, free PKAr binds and activates a cAMP-specific phosphodiesterase that is postulated to have functional homology to the cAMP-specific phosphodiesterase encoded by dunce. The Dictyostelium phosphodiesterase is also activated by bovine PKAr1a, and a synthetic monomeric form of this regulatory subunit is a more potent activator than the dimeric form. (The Dictyostelium PKAr protein lacks a dimerization domain, and its PKA exists as a heterodimer). In this scenario, in the absence of a cAMP signal, PKAc would bind to PKAr, inhibiting this novel activity. Reduction in the level of PKAc, e.g., in a mitotic clone of cells homozygous for a lethal allele of DC0, would lead to free PKAr that would activate Hedgehog signaling (Kiger, 1999 and references).

In an alternative scenario, the effect of PKAr* on Hedgehog target genes could be caused by its ability to deplete nuclear PKAc, a role that cannot be fulfilled by PKI(1-31). Since the normal role of PKI(1-77) is not only to inhibit, but to export, nuclear PKAc, it is possible that PKAc plays another critical role in the nucleus in addition to its catalytic role in phosphorylation. For example, PKAc might function as a corepressor with Ci75 to block transcription of Hedgehog target genes. Consistent with this hypothesis PKI(1-60) has been shown to activate Ci-mediated chloramphenicol acetyltransferase transcription from a model Gli enhancer in Drosophila Kc cells, a finding can be attribute solely to inhibition of proteolysis of cytoplasmic Ci155. It may be that PKAc can function as a corepressor even if its catalytic site is occupied by PKI(1-31). Corepression by PKI(1-31):PKAc and Ci75 might block transcription of target genes, even in the presence of Ci155 produced by concommitant inhibition of Ci155 proteolysis in the cytoplasm. Small changes in the ratio of Ci155 and Ci75 are believed to be critical for activation of Hedgehog target genes. In addition, PKI(1-77) may differ from PKI(1-31) because only the former reduces basal transcription from cAMP-stimulated promoters. If PKAc has such an additional role, then the R224 mutant must have lost this function, as well as its ability to bind PKAr* and PKIF, since PKAcR224F produces no abnormal phenotypes and has no effect on viability. On the other hand, the hypothesized nuclear role of PKAc might be catalytic if nuclear PKAc is in some way inaccessible to nuclear PKI(1-31) (Kiger, 1999 and references).

These considerations suggest that normal Hedgehog signal transduction may require both inhibition of cytoplasmic PKAc activity and export of nuclear PKAc. The Drosophila homolog of PKI(1-77) would be a good candidate for carrying out these functions. The fact that PKI(1-77) seems to play some role in regulating the cell cycle may help to explain why PKI(1-31) has different effects on endoreplicating cells and mitotic cells. Resolving the nature of the roles played by PKAc in the cytoplasm and in the nucleus may lead to simultaneous understanding of the effects seen here on pattern formation and on cell growth (Kiger, 1999 and references).

Direct comparisons of the effects of PKI(1-31) and of PKI(1-77) are needed to provide more insight into how different PKAc inhibitors are functioning. PKAc transgenes with specific catalytic site mutations should provide evidence for or against a noncatalytic nuclear role for PKAc. PKAr* transgenes with domain-specific mutations should provide insight into the role of PKAR* in Hedgehog signaling. Identification of a Drosophila homolog of PKI(1-77) and study of its regulation will be important to achieve a clear understanding of the roles of PKAc. From a practical standpoint, PKI(1-31) transgenes should provide a useful tool for investigating the role of PKA in larval growth regulation, independent of PKA's effects on pattern formation. Mutations that permit larvae to survive the effect of PKI(1-31) and develop to adults should help to identify elements controlling larval growth. Conversely, mutations that sensitize adults or embryos to PKI(1-31) may reveal elements important for pattern formation (Kiger, 1999).


GENE STRUCTURE

At least four RNA species with differing sizes derive from the catalytic subunit gene. The relative abundance of these RNA forms varies at different times during development; the shortest species is particularly prominent in embryos and the longest in adults. The size differences among these RNA species occur principally at the 3' ends, perhaps by the use of different sites of polyadenylation (Kalderon, 1988).

Bases in 5' UTR - 825

Exons - 2


PROTEIN STRUCTURE

Amino Acids - 353

Structural Domains

Pka-C1 is 82% identical to the mammalian catalytic subunit of the cAMP-dependent protein kinase (Kalderon, 1988).

Since a basic surface on the catalytic (C) subunit of mammalian cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Seven specific carboxylates in RIalpha have been identified that are protected from chemical modification in the holoenzyme; each was then replaced with alanine. Of these, mutated rRI amino acids E15, E106, D107, E105, D140, E143, and D258 all are defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha, were mutated to Ala. Replacement of Glu101 has no effect while rRII E99 is very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, D170A and K242A, inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability (Gibson, 1997).

Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. The active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core. Also, the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis. In addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal "tail" (Shaltiel, 1998).


cAMP-dependent protein kinase 1: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
date revised: 21 APR 97 

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