cAMP-dependent protein kinase 1

DEVELOPMENTAL BIOLOGY

Germ cell line

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. A mutation affecting Pka-C1 acts in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of Bicoid and Oskar mRNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition; Oskar RNA fails to accumulate at the posterior, and Bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns previously reported for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells (Lane, 1994).

Pka-C1 is required in fly oogenesis. Intercellular bridges in egg chambers from PKA deficient females are unstable, leading to the formation of multinucleate nurse cells by fusions of adjacent cells. Germline clones of cells homozygous for null mutations of PKA-C1 indicate that PKA acts autonomously in the germline. Highest levels of PKA catalytic subunit protein are associated with germ cell membranes, suggesting that targets of PKA are associated with the membrane or membrane skeleton and contribute to the stabilization of intercellular bridges. The migration of a subset of follicle cells, the border cells, is also disrupted by germline PKA mutations, implying that nurse cell junctions provide an essential path for border cell migrations (Lane, 1995).

The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).

Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptc^mat-zyg+ and smo^mat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).

As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptc^mat- (and to a lesser extent pka^mat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).

Larval

Seventy-six genes have been identified that are strongly expressed in the Drosophila ring gland during development. For nine of these, further studies of expression pattern, mutant phenotype and molecular nature identify the genes as strong candidates to carry out an important role in endocrine functions controlling development. Two of the genes identified encode products that have already been implicated in the functioning of prothoracic glands in other insects. The Calmodulin gene is expressed exclusively and at high levels in the ring gland of third-instar larvae, suggesting an important, presumably endocrine function for calmodulin in that tissue, as has already been suggested for lepidopterans. Calmodulin and other Ca²⁺-binding proteins are integral to the transduction of a wide range of Ca²⁺-dependent signals; there is clear evidence for the Ca²⁺ dependence of ecdysteroid molting hormone (EC) production in the Manduca larval prothoracic gland (PTG), at least for the commitment peak early in the last larval instar. It is known that Ca²⁺ activates prothoracic gland adenylate cyclase both directly and as a complex when bound to calmodulin. Since cAMP phosphodiesterase activity is low at this stage, cAMP is expected to accumulate. Both large and small PTTHs (see Bombyx and Manduca prothoracicotropic hormone) stimulate increased cAMP levels in PTG; a rise in cAMP levels occurs with PTTH-stimulated EC production in early last-instar PTG (Harvie, 1998 and references).

The catalytic subunit of protein kinase A (PKA or cAMP-PK) is also expressed in the Drosophila ring gland. This protein probably functions downstream of cAMP in the Ca²⁺-cAMP-dependent signaling pathway. PKA is activated in M. sexta PTGs by PTTH immediately prior to EC production. This is consistent with the idea that activation of the Ca²⁺-cAMP-dependent signaling pathway by PTTH leads to PKA-dependent phosphorylation of key proteins, including ribosomal protein S6, and that this causes changes in selective translation leading to increased EC production (Harvie, 1998 and references).

The catalytic subunit of cyclic AMP-dependent protein kinase A is required for the correct spatial regulation of dpp expression during eye development. Loss of Pka-C1 function is sufficient to produce an ectopic morphogenetic wave, marked by premature ectopic photoreceptor differentiation and non-autonomous propagation of dpp expression. Pka-C1 lies in a signaling pathway that controls the orderly temporal progression of differentiation across the eye imaginal disc (Strutt, 1995).

Unlike the thoracic discs, the anterior and posterior compartmental organization of the genital imaginal disc is compound, consisting of three primordia ­ the female genital, male genital, and anal primordia. Each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in a compartment-specific manner in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. en and inv are required in the posterior comparment of the genital disc to repress dpp and activate hh. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments (Chen, 1997).

cAMP-dependent protein kinase A and engrailed-invected are genes known to play compartment-specific functions in other discs. The anterior/posterior patterning functions of these genes are conserved in the genital disc. en-inv mutant clones cause posterior to anterior transformations in adult terminalia. Pka is required to repress ptc, dpp and wg expression in the anterior compartment of the genital disc. Pka mutant clones result in pattern duplications in adult terminalia (Chen, 1997).

The adult clonal phenotypes of protein kinase A and engrailed-invected mutants provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. A new model has been proposed to describe the anterior and posterior compartmental organization of the genital disc. Each of the three primordia (female, male and anal) is composed of its own anterior and posterior compartments. Each primordium has a larger anterior compartment and a smaller posterior compartment. Each genital disc is divided into anterior and posterior compartment (Chen, 1997).

Pupal

Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, two mutant phenotypes were undertaken: (1) the batone mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map (batone has not yet been cloned); (2) ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic 'tumors'. Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. It is proposed that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. It is proposed that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing (Kiger, 2001).

This is the first report of hemocytes in the wings of newly eclosed flies. Their presence must have been overlooked because of the debris created by death of the wing epithelium. The power of the GAL4/UAS system to express GFP specifically in hemocytes has now enabled their detection. An apposition of dorsal and ventral wing surfaces occurs after eclosion. Two earlier appositions, followed by separations, of dorsal and ventral wing epithelia have occurred during pupal development. During each of these appositions, hemocytes are believed to secrete extracellular matrix (ECM) that binds the epithelia together. Subsequent separations are believed to be caused by proteolysis and phagocytosis of the ECM by hemocytes. Evidence from Drosophila and from Manduca sexta indicates that components of the ECM are found in hemocytes during pupal development. Therefore, it is reasonable to propose that hemocytes persist between the wing surfaces after eclosion where they phagocytose apoptotic epithelial cells and secrete an ECM that binds dorsal and ventral wing blades together. As a result of the destruction of epithelial cells, this ECM would have to bind directly to the cuticle of the wing surfaces and may contain a protein with chitin-binding domains. Thus, it is likely that the death of the wing epithelia is apoptotic (Kiger, 2001).

The identification of the fluorescent cells in normal wings as hemocytes is an inference based on studies of pupal development and on the detached fluorescent cells observed in wings of flies expressing PKAc, Ricin A, or PanDeltaN, a dominant-negative pangolin transgene. The latter cells fit previous descriptions of hemocytes. The association of PKAc expression with melanotic tumors (known to be caused by hemocytes) in various parts of the body strengthens this identification. The fluorescent cells in normal wings are tightly bound in a strikingly precise array that makes them an integral part of the wing, as might be expected if their role is to secrete ECM. As such, they do not exhibit characteristics that readily identify them as hemocytes. However, PKAc, Ricin A, or PanDeltaN expression disrupts this cellular array and prevents bonding of dorsal and ventral cuticular wing blades without affecting synthesis of the cuticle that forms the wing, demonstrating that the fluorescent cells in the wing are distinct from wing epithelial cells (Kiger, 2001).

Fate mapping places the focus of bae gene activity in the anterior neuroectoderm, a location that could become part of either the brain or the ring gland and distinct from the mesodermal origin of hemocytes. A striking feature of the data is that gynandromorphs either have both wings fully normal or fully mutant. This observation is consistent with a bilateral pair of nervous system primordia that interact in a submissive manner to establish the mutant wing phenotype in a nonautonomous manner. Thus, the role of bae could be to control wing maturation by the release of a hormone that increases blood pressure, causing wing unfolding, and that activates hemocytes to perform their roles of phagocytosis and ECM synthesis. Wing inflation has been ascribed to an unidentified neuroendocrine factor different from the eclosion hormone. A phenotype very similar to that of bae is produced by ectopic expression of UAS-dCBP(nej⁺) using GAL4 strains expressed in specific central nervous system cells (Kiger, 2001).

Comparison of the effects of Ricin A and of PKAc on wing maturation indicates that ectopic PKAc does not simply inactivate hemocytes. Instead, it appears to substitute one normal function of hemocytes for another. Rather than carry out phagocytosis and ECM synthesis, hemocytes enter into an innate immune response in which lamellocytes are differentiated and crystal cells melanize target cells. Evidently, aggregation of lamellocytes within the wing blade interferes with wing expansion, and loss of normal hemocyte function interferes with bonding of dorsal and ventral surfaces. The observation that the effect of ectopic PKAc on the wing is suppressed by overexpression of Pan, the Drosophila homolog of mammalian blood cell transcription factors (lymphocyte enhancer-binding factor 1 and T cell factor), suggests that ectopic PKAc inhibits, or represses synthesis of, Pan, which in turn inhibits Wingless target gene expression. This conclusion is strengthened by the observation that ectopic expression of UAS-dCBP(nej⁺) using GAL4-30A produces phenotypes similar to those caused by ectopic PKAc. Pan is bound and its transcriptional activity inhibited by dCBP. Expression of PanDeltaN, a dominant-negative inhibitor of Wingless target gene expression, elicits what seems to be a massive induction of the cellular innate immune response. Thus, the Wingless signal transduction pathway may be involved in regulating a choice between the innate immune response and the apoptotic/ECM response (Kiger, 2001).

The dominant-lethal interaction between ectopic PKAc and bae is intriguing. When and how death occurs needs closer examination, as does the cellular focus of bae activity. What role PKAc normally plays in regulating hemocyte behavior remains to be investigated. The association of a wing phenotype with altered hemocyte behavior should provide a means of identifying additional genes involved in hemocyte function during wing maturation (Kiger, 2001).

Adult

Involvement of the cAMP cascade in olfactory learning and memory in Drosophila is suggested by the aberrant behavioral phenotypes of the mutants dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase). PKA-C1 is preferentially expressed in the mushroom bodies. Mutants produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing double mutant heteroallelic viable animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes (Skoulakis, 1993).

dunce, rutabaga and Pka-C1 are expressed preferentially in mushroom bodies, neuroanatomical sites that mediate olfactory learning in Drosophila. Interestingly, the PDE (Dunce) and the catalytic subunit of PKA are found primarily in axonal and dendritic compartments of the mushroom body cells, whereas the adenyl cyclase (Rutabaga) is found primarily in the axonal compartment. The reason for this differential compartmentalization is unclear, although the hypothetical role of adenyl cyclase as coincidence detector would predict that conditioned stimuli and unconditioned stimuli (See Dunce site for definition) are integrated in the axonal compartment (Davis, 1995).

In both Drosophila and the honeybee Apis mellifera, cyclic adenosine monophosphate (cAMP)-dependent processes have been implicated in mechanisms of learning. This study characterizes the major target of cAMP in adult animals: the type II cAMP-dependent protein kinase (PKAII). In both species, PKAII (composed of Pka-R2 and Pka-C1 subunits) is restricted to neuronal tissue, where it accounts for more than 90% of total PKA activity. Although the intensity of PKAII immunoreactivity differs between distinct brain regions, labeling is detectable in all neuropiles and most somata. While the visual neuropiles, the antennal lobes, and structures of the central brain exhibit intermediate immunostaining, the mushroom bodies show high labeling and contain a three- to four-fold higher PKA activity, as compared to other neuropiles. Since the mushroom bodies are central sites of olfactory learning mediated via cAMP-dependent signaling, the modulatory functions of transmitters on PKA activity were tested using Kenyon cells from the honeybee. Agents that elevate cytoplasmic Ca2+ levels have no effects on PKA activity in cultured Kenyon cells. Dopamine, serotonin, and octopamine, however, cause an increase in PKA activity in Kenyon cells. The modulation of PKA activity by octopamine, the putative transmitter of the unconditioned stimulus in associative olfactory learning in the honeybee, together with the findings on the central role of the cAMP cascade in Drosophila mushroom bodies, suggests a major implication of PKAII-mediated phosphorylation in learning and memory in both Drosophila and Apis (Muller, 1997).

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