Overall patterns of in vivo expression of Akap200 and DAkap200 were established by performing Western blot analysis on protein samples isolated from flies at various developmental stages. Results obtained for cytosolic and detergent-solubilized, particulate proteins were similar. The Akap200 protein cluster was enriched in pupae (3-4-fold higher than other stages), but substantial levels of these anchor proteins were also evident in embryos, adults, and larvae. DAkap200 is also detected at all phases of the Drosophila life cycle. This indicates that alternative splicing of Akap200 gene transcripts is operative during the progression of embryonic and postembryonic development. The concentration of DAkap200 in the adult head, which is enriched in neurons, is ~7-fold higher than that observed in body parts. Therefore, alternative excision of exon 5 from Akap200 mRNA and/or stability of DAkap200 protein may be differentially regulated in a cell/tissue specific fashion in mature Drosophila (Li, 1999).
Analyses of abnormal nurse cell fusions during Drosophila oogenesis have revealed that genes regulating cytoskeleton function and genes involved in cAMP metabolism are required to preserve the structural integrity of the nurse cells. It was hypothesized that the anchoring of PKA holoenzyme near the cortex of cells may regulate cytoskeletal function. To test this hypothesis, the distribution of AKAPs was examined in wild-type ovaries. One of these, Akap200, can be detected near the membranes of the germline cells throughout oogenesis and is therefore a candidate for regulating PKA activity near the subcortical cytoskeleton. In somatic follicle cells, Akap200 is also associated with membranes during the early stages of oogenesis. Expression decreases in the oocyte-associated follicle cells as they begin their migration over the oocyte at approximately stage nine of oogenesis. Akap200 expression remains in the follicle cells associated with the nurse cells and in the nurse cells themselves. Akap200 is enriched on the ring canals of the nurse cells. Ring canals have an inner and outer rim, and although some proteins associate only with inner rims, specific functions have not yet been attributed to either the inner or the outer rim. Nevertheless, costaining with Hts-RC antibodies, which recognize inner-rim-specific epitopes of the hu-li tai shao gene product, suggests that Akap200 localizes to the outer rim of the ring canals. Finally, in addition to the membrane and ring canal association, Akap200 immunoreactivity is also associated with cytoplasmic puncta at all observable stages (Jackson, 2002).
The catalytic subunit of Pka-C is localized to the membranes of germline cells in wild-type ovaries. The subcellular localization of the regulatory subunits has not yet been described however, and because the catalytic subunits dissociate from the regulatory subunits in the presence of cAMP, the subcellular localization of the regulatory and catalytic subunits may not necessarily overlap. Therefore wild-type ovaries were stained with antibodies prepared against the Drosophila Pka-RII subunit. In the ovary, the localization of Pka-RII resembles Akap200. A majority of Pka-RII is associated with membranes of both the follicle cells and the germline-derived cells throughout oogenesis. At the anteriormost region of the germarium (region 1), Pka-RII is found on the membranes of the germline stem cells. Pka-RII levels appeared to be higher in the follicle cells than in the germline cells. In the middle stages of oogenesis, Pka-RII expression increases in the anteriormost group of follicle cells that will become border cells. Pka-RII is also found on the outer rim of ring canals of nurse cells. It persists on the ring canals until oogenesis is completed. Pka-RI localization is diffuse throughout the cytoplasm in all the cells in the ovary, and does not appear to be localized to particular structures in wild-type egg chambers (Jackson, 2002).
To evaluate the significance of Akap200 localization and to test its function in PKA anchoring, a P element insertion in the Akap200 locus (l(2)k07118k07118), identified and mapped by the Berkeley Drosophila Genome Project, was characterized. This P element is located in the large first intron of the Akap200 gene, and although the mutation was originally described as lethal, an unrelated background mutation found on the chromosome accounted for a majority of the lethality. Once this background lethal mutation was removed, Akap200 homozygotes could be recovered at slightly less than Mendelian-predicted frequencies; this allele is therefore referred to as Akap200k07118. Nevertheless, in all experiments, the phenotypes of hemizygous Akap200 mutants (Akap200/Df(2L)N22-14) were examined to reduce the contribution of other recessive mutations on the original P element chromosome. To verify that the P element is responsible for Akap200 phenotypes, and to generate other alleles, the P element was mobilized and multiple, independent excision lines were established. Precise excision reverted all the examined phenotypes, demonstrating that the P element is responsible for the observed abnormalities. Several lines exhibited decreased viability and/or the presence of extra notal bristles when trans to the Df(2L)N22-14 chromosome; four of these lines were chosen for further analysis. Although all of these lines produce extra notal bristles, neither the original insertion allele nor any of the excision lines demonstrated a complete penetrance in the reduced viability or bristle phenotype (Jackson, 2002).
Cuticles and other adult structures from Akap200k07118/Df(2L)N22-14 hemizygotes have a mostly wild-type morphology. In approximately a fifth of the adult flies, however, extra notal macrochaetae were present. These extra macrochaetae were accompanied by a socket cell and were either directly adjacent to the normal macrochaete or midway between the proximal and distal macrochaetae. Extra bristles were not observed in other locations, however, and the presence of more than one extra bristle on a single notum was rare. These observations suggest that Akap200 could be involved in the choice between the alternate cell fates of epidermis or sensory organ (Jackson, 2002).
Based on the similar staining patterns, it was hypothesized that Akap200 is responsible for the Pka-RII membrane sequestration and therefore, disrupting Akap200 function would redistribute Pka-RII to other areas. To test this hypothesis, Pka-RII localization was examined in Akap200 mutants. In control germaria, Akap200 and Pka-RII are associated with membranes of both the germline and follicle cells. In Akap200k07118 mutant ovaries, almost all the Akap200 immunoreactivity was abolished. This low but detectable residual staining suggested that the Akap200k07118 allele is not null. The effects of loss of Akap200 function are most evident in the germline cells, in which Pka-RII membrane staining decreases and cytoplasmic staining increases. Membrane association persists in the follicle cells however, and to a lesser degree in later-staged nurse cells. This persistent membrane staining may result from partial Akap200 activity or because other AKAPs anchor Pka-RII to the membranes of these cells. In the excision allele Akap200ix4, a partial restoration of Akap200 immunoreactivity correlates with a partial restoration of Pka-RII membrane localization, with a concomitant decrease in cytoplasmic staining. In Akap200D7 flies, Akap200 immunoreactivity is undetectable and is indistinguishable from controls with secondary antibody only. As a result, Pka-RII membrane association is reduced in the germline stem cells and in other cells of the ovary. Notably, the total amount of Pka-RII protein is unchanged in each of the Akap200 mutants. Akap200 is therefore responsible for sequestering a majority of Pka-RII to the membranes of germline cells during the early stages of oogenesis (Jackson, 2002).
It was predicted that if PKA anchoring is important for preserving nurse cell membrane and actin integrity, then disrupting anchoring should produce binucleate nurse cells. Both loss-of-function and gain-of-function approaches were used to test this prediction. The effects of Akap200 mutations on the morphology of the egg chamber were analyzed by staining mutant egg chambers with fluorescent-phalloidin conjugates, to examine actin-rich ring canals and subcortical actin, and DAPI, to visualize the nuclei. Females mutant for Akap200 produce egg chambers that have multinucleate cells with ring canal remnants. As is the case with most other mutations that produce multinucleate cells, these remnants stain with Hts-RC antibodies, suggesting that cytokineses and ring canal formation is initially normal in these mutants (Jackson, 2002).
It was reasoned that loss of Akap200 activity and the resulting failure to sequester PKA holoenzyme would produce a change in the local concentration of available Pka-C. This hypothesis was tested directly by expressing a constitutively active Pka-C mutant subunit (Pka-C*) throughout the germline cells. This transgene, driven by a UAS promoter, has a point mutation that diminishes its ability to bind to PKA regulatory subunits; it therefore cannot be sequestered. Two lines that express GAL4 at different levels in the germline [nos-GAL4-VP16 and NGT40, were used to determine the relative dosage effects of active Pka-C. When Pka-C* was expressed in the germline, binucleate cells were produced at a frequency that correlated with the levels of GAL4 expression (NGT40/+ < nos-GAL4-VP16/+). Although both NGT40 and nos-GAL4-VP16 direct expression during the earliest stages of oogenesis, binucleate cells were only observed after approximately stage five or six. A similar phenotype was observed when NGT40 was used to drive wild-type Pka-C instead of Pka-C*, although fewer affected egg chambers resulted. Nevertheless, to ensure the analysis of all potential mutant egg chambers, the frequency of binucleate cells was counted at all stages between 2 and 11, resulting in binucleate cell frequencies of less than 10% for all egg chambers. No binucleate cells were observed with a peptide inhibitor of Pka-C (UAS-PKIF) or a mutant that fails to bind to Pka-C (UAS-PKIG19,20), demonstrating that the binucleate cell phenotype is not due to GAL4 expression in the germline. Therefore, ectopic Pka-C activity disrupts the morphology of the germline cells (Jackson, 2002).
Models of AKAP function have suggested two distinct roles for AKAPs: they either control the local activity of Pka-C by sequestering inhibitory subunits to specific subcellular locales, or they act as a scaffold, bringing PKA signaling components together. The first model predicts that increased Pka-C activity would either have no effect or enhance AKAP loss of function phenotypes, whereas in the scaffold model, increased Pka-C activity would overcome the loss of AKAP function. To distinguish between these roles of Akap200 in regulating actin morphology, Pka-C* was expressed in Akap200 loss of function ovaries. The frequency of multinucleate cells increased when Pka-C* was expressed in an Akap200/Df(2L)N22-14 mutant background. These results are consistent with the hypothesis that Akap200 regulates the activity of the Pka-C subunit in the vicinity of subcortical actin in the nurse cells by localizing Pka-RII to these regions (Jackson, 2002).
Interestingly, it was noticed during these studies that Akap200 mutations also affect the size of the ring canals in the intact, mononucleate nurse cells. Ring canal size has been measured throughout oogenesis, and although variability is found because of the age of the ring canal, the size falls within a stereotypical range for a particular stage. In the strong Akap200 alleles, the ring canals appear to be larger and thinner than wild type. Ring canals were stained with phalloidin, anti-Hts-RC and anti-phosphotyrosine, then the inner and outer ring canal diameter was measured between stages seven and ten in mutant and wild-type egg chambers. At each stage and with each epitope, both the inner and outer rims are larger in the mutants. Although the ring canals are large and thin, phosphotyrosine, Hts-RC, Kelch and Filamin all appeared to localize normally to the mutant ring canals. The Akap200D7 allele also produces large, thin ring canals that are similar in size to Akap200k07118 ring canals. By contrast, the weaker Akap200ix4 allele, which retains some Akap200 function, has an approximately normal ring canal size, even though it produces multinucleate nurse cells (Jackson, 2002).
Because loss of Akap200 function produces large ring canals, it was hypothesized that the gain of Akap200 function might result in smaller ring canals. Therefore Akap200 was overexpressed in the nurse cells by crossing a GAL4-dependent 'enhancer piracy' line, EP2254, which maps to the Akap200 locus, to flies containing a germline-expressing GAL4 transgene (nos-GAL4-VP16). Ovaries from females carrying both nos-GAL4-VP16 and UAS-Akap200 produce egg chambers in which ring canals are smaller and thicker than wild type. These females are fertile however, and the small ring canals do not interfere with dumping (the transfer of nurse cell contents to the oocyte). Although nos-GAL4-VP16 is expressed at the earliest stages of oogenesis, no differences were detected in ring canal morphology prior to stage four, nor were other defects observed that could be correlated with altered germline stem cell function (i.e. extra or fewer divisions, changes in fate). Overexpressing Akap200 in the follicle cells results in ovaries that are indistinguishable from wild-type (Jackson, 2002).
Although many genes have been identified that alter ring canal function, altered ring canal size has only been observed in mutants of the non-receptor tyrosine kinases Src64B and Tec29. In these mutants, ring canals are smaller, a phenotype opposite that of the Akap200 mutants. Ring canals were examined in Src64B mutant ovaries when Akap200 gene dose is reduced, to determine whether Akap200 could antagonize Src64B in regulating ring canal size. The hypomorphic Src64BPI allele is homozygous viable and produces ring canals that are smaller than wild type. Akap200k07118 acted as a dominant suppressor of the small ring canal size phenotype produced by these mutants. Females of genotype Akap200k07118/+; Src64BPI/Src64BPI produce ring canals that are almost wild type in size and have near normal amounts of phosphotyrosine. Reducing Src64B gene dose by half, however, fails to affect the size of the ring canals produced by Akap200k07118 mutants. Ring canals produced by Akap200k07118/Akap200k07118; Src64BPI/+ are slightly smaller than those produced by Akap200k07118 mutants. These results suggest that Src64B and Akap200 act antagonistically to regulate ring canal growth. Akap200 protein localization is not altered detectably in Src64BPI mutant egg chambers, nor is Src64B localization changed visibly in Akap200 mutant ovaries. These observations suggest that post-translational mechanisms may be responsible for the genetic interaction (Jackson, 2002).
Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screened was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were recovered; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).
One of the misexpression interactors, MESR2, was an insertion upstream of the Akap200 locus. Akap200 refers to Drosophila A kinase anchor protein of molecular weight 200 kd, and binds the regulatory II (rII) subunit of cyclic AMP-dependent protein kinase (PKA). The Akap200 gene produces two different transcripts, one that contains the binding site for RII and one where the exon encoding for the RII binding site is spliced out to generate a protein that does not interact directly with PKA. Both isoforms of AKAP200 are expressed at relatively similar levels throughout development as well as in adult heads (Huang, 2000).
PKA is the principal mediator of signals that activate adenylate cyclase. cAMP signals are often targeted to effectors that accumulate to discrete intracellular locations. This targeting is due to a nonuniform distribution of PKA molecules within cells. In Drosophila, PKA has been implicated in normal developmental events in all imaginal tissues through the Hedgehog signaling pathway and is involved in signaling pathways that generate cell polarity: this requires that Hh be localized to distinct intracellular locations. Subcellular localization of PKA occurs through association with AKAPs. AKAPs are a functionally related family of proteins, defined by their ability to associate with PKA. Each AKAP contains a unique targeting domain that directs the complex to a defined intracellular location where PKA is placed proximal to both a signal generator (adenylate cyclase) as well as to potential PKA effector molecules. Coordinate binding of specific combinations of enzymes can allow such complexes to respond to distinct second messenger-mediated signals (Huang, 2000).
Studies in mammalian cells have suggested that PKA signaling via Rap1, another small molecular weight GTP-binding protein, antagonizes RAS1 signaling by competing for RAS pathway components such as B-Raf and MAPK. However, more recent studies suggest no genetic interaction between Drosophila Rap1 and RAS1. In Drosophila, overexpression of Rap1 in a heterozygous RAS1 mutant background has no effect on photoreceptor determination, suggesting no interaction between the two gene functions. A heterozygous Rap1 mutation does not reduce the number of R7 cells in a sev-RAS1V12 rough eye, also suggesting that the two pathways do not interact. Although there is no direct evidence linking PKA activation to MAPK activation via Rap1, there may be a still unknown pathway by which these molecules can signal (Huang, 2000).
The screen isolated Akap200 as a misexpression enhancer of KDN and suppressor of RAS1V12. This suggests that overexpression of this AKAP decreases signaling through RAS1. Overexpression of an AKAP might cause mislocalization of PKA molecules to the plasma membrane. This could activate a signaling pathway that normally is not utilized in this cell or at this time in development. If PKA and Rap1 are involved in RAS signaling, why were they not uncovered in previous loss-of-function screens? One possibility is that mutations in either gene may not be dose sensitive and therefore be unable to dominantly modify a rough eye phenotype. Another is the possibility that overexpression of an AKAP causes abnormal targets of PKA to become activated. Whether PKA signals through Rap1 is still unclear; however, the reported effects of attenuating RAS1/MAPK signaling are supported by this study. The enhancement of the KDN rough eye phenotype could be due to the additive effects of inefficient signaling due to KDN as well as the attenuation of MAPK by mislocalized PKA. In the activated RAS1V12 background, the attenuating effects of activated PKA due to mislocalization to the plasma membrane might reduce the amount of signaling through the pathway to suppress the RAS1-dependent rough eye phenotype (Huang, 2000).
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date revised: 12 October 2002
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