Protein kinase A localization affects the morphology of actin structures in the Drosophila ovary. S.M. Jackson , C.A. Berg. Dept Genetics, Univ Washington, Seattle, WA.

   Protein kinase A holoenzyme (PKA) is anchored to specific regions of the cell through interactions between regulatory subunits (Pka-R) and a large family of A-kinase anchoring proteins (AKAPs). We are investigating the role of PKA anchoring during oogenesis in Drosophila by analyzing DAKAP200, a bipartite protein that contains a Pka-RII binding domain and a separate MARCKS (myristoylated alanine-rich C-kinase substrate) domain. In wild-type ovaries, Pka-RII and DAKAP200 colocalize with subcortical actin and with the outer rim of the actin-rich ring canals. We isolated several DAKAP200 loss-of-function mutations. In mutant ovaries, most of the Pka-RII subunit was redistributed from subcortical areas of the cells to cytoplasmic regions. Mutant ovaries had binucleate or multinucleate cells, suggesting that a loss of cytoskeletal integrity occurred that resulted in fusions between adjacent cells. Moreover, most mutant alleles produced enlarged, thinned ring canals. Conversely, overexpression of DAKAP200 in the germline resulted in thicker, smaller ring canals. Interestingly, one DAKAP200 mutant allele affected the structural integrity of cells but did not affect ring canal size. Finally, expressing a mutant PKA catalytic subunit (Pka-C), incapable of binding to regulatory subunits and therefore incapable of being localized resulted in cell fusions at frequencies correlated with the dose of expression. Ring canal size, however, was unaffected. We therefore hypothesize that DAKAP200 has two separable functions. One function anchors Pka-RII and affects subcortical cytoskeletal integrity, while the other function regulates ring canal growth. Our results provide evidence that PKA localization is required for morphogenesis of specific cytoskeletal structures during development.