The Btk29A kinase is the only Drosophila member of a subfamily of tyrosine kinases that includes the mammalian kinases Btk, Itk and Tec. The mammalian proteins are required for hematopoiesis and the immune response, and are activated in response to signaling from the B-cell and T-cell receptors. Although Btk29A is expressed in a dynamic and complex pattern that includes the blood cells, there has been little investigation of the requirements for Btk29A in Drosophila. The PI recently identified Btk29A mutants and was able to show that it is essential for embryogenesis, for formation of bristles in the eye, and for the late stages of oogenesis. In addition, the PI has shown a genetic interaction between Btk29A and Src64B, which encodes a Src-family tyrosine kinase closely related to those that activate Btk, Itk and Tec. The PI now plans to examine this interaction more closely and search for additional genes in the Btk29A pathway. To get a more complete idea of the roles of Btk29A in embryogenesis, eye development, and oogenesis, the PI will isolate Btk29A null mutants and construct a Btk29A transposon that rescues the mutant phenotypes. Both Btk29A and Src64B are required for ring canal growth in developing egg chambers and interact genetically to give a more extreme phenotype. The PI will test the effects of these mutants on phosphotyrosine incorporation into the ring canals and will test whether the two kinases interact directly. The PI has recently shown that ring canals carry phosphothreonine as well as phosphotyrosine and that the phosphothreonine is also dependent on Btk29A. The PI plans to test whether the phosphotyrosine signal is transduced to phosphothreonine via the Ras or Rho families of small GTPases. Btk29A and Src64B are also required for dorsoventral patterning of the follicle cells. The PI plans to test whether they work in the Egfr-Ras pathway in these cells. In the absence of Btk29A the interommatidial bristles of the eye are truncated. The PI plans to compare growth of normal and mutant bristles using labeled phalloidin and antibodies to Btk29A, phosphotyrosine and phosphothreonine. Then the PI will use a sensitized screen to isolate mutations that enhance or suppress the bristle phenotype. Among these the PI hopes to find mutations in new members of Btk29A signaling pathways.
