The role of D-raf in the terminal class signal transduction pathway in Drosophila melanogaster

This dissertation describes the characterization of D-raf-mediated signal transduction in the establishment and elaboration of pattern in Drosophila melanogaster. D- raf is the Drosophila homologue of the human Raf-1 proto-oncogene. As a member of the Drosophila Torso (Tor) signal transduction cascade, D-raf acts to define cellular fates at the embryonic poles. Here we show that the serine/threonine kinase activity of D-raf is required for propagation of the Tor signal and that human Raf-1 can substitute for D-raf in this pathway. We identified two serine phosphorylation sites (S388 and S743) and found that serine or its phosphorylation at site 743 is essential for D-raf function. Serine-to-alanine substitution at residue 388, amino-terminal truncation, or membrane-targeted D-raf resulted in constitutive, Tor-independent signal transduction at the embryonic poles. Membrane targeted D-raf, but not amino-terminal truncated or serine substituted D-raf (S388A), could induce the development of terminal structures in the center of embryos. Thus, localization of D-raf to the membrane appears to be an essential step in promoting Tor signal transduction in the embryonic center;We also found that maternal D-raf activation takes place between nuclear cycle 4 and 12 and zygotic expression of the D-raf gene occurs at nuclear cycle 13, 14, and blastoderm stages. Western blot analysis revealed that the accumulation of D-raf protein in embryos at 0-2 and 2-4 hours after egg laying was observed in wild-type embryos, and those derived from D-raf[superscript]400B8, D-raf[superscript] C2Z2, D-raf[superscript] DF903, D-raf[superscript] raf2, D-raf[superscript] PB26 and D-raf[superscript] DC817 but is absent from D-raf[superscript]11-29, D-raf[superscript] EA75, D-raf[superscript]107, and D-raf[superscript] raf1 mutant germlines. We also show that maternal D-raf[superscript] 400B8 and D-raf[superscript] C2Z2 proteins exhibit dominant negative effects and these effects can be surpressed by a membrane targeted D-raf. This suggests that D-raf requires interaction with its substrate(s) at a membrane associated site