THE DROSOPHILA P70(S6K) HOMOLOG EXHIBITS CONSERVED REGULATORY ELEMENTS AND RAPAMYCIN SENSITIVITY

The protein p70(s6k)/p85(s6k) lies on a mitogen-stimulated signaling p athway and plays a key role in G(1) progression of the cell cycle, Act ivation of this enzyme is mediated by a complex set of phosphorylation events, which has largely contributed to the difficulty in identifyin g the upstream kinases that mediate p70(s6k) activation, Genetics has proved a powerful complementary approach for such problems, providing an alternative means to identify components of signaling cascades and their functional end targets, As a first step toward implementing such an approach, we have cloned cDNAs encoding the Drosophila melanogaste r p70(s6k) homolog (Dp70(s6k)), Dp70(s6k) is encoded by a single gene, which generates three mRNA transcripts and exhibits an overall identi ty of 78% in the catalytic domain with its mammalian counterpart, Impo rtantly, this high identity extends beyond the catalytic domain to the N terminus, linker region, and the autoinhibitory domain, Furthermore , all the critical phosphorylation sites required for mammalian p70(s6 k) activation are conserved within these same domains of Dp70(s6k), Ch ief amongst these conserved sites are those associated with the select ive rapamycin-induced p70(s6k) dephosphorylation and inactivation, Con sistent with this observation, analysis of total S6 kinase activity in fractionated Drosophila Schneider line 2 cell extracts reveals two pe aks of activity, only one of which is rapamycin sensitive, By employin g a monospecific polyclonal antibody generated against Dp70(s6k), we s how that the cloned Dp70(s6k) cDNA has identity with only the rapamyci n sensitive peak, suggesting that this biological system would be usef ul in determining not only the mechanism of p70(s6k) activation, but a lso in elucidating the mechanism by which rapamycin acts to inhibit ce ll growth.