Identification of insulin-induced sites of ribosomal protein S6 phosphorylation in Drosophila melanogaster

Insulin treatment of Drosophila melanogaster Kc 167 cells induces the multi ple phosphorylation of a Drosophila ribosomal protein, as judged by its dec reased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cyclohexim ide and blocked by pretreatment with rapamycin. Isolation and mass spectrom etric analysis revealed that the multiply phosphorylated protein was the la rger of two Drosophila melanogaster orthologues of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A derived from sti mulated Kc 167 cells with the endoproteinase Lys-C released a number of pep tides, one of which contained all the putative phosphorylation sites. Conve rsion of phosphoserines to dehydroalanines with Ba(OH)(2) showed that the s ites of phosphorylation reside at the carboxy terminus of DS6A. The sites o f phosphorylation were identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(23 9), Ser(242), and Ser(245) Finally, phosphopeptide mapping of individual ph osphoderivatives, isolated from two-dimensional polyacrylamide gels, indica ted that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion. The importance of these observati ons in cell growth and development is discussed.