Identification of insulin-induced sites of ribosomal protein S6 phosphorylation in Drosophila melanogaster.
Insulin treatment of Drosophila melanogaster Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis revealed that the multiply phosphorylated protein was the larger of two Drosophila melanogaster orthologues of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A derived from stimulated Kc 167 cells with the endoproteinase Lys-C released a number of peptides, one of which contained all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) showed that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation were identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245). Finally, phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicated that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion. The importance of these observations in cell growth and development is discussed.
Radimerski
T
T
Friedrich Miescher Institute, Basel, Switzerland.
Mini
T
T
Schneider
U
U
Wettenhall
R E
RE
Thomas
G
G
Jenö
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004331:Drosophila melanogaster / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D018402:Manduca; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis
D007328:Insulin; D010748:Phosphopeptides; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins