Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae

With ribosomal P protein as a substrate, five peaks of protein kinase activ ity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose, Two of them correspond to CK-II and the other th ree have been called RAP-1, RAP-II, and RAP-III, RAP-I was previously chara cterized. RAP-III is present in a very small amount, which hindered its pur ification. RAP-II was further purified on phosphocellulose, heparin-Sepharo se, and P protein-Sepharose, studied in detail, and compared with other aci dic protein kinases, including RAP-I, CK-II, and PK60, RAP-II is shown by S DS-PAGE and centrifugation on glycerol linear density gradients to have a m olecular mass of around 62 kDa and it is immunologically different from RAP -I and PK60, RAP-II phosphorylates the P proteins in the last serine residu e at the highly conserved carboxyl terminal domain as other P-protein kinas es, The ribosome-bound stalk P proteins are not equally phosphorylated by t he different kinases, Thus, RAP-II and PK60 mainly phosphorylate P1 beta an d P2 alpha whereas RAP-I and CK-II modify all of them. A comparative study of the K-m and V-max of the phosphorylation reaction by the different kinas es using individual purified acidic proteins suggests changes in the substr ate susceptibility upon binding to the ribosome, All the data available rev eal clear differences in the characteristics of the various P protein kinas es and suggest that the cell may use them to differentially modify the stal k depending, perhaps, on metabolic requirements. (C) 2000 Academic Press.