With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose. Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III. RAP-I was previously characterized. RAP-III is present in a very small amount, which hindered its purification. RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60. RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular 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 residue at the highly conserved carboxyl terminal domain as other P-protein kinases. The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases. Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha 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 kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome. All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements.

• PMID: 10683252
• DOI full text
• PubMed

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Comparative Study | Journal Article | Research Support, Non-U.S. Gov't

Authors

Bou G, Remacha M, Ballesta JP

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