SUBSTRATES FOR PROTEIN-KINASE CK2 IN INSULIN-RECEPTOR PREPARATIONS FROM RAT-LIVER MEMBRANES - IDENTIFICATION OF A 210-KDA PROTEIN SUBSTRATEAS THE DIMERIC FORM OF ENDOPLASMIN

Chromatography of extracts from rat liver membranes on wheat-germ lect in-Sepharose resulted in a partial resolution of the insulin receptor from other phosphorylatable proteins, Among the latter, a protein (p21 0, with an apparent M-r of 210 kDa on SDS/PAGE under nonreducing condi tions) was found to be phosphorylated by protein kinase CK2 on Thr and Ser residues, Under reducing conditions p210 was resolved into two ph osphopolypeptides with apparent M-r of 95 and 105 kDa. Neither the 95- kDa nor the 105-kDa polypeptides were recognized by antibodies against the beta-subunit of the insulin receptor, Both polypeptides gave iden tical phosphopeptide maps after protease V8 digestion and contained th e same N-terminal amino acid sequence, This sequence coincided with th at of endoplasmin, and both polypeptides as well as p210 were recogniz ed by antibodies against this protein, This shows that p210 correspond s to the dimeric form of rat liver endoplasmin, DEAE-Sepharose chromat ography of p210 preparations removed most other contaminating proteins and revealed the presence of a protein kinase activity that coeluted with p210, This protein kinase possessed the properties (substrate spe cificity and inhibition by heparin) that are characteristic of the pro tein kinase CK2 enzymes, Furthermore, phosphoamino acid analysis and p hosphopeptide maps of the 95/105-kDa polypeptides phosphorylated eithe r by the endogenous protein kinase or by exogenous protein kinase CK2 gave similar results, The phosphorylation of p210/endoplasmin by prote in kinase CK2 and its coelution gives support to the involvement of th is protein kinase in membrane-associated processes. (C) 1997 Academic Press.