Substrates for protein kinase CK2 in insulin receptor preparations from rat liver membranes: Identification of a 210-kDa protein substrate as the dimeric form of endoplasmin

Ramon Trujillo, Francesc Miró, Maria Plana, Marta José, Mathieu Bollen, Willy Stalmans, Emilio Itarte

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Resum

Chromatography of extracts from rat liver membranes on wheat-germ lectin-Sepharose resulted in a partial resolution of the insulin receptor from other phosphorylatable proteins. Among the latter, a protein (p210, with an apparent M(r) of 210 kDa on SDS/PAGE under nonreducing conditions) was found to be phosphorylated by protein kinase CK2 on Thr and Ser residues. Under reducing conditions p210 was resolved into two phosphopolypeptides with apparent M(r) of 95 and 105 kDa. Neither the 95-kDa nor the 105-kDa polypeptides were recognized by antibodies against the βsubunit of the insulin receptor. Both polypeptides gave identical phosphopeptide maps after protease V8 digestion and contained the same N-terminal amino acid sequence. This sequence coincided with that of endoplasmin, and both polypeptides as well as p210 were recognized by antibodies against this protein. This shows that p210 corresponds to the dimeric form of rat liver endoplasmin. DEAE- Sepharose chromatography 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 specificity and inhibition by heparin) that are characteristic of the protein kinase CK2 enzymes. Furthermore, phosphoamino acid analysis and phosphopeptide maps of the 95/105-kDa polypeptides phosphorylated either by the endogenous protein kinase or by exogenous protein kinase CK2 gave similar results. The phosphorylation of p210/endoplasmin by protein kinase CK2 and its coelution gives support to the involvement of this protein kinase in membrane-associated processes.
Idioma originalAnglès
Pàgines (de-a)18-28
RevistaArchives of Biochemistry and Biophysics
Volum344
DOIs
Estat de la publicacióPublicada - 1 d’ag. 1997

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