Characterization of calreticulin as a protein interacting with protein kinase C.
A protein kinase C (PKC)-binding protein was purified to homogeneity from the Triton-insoluble fraction from rat hepatocytes homogenates. The protein was identified as the mature calreticulin chain by N-terminal amino acid sequencing and by its immunoreactivity with anti-calreticulin antibody raised against the C-terminal KDEL (single-letter code) sequence. The calculated molecular mass was 46. 6 kDa but the protein migrates in SDS/PAGE as a doublet with apparent molecular masses of 60 and 55 kDa. Studies in vitro with purified calreticulin with the use of an overlay assay approach demonstrated that it binds to activated PKC isoenzymes expressed in rat hepatocytes. Phosphorylation of purified calreticulin with a PKC isoenzyme-specific immune complex kinase assay showed that it is also a very good substrate for all PKC isoforms in vitro. The treatment of intact cells with phorbol ester or with adrenaline (epinephrine) plus propranolol increased calreticulin phosphorylation, which was blocked by the pretreatment of cells with the PKC-specific inhibitor Ro 31-8220. The analysis of calreticulin immunoprecipitates from control or treated cells indicated that PKCalpha, PKCbeta, PKCtheta;, PKCzeta and PKCmu, but not PKCdelta or PKCepsilon, co-immunoprecipitated with calreticulin. Taken together, our results indicate that PKC interacts in vivo with calreticulin and suggest that they can operate in common signalling pathways.
Rendón-Huerta
E
E
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. postal 70-159, D.F. 04510, México.
Mendoza-Hernández
G
G
Robles-Flores
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D021382:Protein Sorting Signals; D051381:Rats; D017208:Rats, Wistar; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D007527:Isoenzymes; D009842:Oligopeptides; D021382:Protein Sorting Signals; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D012261:Ribonucleoproteins; C073762:lysyl-aspartyl-glutamyl-leucine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D004837:Epinephrine