Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.
A 70-kDa protein is phosphorylated in cell-free preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the 70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol of normal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed.
Laumas
S
S
Division of Biological Sciences, Cornell University, Ithaca, NY 14853.
Abdel-Ghany
M
M
Leister
K
K
Resnick
R
R
Kandrach
A
A
Racker
E
E
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000494:pharmacology*; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine
D000470:Alkaloids; D010750:Phosphoproteins; D011480:Protease Inhibitors; D004533:Egtazic Acid; D011493:Protein Kinase C; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat; D019311:Staurosporine; D005033:Ethylmaleimide