Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Abstract

A 70-kDa protein is phosphorylated in cellfree 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 ofnormal 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.

MARCKS-related Protein (MRP) Is a Substrate for the Leishmania major Surface Protease Leishmanolysin (gp63)

“…These data are in line with those of Spizz and Blackshear (12,13), who found that PKC-phosphorylated MARCKS was a poor substrate for cathepsin B. Similarly, decreased susceptibility of MARCKS to cathepsin L after phosphorylation by PKC has been reported (33), and regulation of proteolysis by substrate phosphorylation was also described for the related GAP-43 protein (34).…”

Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment

“…P2, P5, P10, and P12) may be generated by either direct endocleavage at atypical sites, or by endocleavage at cathepsin L typical sites followed by a carboxypeptidase activity at the peptide C terminus. The mass spectrometry analysis also demonstrated that the linker segment is preferentially phosphorylated on serine and tyrosine residues, suggesting their possible regulatory role in heparanase processing through an effect on the accessibility of particular cathepsin L cleavage sites, as reported for HSP-70 (41).…”

Down-regulation of MARCKS-related Protein (MRP) in Macrophages Infected with Leishmania

“…MARCKS mRNA and protein expression can be decreased in fibroblasts through either PKC-dependent or -independent pathways by a post-transcriptional mechanism (24,25). MARCKS concentrations may also be regulated by specific proteolytic cleavage of the unphosphorylated protein by a cysteine protease (26,27), which has recently been identified as cathepsin B (28). To our knowledge, no reports concerning the down-regulation of MRP are avail-able.…”