Protein kinase C-mediated phosphorylation of the myristoylated alanine-rich C-kinase substrate protects it from specific proteolytic cleavage.
The myristoylated alanine-rich C kinase substrate (MARCKS) is a major cellular substrate of protein kinase C. Its concentration in cells is important for the normal development of the central nervous system, and perhaps other physiological processes. We found that MARCKS concentrations in cells were regulated in part by a specific proteolytic cleavage; this resulted in two fragments, each representing about half of the intact protein, that co-existed with MARCKS in cells and tissues. These fragments were present in significant concentrations in quiescent fibroblasts; they disappeared, and the amount of intact MARCKS increased, within 15 s of activation of protein kinase C by serum. In vitro experiments demonstrated that phosphorylated MARCKS was a poor substrate for a protease activity present in cell extracts, whereas dephosphorylated MARCKS was a good substrate. Both the protease activity and the specific MARCKS cleavage products were essentially absent in brain, but present in many other cells and tissues. The protease activity, which had the characteristics of a cysteine protease, cleaved MARCKS between Asn147 and Glu148 of the bovine sequence, three amino acids to the amino-terminal side of the MARCKS phosphorylation site domain. These studies demonstrate that MARCKS is subjected to specific cleavage by a cellular protease, in a manner dependent on the phosphorylation state of the substrate. This represents a novel means of regulating cellular MARCKS concentrations; these data also raise the interesting possibility that MARCKS is involved in regulating the activity of this novel cellular protease.
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Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
Blackshear
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D001224:Aspartic Acid; D012694:Serine; D011493:Protein Kinase C; D003546:Cysteine Endopeptidases