Evidence for a non-myristoylated pool of the 80 kDa protein kinase C substrate of rat brain.
A protein of 80 kDa apparent molecular mass was found to be specifically myristolylated in rat brain cytosols derived from either whole brain or synaptosomes. The attachment of the fatty acid took place in the absence of protein synthesis, since the cytosols did not incorporate [14C]lysine into protein, nor did cycloheximide affect the incorporation of the myristic acid into the protein. The fatty acid was incorporated into the protein via an acid-labile/alkali-resistant band, and Pronase digestion of the labelled protein showed that the lipid was covalently linked to a glycine residue. Together, these data suggested that the myristic acid was amide-linked to the N-terminal residue of the protein. The protein was identified as one of the major protein kinase C substrates, the MARCKS (myristoylated alanine-rich C kinase substrate) protein, by showing that Ca2+ stimulated its phosphorylation, by its heat stability and by immune precipitation (using an antiserum to the MARCKS protein). Incorporation of myristic acid into intact protein continued for up to 12 h, despite the fact that over this period some degradation of the protein could be demonstrated. In pulse-chase experiments, the pattern of loss of the incorporated fatty acid was similar to that of the protein itself, and therefore the loss of radioactivity probably reflects protein degradation rather than specific de-acylation of the protein. Together, these results suggest that there is a pool of unacylated MARCKS protein in the rat brain.
McIlhinney
R A
RA
Department of Pharmacology, University of Oxford, U.K.
McGlone
K
K
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013229:Stearic Acids / Q000378:metabolism; D013379:Substrate Specificity; D013574:Synaptosomes / Q000201:enzymology / Q000378:metabolism
D005227:Fatty Acids; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D009227:Myristic Acids; D010169:Palmitic Acids; D011506:Proteins; D013229:Stearic Acids; D019814:Myristic Acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019308:Palmitic Acid; C031183:stearic acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D011493:Protein Kinase C