SPECIFICITY OF THE HIGH-AFFINITY INTERACTION OF PROTEIN-KINASE-C WITHA PHYSIOLOGICAL SUBSTRATE, MYRISTOYLATED ALANINE-RICH PROTEIN-KINASE-C SUBSTRATE

Although myristoylated alanine-rich C kinase substrate (MARCKS), has b een employed as an indicator for the activation of protein kinase C (P RC) in intact cells, little is known about its specificity for PKC fam ily members. To address this question, we partially purified human MAR CKS from baculovirus-infected cells and compared the kinetic parameter s for phosphorylation by PKC isozymes, conventional PKC alpha (cPKC al pha), novel PKC delta (nPKC delta), nPKC epsilon, and atypical PKC zet a (aPKC zeta), all of which are distributed in a wide variety of cells . cPKC alpha, nPKC delta, and nPKC epsilon efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKC alpha, nPKC delta, and nPKC epsilon was extremely high and decreased in the order alpha > delta > epsilon with K-m values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the sa me order. In contrast, aPKC zeta did not phosphorylate MARCKS efficien tly, and we were unable to estimate the kinetic parameters. These resu lts suggest that cPKC alpha, nPKC delta, and nPKC epsilon but not aPKC zeta are enzymes that phosphorylate MARCKS in response to PHC activat ors in intact cells. The structural requirements of MARCKS for efficie nt phosphorylation by these PKC members were then examined using a pep tide that surrounds the phosphorylation site of MARCKS (peptide MARCKS ). Interestingly, intact MARCKS showed a 90-150 times lower rate of ph osphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PI(C members. This im plies that intact MARCKS protein retains a very high affinity for PRC with the sacrifice of its phospho-accepting activity. The structural r equirements of PI(C were then examined using a calpain-cleaved active fragment of nPKC delta. MARCKS was phosphorylated by the active cataly tic fragment as efficiently as by intact nPKC delta, indicating that t he kinase domain is sufficient far the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nP KC delta and its regulatory domain bind to MARCKS, suggesting that bot h the kinase and regulatory domains of nPKC delta are involved in the high affinity interaction with intact MARCKS protein.