BINDING OF MYRISTOYLATED ALANINE-RICH PROTEIN-KINASE-C SUBSTRATE TO PHOSPHOINOSITIDES ATTENUATES THE PHOSPHORYLATION BY PROTEIN-KINASE-C

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a peripheral membrane protein that undergoes phosphorylation-dependent translocation between membrane and cytosol, MARCKS binds to acidic ph ospholipids with high affinity (K-d less than 0.5 mu M) but binds poor ly to neutral phospholipids. Although interaction of MARCKS with acidi c phospholipids lacks specificity when determined by binding assay, th ese phospholipids exert distinctive effects on the phosphorylation of this protein by protein kinase C (PKC), Preincubation of MARCKS with p hosphatidylserine (PS) or phosphatidylglycerol enhanced the phosphoryl ation; whereas with phosphatidic acid, phosphatidylinositol (PI), phos phatidylinositol-4-phosphate, or phosphatidylinositol-4,5-bisphosphate inhibited the phosphorylation of this substrate by PKC, Phosphoinosit ide inhibition of MARCKS phosphorylation was apparently directed at th e substrate rather than at the kinase as the phosphorylation of two ot her phospholipid-binding PKC substrates, neuromodulin and neurogranin, exhibited different responses from those of MARCKS. Furthermore, the inhibition of phosphoinositides on MARCKS phosphorylation was seen wit h PKC isozymes alpha, beta, gamma, and delta and with the catalytic fr agment of PKC, protein kinase M. A 25-amino-acid synthetic peptide cor responding to the phosphorylation site domain (PSD) of MARCKS, but not to the myristoylated N-terminal peptide, competed equally effectively with MARCKS in binding to either PS- or PI-containing vesicles, sugge sting that both phospholipids bind to the PSD of MARCKS, Binding of PI to MARCKS inhibited PKC phosphorylation of all three phosphorylation sites, These results suggest that phosphoinositides and PS bind at dif ferent residues within the MARCKS PSD, so that the resulting phospholi pid/MARCKS complexes are differentially phosphorylated by PKC. (C) 199 6 Academic Press, Inc.