MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE (MARCKS) PRODUCES REVERSIBLE INHIBITION OF PHOSPHOLIPASE-C BY SEQUESTERING PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IN LATERAL DOMAINS

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PRC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies su ggest that this binding requires both hydrophobic insertion of its myr istate chain into the bilayer and electrostatic interaction of its clu ster of basic residues with acidic lipids. Phosphorylation of MARCKS b y PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocat ion of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 mu M) inhibit phospholipa se C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphospha te (PIP2) in phospholipid vesicles. A peptide corresponding to the bas ic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta(1) and -beta(1). Direct fluorescence micr oscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis . Phosphorylation of MARCKS(151-175) by PKC releases the inhibition an d allows PLC to produce a burst of inositol 1,4,5-trisphosphate and di acylglycerol.