title: Binding of myristoylated alanine-rich protein kinase C substrate to phosphoinositides attenuates the phosphorylation by protein kinase C.
abstract: The myristoylated aline-rich protein kinase C substrate (MARCKS) is a peripheral membrane protein that undergoes phosphorylation-dependent translocation between membrane and cytosol. MARCKS binds to acidic phospholipids with high affinity (Kd less than 0.5 microM) but binds poorly to neutral phospholipids. Although interaction of MARCKS with acidic phospholipids lacks specificity when determined by binding assay, these phospholipids exert distinctive effects on the phosphorylation of this protein by protein kinase C (PKC). Preincubation of MARCKS with phosphatidylserine (PS) or phosphatidylglycerol enhanced the phosphorylation; whereas with phosphatidic acid, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, or phosphatidylinositol-4,5-biphosphate inhibited the phosphorylation of this substrate by PKC. Phosphoinositide inhibition of MARCKS phosphorylation was apparently directed at the substrate rather than at the kinase as the phosphorylation of two other phospholipid-binding PKC substrates, neuromodulin and neurogranin, exhibited different responses from those of MARCKS. Furthermore, the inhibition of phosphoinositides on MARCKS phosphorylation was seen with PKC isozymes alpha, beta, gamma, and delta and with the catalytic fragment of PKC, protein kinase M. A 25-amino-acid synthetic peptide corresponding 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, suggesting 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 different residues within the MARCKS PSD, so that the resulting phospholipid/MARCKS complexes are differentially phosphorylated by PKC.
authors.affiliation: Section on Metabolic Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
authors.forename: K
authors.initials: K
authors.lastname: Seki
authors.forename: F S
authors.initials: FS
authors.lastname: Sheu
authors.forename: K P
authors.initials: KP
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619712:Marcks protein, rat; D008563:Membrane Lipids; D008565:Membrane Proteins; D009227:Myristic Acids; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D010743:Phospholipids; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C