title: Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C.
abstract: We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.
authors.affiliation: Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: J M
authors.initials: JM
authors.lastname: Graff
authors.forename: T N
authors.initials: TN
authors.lastname: Young
authors.forename: J D
authors.initials: JD
authors.lastname: Johnson
authors.forename: P J
authors.initials: PJ
authors.lastname: Blackshear
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D003432:Cross-Linking Reagents / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013388:Succinimides / Q000378:metabolism
chemical_list: D002147:Calmodulin; D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D010455:Peptides; D011506:Proteins; D013388:Succinimides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C019358:disuccinimidyl suberate