title: Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate.
abstract: Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKC alpha (cPKC alpha), novel PKC delta (nPKC delta), nPKC epsilon, and atypical PKC zeta (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 Km values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, a PKC zeta did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKC alpha, nPKC delta, and nPKC epsilon but not a PKC zeta are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKC delta. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKC delta, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKC delta and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKC delta are involved in the high affinity interaction with intact MARCKS protein.
authors.affiliation: Department of Molecular Biology, Yokohama City University School of Medicine, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Fujise
authors.forename: K
authors.initials: K
authors.lastname: Mizuno
authors.forename: Y
authors.initials: Y
authors.lastname: Ueda
authors.forename: S
authors.initials: S
authors.lastname: Osada
authors.forename: S
authors.initials: S
authors.lastname: Hirai
authors.forename: A
authors.initials: A
authors.lastname: Takayanagi
authors.forename: N
authors.initials: N
authors.lastname: Shimizu
authors.forename: M K
authors.initials: MK
authors.lastname: Owada
authors.forename: H
authors.initials: H
authors.lastname: Nakajima
authors.forename: S
authors.initials: S
authors.lastname: Ohno
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C