The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C

Herget, T., Oehrlein, S. A., Pappin, D. J., Rozengurt, E., Parker, P. J. (October 1995) The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C. Eur J Biochem, 233 (2). pp. 448-57. ISSN 0014-2956 (Print)0014-2956 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/7588787
DOI: 10.1111/j.1432-1033.1995.448_2.x

Abstract

The myristoylated alanine-rich C-kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC beta 1, novel (n)PKC delta and nPKC epsilon efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC delta (Km = 0.06 microM) was higher than that of nPKC epsilon and cPKC beta 1 (Km = 0.32 microM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC epsilon > cPKC beta 1 > nPKC delta. Atypical (a)PKC zeta did not phosphorylate the intact MARCKS protein. However, a 25-amino-acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC zeta as well as by the other three PKC. Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.

Item Type: Paper
Uncontrolled Keywords: Intracellular Signaling Peptides and Proteins Isoenzymes/ physiology Kinetics Membrane Proteins Phosphorylation Protein Kinase C/ physiology Proteins/ metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > phosphorylation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase > Protein kinase C
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Jessica Koos
Date: 15 October 1995
Date Deposited: 11 Aug 2014 15:56
Last Modified: 11 Aug 2014 15:56
URI: http://repository.cshl.edu/id/eprint/30604

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