Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

J. M. Graff, T. N. Young, J. D. Johnson, P. J. Blackshear

Research output: Contribution to journalArticle

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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 M(r) 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.

Original languageEnglish (US)
Pages (from-to)21818-21823
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number36
StatePublished - 1989

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Phosphorylation
Calmodulin
Protein Kinase C
Substrates
Calmodulin-Binding Proteins
Peptides
Proteins
Chickens
Chemical activation
Cyclic Nucleotides
Phosphoric Diester Hydrolases
Electrophoresis
Sodium Dodecyl Sulfate
Serine
Inhibitory Concentration 50
Gels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. / Graff, J. M.; Young, T. N.; Johnson, J. D.; Blackshear, P. J.

In: Journal of Biological Chemistry, Vol. 264, No. 36, 1989, p. 21818-21823.

Research output: Contribution to journalArticle

Graff, J. M. ; Young, T. N. ; Johnson, J. D. ; Blackshear, P. J. / Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 36. pp. 21818-21823.
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