Substrate specificity of a multifunctional calmodulin-dependent protein kinase

R. B. Pearson, J. R. Woodgett, P. Cohen, B. E. Kemp

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    Abstract

    The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide corresponding to the NH2-terminal 10 residues of glycogne synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylatd with a V(max) of 12.5 μmol·min-1·mg-1 and an apparent K(m) of 7.5 μM compared to values of 1.2 μmol·min-1·mg-1 and 3.1 μM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a K(M) of 4 μM and a V(max) of 5.4 μmol·min-1·mg-1. The importance of the arginine 3 redisues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased V(max) 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substratesa and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other 'arginine-requiring' protein kinases.

    Original languageEnglish
    Pages (from-to)14471-14476
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume260
    Issue number27
    Publication statusPublished - 1 Dec 1985

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    Pearson, R. B., Woodgett, J. R., Cohen, P., & Kemp, B. E. (1985). Substrate specificity of a multifunctional calmodulin-dependent protein kinase. Journal of Biological Chemistry, 260(27), 14471-14476.