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Originally published In Press as doi:10.1074/jbc.M009366200 on February 20, 2001

J. Biol. Chem., Vol. 276, Issue 21, 17836-17843, May 25, 2001
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Specific Phosphorylation of Threonine by the Dictyostelium Myosin II Heavy Chain Kinase Family*

Xia Luo, Scott W. Crawley, Paul A. SteimleDagger §, Tom T. EgelhoffDagger , and Graham P. Côté

From the Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada and the Dagger  Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.

* This work was supported in part by Canadian Institutes of Health Research Grant MOP8603 (to G. P. C.) and National Institutes of Health Grant GM50009 (to T. T. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by an American Cancer Society Post-doctoral Fellowship.

To whom all correspondence should be addressed: Dept. of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2998; Fax: 613-533-2497; E-mail: coteg@post.queensu.ca.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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