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Originally published In Press as doi:10.1074/jbc.M202761200 on June 21, 2002

J. Biol. Chem., Vol. 277, Issue 37, 34048-34054, September 13, 2002
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A Conserved Serine Juxtaposed to the Pseudosubstrate Site of Type I cGMP-dependent Protein Kinase Contributes Strongly to Autoinhibition and Lower cGMP Affinity*

Jennifer L. BuschDagger , Emmanuel P. Bessay, Sharron H. Francis, and Jackie D. Corbin§

From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta ), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha , basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the Ka and KD values for cGMP are 127 and 36 nM, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases ~10-fold in both Ka and KD, and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta . The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.

* This work was supported by National Institutes of Health Grant DK 40029 (to J. D. C.) and Molecular Endocrinology Training Program Grant T32DK07563 (to J. L. B.).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.

Dagger Recipient of a Dissertation Enhancement Award from Vanderbilt University Graduate School.

§ To whom correspondence should be addressed. Tel.: 615-322-4384; Fax: 615-343-3794; E-mail: jackie.corbin@mcmail.vanderbilt.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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