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J. Biol. Chem., Vol. 281, Issue 11, 6977-6984, March 17, 2006

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Isolated Regulatory Domains of cGMP-dependent Protein Kinase I and I Retain Dimerization and Native cGMP-binding Properties and Undergo Isoform-specific Conformational Changes^*

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

Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGI-(349–670) and PKGI-(364–685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 Å, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K_D values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGI-isolated R domain and contraction of the PKGI-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGI holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGI R domain may partially account for the elongation of wild type PKGI associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.

Received for publication, October 5, 2005 , and in revised form, December 20, 2005.

^* This work was supported by National Institutes of Health Research Grants DK40029 and DK58277. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Wheaton College, Biology Dept., 501 East College Ave., Wheaton, IL 60187.

² Present address: Tulane University School of Medicine, New Orleans, LA 70112.

³ To whom correspondence should be addressed: Light Hall Rm. 702, Dept. of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Tel.: 615-322-4383; Fax: 615-343-3794; E-mail: Sharron.francis{at}vanderbilt.edu.

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