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

Abstract

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.Mammalian PKGs 4 (PKGI␣, PKGI␤, and PKGII) are major intracellular receptors for cGMP (1, 2). PKGI␣ and PKGI␤, products of alternative splicing, differ only in the first ϳ100 AA (3-6), but PKGII is a separate gene product (7,8). Cyclic GMP-mediated activation of PKGs in response to nitric oxide, natriuretic peptides, or guanylins is integrally involved in myriad physiological processes, including modulation of vascular smooth muscle tone, water, and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation, and bone formation. Despite the central role of these enzymes in both physiological and pathophysiological processes such as erectile dysfunction, pulmonary hypertension, or systemic hypertension, little is known about the molecular mechanism whereby these kinases are activated.PKGs are chimeric proteins composed of regulatory (R) and catalytic (C) domains (9). The C domain, located in the carboxyl-terminal region of the polypeptide, contains an ATP-binding subdomain and a protein substrate-binding subdomain. The C domain is the most highly conserved region among the PKGs and catalyzes transfer of the ␥-phosphate from ATP to specific serines or threonines in consensus sequences in protein substrates. The R domain, located in the aminoterminal segment of PKG, also contains a number of critical functional subdomains; the amino-terminal 100 AA, which have only 35% identity in PKGI␣ and PKGI␤, include a dimerization subdomain, autoinhibitory subdomain, and autophosphorylation sites. The remainder of the R domain seque...

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