Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding.

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.

cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action.

To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.

Cyclic GMP specifically suppresses Type-Ialpha cGMP-dependent protein kinase expression by ubiquitination.

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-alpha, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Ialpha, but not PKG-Ibeta. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Ialpha to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Ialpha in smooth muscle.

Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes.

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 PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) 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 A, 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 PKGIalpha-isolated R domain and contraction of the PKGIbeta-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 PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity.

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 K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), 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.

Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases.

Cyclic AMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are autoinhibited through multiple interactions between their respective regulatory and catalytic domains. A large portion of this autoinhibition occurs through interactions between residues within the catalytic domain and those within either a substrate-like sequence (-RRXSX-) or pseudosubstrate sequence (-RRXAX-) in the regulatory domains. These contacts effectively inhibit catalysis by blocking substrate binding. Particularly important contacts involve the P-2, P-3, and P+1 residues where either serine, which is potentially autophosphorylated, or alanine occupies the P0 position. The primary sequence is apparently less important for autoinhibition in PKGs than in PKAs, and a conserved serine at P+2 in PKGs is important for autoinhibitory contacts. Elements outside the substrate-related sequences also contribute to autoinhibition in both PKA and PKG. For example, synthetic peptides with relatively short pseudosubstrate sequences are weak inhibitors; while heat-denatured RII subunit does not inhibit catalytic subunit, it is still rapidly autophosphorylated; and truncated PKGs lacking the substrate-like sequence are still partially autoinhibited. Thus, capacity for autoinhibition of PKA or PKG is provided by contacts involving direct interactions with the catalytic site and by contacts that stabilize an inactive conformation.