A Conserved Serine Juxtaposed to the Pseudosubstrate Site of Type I cGMP-dependent Protein Kinase Contributes Strongly to Autoinhibition and Lower cGMP Affinity

Abstract

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type I␣ and type I␤ (PKG-I␣ and PKG-I␤), 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-I␣ (S64A, S64T, S64D, and S64N) and PKG-I␤ (S79A). In wild-type PKG-I␣, 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-I␣ basal kinase activity ratio increases 2-fold, cGMP binding affinity increases ϳ10-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-I␤ (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-I␤. 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.Cyclic GMP-dependent protein kinase type I (PKG-I) 1 exists as a homodimer and is comprised of multiple functional domains. Each monomer contains a dimerization component, an autoinhibitory region, two cGMP-binding sites, and a catalytic domain (1, 2). There are two splice variants of PKG-I, PKG-I␣ and PKG-I␤ (3-6), which show broad variation in tissue distribution (7-10). Although the amino acid sequences of their cGMP-binding sites and catalytic domains are identical, the variants share only 36% homology in their 88 and 103 aminoterminal residues, respectively (1, 4 -6). This region of variable sequence includes the autoinhibitory domain and the autophosphorylation sites, which impart distinct functional characteristics to the kinases. The autoinhibitory domain contains a sequence that mimics the substrate sequence ((R/K)(R/K) X(S/T)X) except that an alanine instead of a phosphorylatable serine or threonine is located at P 0 , the position of phosphorylation. Thus, the sequence is referred to as a pseudosubstrate site. Interactions involving residues in the pseudosubstrate site have been demonstrated to be important in maintaining these kinases in an inactive state, and it has been proposed that this simulates interaction of this sequence with the substrate-binding site in the catalytic domain (11, 12). The interaction between the pseudosubstrate site and the catalytic domain maintains the enzyme in an autoinhibited state. Removal of the pseudosubstrate site by proteolysis relieves much of the autoinhibition (13,14), and perturbation of certain residues within the pseudosubstrate sequence of synthetic PKG peptides interferes with the inhibitory potencies of the peptides (15). ...

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

“…2B). The K D values obtained for WT PKGI␣ and PKGI␤ were somewhat lower than those obtained previously, although the ϳ6-fold difference in affinity for cGMP between the two isoforms is consistent with previous results (25,26). One possible explanation for the lower values is that the concentration of PKG proteins used for the K D determination was lower than concentrations used previously and therefore may result in a more accurate K D determination.…”

Molecular mechanisms that could contribute to prolonged effectiveness of PDE5 inhibitors to improve erectile function

“…11,[35][36][37] Cyclic GMP-activated PKG undergoes autophosphorylation, which slightly elevates the enzyme's activity even in the absence of cGMP and increases its sensitivity to subsequent increases in cGMP (Figure 3). [38][39][40][41] Autophosphorylation also causes an apparent conformational change in the enzyme and promotes higher affinity for cGMP binding compared to unphospho-PKG, thereby retaining cGMP at the allosteric cGMP-binding sites; 38,42 since the cGMP-binding allosteric sites of PKG are positively cooperative, 43 this would tend to 'prime' PKG for full activation by a subsequent small increase in cGMP. Some proteins that have been phosphorylated by PKG may subsequently be covalently modified by other posttranslational modifications such as oxidation, ubiquitination or phosphorylation by other kinases that could prolong or otherwise impact the consequences of cGMP signaling.…”

Autophosphorylation of cGMP-dependent Protein Kinase Type II

“…On the other hand, the presence of such a consensus sequence in the case of Ser 110 might explain the preferential autophosphorylation of this residue in cGK II, occurring for a large part even under basal conditions. The pseudosubstrate region is relatively well conserved among the cGKs and cAMP-dependent protein kinases and is thought to be critical for autoinhibition of the catalytic activity of cAK and cGK in the basal state (3,14,19 (18,20). The effect of mutating Ser 126 in cGK II to Ala on cGMP affinity is also similar to the effect of the equivalent mutations in cGK I␣ and in cGK I␤ (18).…”

“…The pseudosubstrate region is relatively well conserved among the cGKs and cAMP-dependent protein kinases and is thought to be critical for autoinhibition of the catalytic activity of cAK and cGK in the basal state (3,14,19 (18,20). The effect of mutating Ser 126 in cGK II to Ala on cGMP affinity is also similar to the effect of the equivalent mutations in cGK I␣ and in cGK I␤ (18). The preferential autophosphorylation of residues in the vicinity of the pseudosubstrate site also suggests that this region is in close contact with the catalytic head.…”