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 Busch,Emmanuel P Bessay,Sharron H Francis,Jackie D Corbin

doi:10.1074/jbc.m202761200

Abstract

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.

Highlights

Cyclic GMP-dependent protein kinase type I (PKG-I)1 exists as a homodimer and is comprised of multiple functional domains
Basal Activity Ratios of PKG-I␣ Ser64 Mutants—It can be seen in Fig. 1 that the only invariant residues in the amino acid sequence surrounding the pseudosubstrate site of PKA regulatory subunit (RI) and PKG is an SAE triplet which begins at Ser64 of PKG-I␣
Basal Kinase Activity Ratios and cGMP Binding Affinities of PKG-I␤ Mutants—Because a serine at this position is conserved in all known PKGs, it was of interest to determine whether this residue serves a similar function in the PKG-I␤ isoform

Summary

Introduction

Cyclic GMP-dependent protein kinase type I (PKG-I)1 exists as a homodimer and is comprised of multiple functional domains. 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␤.

Results

Conclusion