Role of the Glycine Triad in the ATP-binding Site of cAMP-dependent Protein Kinase

Abstract

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both K m and k cat values consistent with the prediction of the importance of the tip of the glycinerich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in K m (ATP) , an 8 -12-fold increase in K m (peptide) , and a 3-5-fold drop in k cat . The K m (ATP) and K m (peptide) values of C(G52S) were increased 8-and 18-fold, respectively, and the k cat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.The eukaryotic protein kinases specific for serine, threonine, and tyrosine all share a conserved catalytic core that folds into a topologically similar three-dimensional structure (2-5). Particularly conserved in this core are the amino acids involved in nucleotide binding and catalysis with one of the most highly conserved features being a GXGXXG motif near the amino terminus. The first and second glycines of this triad are essentially invariant, whereas the third is somewhat more variable but is always a small residue. Our goal is to understand the functional importance of this motif, a hallmark of the protein kinase family.The catalytic (C) 1 subunit of cAMP-dependent protein kinase (cAPK), one of the most extensively investigated members of the protein kinase family, has served repeatedly as a prototype for studying structure-function relationships in this enzyme family (6, 7). It is a relatively simple protein kinase, and both its regulatory and catalytic subunits can be purified readily as active proteins in Escherichia coli. The glycine-rich motif in the C-subunit, (LG 50 TG 52 SFG 55 RV), displays at each position the amino acid residue found in the majority of protein kinases (3, 4). The first crystal structure of a protein kinase was a binary complex of the C-subunit and a peptide inhibitor (2, 8). Based on this structure and on subsequent structures of other protein kinases, the conserved features of the unique protein kinase nucleotide...

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays

“…Interestingly, one locus that showed increased exchange in EnvZc in the presence of nonembedded nanodiscs was the glycinerich loop (Fig. 4 D), a critical motif that is associated with ATP binding and phosphotransfer in all kinases (33). Importantly, the glycine-rich motif also exhibited increased intrinsic exchange in both nanodisc and detergent-solubilized EnvZ.…”

Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

“…Mutants were further resolved on the basis of differing phosphorylation states by chromatography on Mono S FPLC (29). For these studies, isoforms II and III were used which correspond to the enzyme that is phosphorylated at Ser10, Thr197, and Ser339 (isoform II) and Ser338 and Thr197 (isoform III) (23). Pooled fractions were concentrated by Amicon PM10 ultrafiltration.…”

“…Glycine mutations were introduced into the mouse C-subunit gene by an oligonucleotide-directed method (23). Transformed bacteria were cultured in YT media containing 200 µg/mL ampicillin, and gene expression was induced with 1 mM IPTG once the culture's optical density was 0.6 AU at 600 nm.…”

“…Although the three glycines in the glycine-rich loop of the C-subunit were mutated previously to both Ala and Ser (23), only the Ser mutants were characterized in this study owing to partial solubility problems with some of the alanine mutants. With a comparison of the same substitution at each site, the results can be evaluated equivalently.…”

“…Although purifications and perturbations in steady-state kinetic parameters were described previously for mutants of the glycine-rich loop of the C-subunit, the exact effects of these mutations on individual steps in the reaction mechanism were not reported (23). The general effects of mutations in the glycine-rich loop of the phosphorylase kinase γ-subunit were also described previously (24).…”