Abstract
The substrate specificity of the cAMP-dependent protein kinase (cAPK) from Saccharomyces cerevisiae has been investigated using synthetic peptides corresponding to the local phosphorylation site sequence around Ser-230 in the yeast transcriptional activator ADR1. ADR1 is required for the expression of the glucose-repressible alcohol dehydrogenase. Yeast cAPK (encoded by the TPK1 gene) phosphorylated Ser-230 in the synthetic peptide ADR1-217-234, VRKRYLKKLTRRASFSAQ-NH2, with a Km of 5.3 microM compared with 46 microM for LRRASLG (Kemptide). Porcine heart cAPK phosphorylated the ADR1 peptide and Kemptide with the considerable lower Km values of 0.23 and 1.6 microM, respectively. These results indicate that the ADR1 peptide is an excellent substrate for cAPK. Both the yeast and mammalian protein kinases qualitatively shared a number of substrate specificity determinants in common involving residues on the proximal NH2-terminal side and up to the +4 position of the COOH-terminal side of the phosphoacceptor. The mammalian enzyme, however, had a much higher affinity for its substrates than did the yeast enzyme. In addition, the yeast and mammalian enzymes displayed several quantitative differences in their preferences for particular peptide substrates. In particular, the mammalian enzyme strongly preferred substrates with NH2-terminal extensions beyond the -4 position relative to the phosphoacceptor. These results suggest that all eukaryotic cAPKs recognize similar but not identical substrate specificity determinants. They also suggest that the different affinities for substrates that inhere to the individual enzymes could influence their physiological roles.
Highlights
The substrate specificity of the CAMP-dependent domains [1].Whereas the substrate specificity determinants protein kinasefrom Saccharomyces cerevisiae for mammaliancAPK havebeenanalyzedextensively the has been investigated using synthetic peptides corresponding to the local phosphorylation site sequence around Ser-230 in the yeast transcriptional activator ADR1
Yeast cAPK phosphorylated Ser-230 in the synthetic peptide ADRl-217-234, VRKRYLKKLTRRASFSAQ-NH2,with aK, of 5.3 p~ compared with 46 pM-fOr LRRASLG (Kemptide).Porcine heart cAPK phosphorylatedthe ADRl peptide and Kemptide with the considerably lower K, values of 0.23 and 1.6 p ~ re, spectively
These results indicate that the ADRl peptide is an excellent substrate for cAPK
Summary
IK:19~91 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266, No 27, Issue of September 25, pp. 17932-17935, 1991 Printed in U.S.A. Yeast cAPK (encoded by the T P K l gene) phosphorylated Ser-230 in the synthetic peptide ADRl-217-234, VRKRYLKKLTRRASFSAQ-NH2,with aK , of 5.3 p~ compared with 46 pM-fOr LRRASLG (Kemptide).Porcine heart cAPK phosphorylatedthe ADRl peptide and Kemptide with the considerably lower K , values of 0.23 and 1.6 p ~ re, spectively These results indicate that the ADRl peptide is an excellent substrate for cAPK. The mammalian and yeasetnzymes phosphorylate Kemptide with similar kcatvalues, but the mammalian cAPK phosphorylates it witah K , that is 25-fold lower than thatfound using the yeast enzyme [2] This potential difference in substrate affinity between the two enzymes suggests that other differences in substrate specificity might be present. The mammalianenzyme strongly preferred substrates with NH2-terminalextensions beyond the -4 position relative to the phosphoacceptor These results suggest that all eukaryotic cAPKs recognize similar but not identipeptides serve as excellent substrates for yeast and mammalian cAPK. They suggest that the different affinities for substrates that inhere to the individual enzymes could influence their physiological roles
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