Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical.
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.
St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
C L
CL
Denis
B E
BE
Kemp
M J
MJ
Zoller
D000818:Animals; D007700:Kinetics; D008322:Mammals; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D015534:Trans-Activators
D015534:Trans-Activators; D011494:Protein Kinases