Abstract
We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.
Highlights
From the Departments of Chemistry and Medicinal Chemistry, Natural Sciences and Mathematics Complex, State University of New York, Buffalo, New York 14260
We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine
We have recently shown that the active site substrate specificity of both the cAMP-dependent protein kinase ("PKA") as well as protein kinase C ("PKC") is not limited to serine and threonine moieties (4-9)
Summary
From the Departments of Chemistry and Medicinal Chemistry, Natural Sciences and Mathematics Complex, State University of New York, Buffalo, New York 14260. It may be possible to target inhibitors for specific protein kinases by utilizing peptides containing the appropriate primary sequence. We have found that protein kinases will phosphorylate a diverse array of alcoholcontaining non-amino acid residues (4-9) Most importantly, this approach allows us to address the specific issues noted above concerning protein kinase inhibitor design. The active site substrate specificity of protein kinases, coupled with their characteristic sequence specificities, may provide a useful method for targeting protein kinases in the cell. Up until this point, our work has dealt exclusively with serine/threonine-specific protein kinases. We have found that the specificity of pp60c-src is extraordinarily broad and includes a unusually diverse structural array of alcohol-containing residues
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