Kinase-mediated protein phosphorylation is a crucial component of the signal transduction pathways by which hormones and related species influence their target cells. There is now ample evidence to implicate many protein kinases in the molecular events that constitute carcinogenesis. An in depth analysis of the active site constraints that control substrate specificity will be helpful in ascertaining the fashion by which protein kinases recognize substrates and thereby influence carcinogenesis. We have found that such an analysis can also provide a useful structural framework upon which the design of potent reversible and irreversible inhibitors can be based. The work described herein focuses on the cAMP-dependent protein kinase (""""""""A-kinase""""""""), a species that is readily available in homogeneous form. This particular enzyme provides us with the opportunity to explore in great detail the active site noncovalent interactions that are crucial to substrate recognition. Since striking structural homologies exist among all protein kinases and these homologies are most evident within the sequence that encompasses the catalytic site, much of what we learn in terms of active site structure for the A-kinase should be applicable to other protein kinases as well. The following objectives constitute the major goals of the research described in this proposal: (1)Our initial efforts will extend our work concerning the manner in which noncovalent active site interactions influence the kinetic mechanism of A-kinase-catalyzed phosphorylation of peptide substrates. Our study in this area will focus on substrates and reversible inhibitors that are appropriately designed to interact with the A-kinase in a specified manner. (2) A series of synthetic hydroxyl-bearing amino acids (contained within an active site-directed peptide) will be employed to probe the substrate specificity of the A-kinase. These studies will provide information concerning the size and shape of the active site.(3) The results obtained in (1) and (2) will enable us to create inhibitors that can function within the structural constraints that are inherent in the active site. In this regard, such an approach has already proven successful, as we have prepared the first example of a pure peptide-based affinity label. We now wish to extend this work into the area of mechanism-based inhibitors.
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