The active site substrate specificities of v-Abl and cSrc are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a "dual specificity" with respect to the ability to utilize D-and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.Protein phosphorylation plays a pivotal role in signaling pathways, which regulate such fundamental processes as cell growth, differentiation, and division (1, 2). Consequently, protein kinase inhibitors that can be precisely directed to specific members of the kinase family could prove to be of decided utility in understanding and controlling these signaling pathways. Much of the effort devoted to the design and construction of agents that are targeted to individual protein kinases has focused on the identification of characteristic substrate sequence specificities. Because most protein kinases utilize short peptides as substrates (3, 4), it may be possible to create inhibitors for specific protein kinases by employing peptides containing appropriate primary sequences. Unfortunately, this approach suffers from the limitation that many kinases exhibit broad overlapping sequence specificities with other family members. One way to overcome this obstacle is to employ additional recognition motifs in conjunction with the specific primary sequence to narrow the range of kinases that are impaired by inhibitory species.We have developed a facile method to rapidly assess the active site substrate specificity of protein kinases (5-11). Although protein kinases will generally phosphorylate only serine, threonine, and/or tyrosine residues in intact protein substrates, we have found that these enzymes will phosphorylate a diverse array of alcohol-containing non-amino ...