No abstract

Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.Phosphorylation via protein kinases is responsible for a large part of cellular signal transduction and is described as a universal regulatory mechanism (1, 2). Perturbation of kinase-mediated signaling pathways results in a number of diseases, including diabetes, cancer, and inflammation (3, 4). Because most protein kinases reside in the cell in an inactive state and are activated by signal transduction processes, many diseases are triggered by overactivation of protein kinases via mutation, overexpression, or malfunctioning cellular inhibition.The human genome encodes some 518 protein kinases (5) that are notably different in how their catalysis is regulated but share a catalytic domain conserved in sequence and structure (6, 7). The latter consists of 250 -300 amino acids, binds substrate and cosubstrate, and catalyzes the phosphorylation reaction.This catalytic domain, together with less conserved surrounding sites, has been the focus of inhibitor design that has exploited differences in kinase structure and pliability to achieve selectivity. Many drugs that target protein kinases are in clinical trials, and some have already been approved, such as the Abl kinase inhibitor Gleevec for therapy against chronic myelogenous leukemia (8) and Tarceva (erlotinib) against nonsmall cell lung cancer (9). The first protein kinase inhibitor that passed the cli...

An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.