Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

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...

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

“…Conversion of amino acids in the active site of PKA to their equivalents in PKB conferred greater selectivity for balanol-like derivatives that preferentially inhibited PKB (Breitenlechner et al, 2005b). In another study, residues in the P-loop, hinge region, and activation loop of PKA were converted to their equivalents in Rho kinase, and two mutants, T183A and L49I (corresponding to Ser334 and Ile197 in GRK2), were shown to increase the affinity of Rho kinaseselective inhibitors for PKA (Bonn et al, 2006). Sequence alignment between the various GRK isoforms likewise reveals nonconserved residues in close proximity to bound CMPD103A and CMPD101 (Fig.…”

“…However, when tested in phosphorylation assays against bROS, none of the mutations greatly altered the affinity for inhibitors, suggesting that the identity of the amino acids surrounding the binding site do not strongly contribute to selectivity among GRKs. This is in stark contrast to a previous studies with PKA, in which the L49I mutation (corresponding to GRK2-I197L) decreased the affinity of the staurosporine-like inhibitor (9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3Ј,2Ј,1Ј-kl]pyrrolo [3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester (KT5720) by ϳ180 fold (Bonn et al, 2006).…”

Prediction of Cancer Driver Mutations in Protein Kinases

“…One of them, L49, provides an additional example of the importance of residues involved in determining the size and shape of the nucleotide binding pocket (40). The other two, K105 and S109, lie in the aC-h4 region; do not seem to be conserved; are not positioned to disrupt the K72-E91 salt bridge, which forms on activation; and their side chains extend away from the nucleotide binding pocket.…”

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

“…4b) since the position of N7-atom of fasudil was not largely shifted. A distance between fasudil and Ala231, which is considered as a key residue relevant to selectivity between Rho-kinase and PKA [20], was changed by about 2 Å and the shift of fasudil occurred around 800 ps (Fig. 5b).…”

“…A distance between Leu111 and fasudil was retained (data not shown). Both were considered as a key residue for selectivity [20]. Around the binding site of the homopiperazine ring, the interactionmanner of the N4 0 -atom of fasudil with acidic residues in the ATP binding site was unchanged between the 0-ns and 2-ns structures (Fig.…”