title: 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.
authors.affiliation: Group of Structural Biochemistry, German Cancer Research Center, 69120 Heidelberg.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bonn
authors.forename: Saturnino
authors.initials: S
authors.lastname: Herrero
authors.forename: Christine B
authors.initials: CB
authors.lastname: Breitenlechner
authors.forename: Andrea
authors.initials: A
authors.lastname: Erlbruch
authors.forename: Wolf
authors.initials: W
authors.lastname: Lehmann
authors.forename: Richard A
authors.initials: RA
authors.lastname: Engh
authors.forename: Michael
authors.initials: M
authors.lastname: Gassel
authors.forename: Dirk
authors.initials: D
authors.lastname: Bossemeyer
mesh_terms: D000818:Animals; D001549:Benzamides; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000069347:Erlotinib Hydrochloride; D000068877:Imatinib Mesylate; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D011799:Quinazolines / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D054460:rho-Associated Kinases
chemical_list: D001549:Benzamides; D047908:Intracellular Signaling Peptides and Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D011993:Recombinant Fusion Proteins; D000068877:Imatinib Mesylate; D000069347:Erlotinib Hydrochloride; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases