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Structural analysis of protein kinase A mutants with Rho-kinase inhibitor specificity.

Bonn S, Herrero S, Breitenlechner CB, Erlbruch A, Lehmann W, Engh RA, Gassel M, Bossemeyer D
J Biol Chem 281 24818-30 (2006)
Related entries: 1q8t, 1q8u, 1q8w, 2gfc, 2gnf, 2gnh, 2gni, 2gnj, 2gnl

Cited: 19 times
EuropePMC logo PMID: 16699172

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.

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  2. Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors. Thal DM, Yeow RY, Schoenau C, Huber J, Tesmer JJ. Mol Pharmacol 80 294-303 (2011)
  3. Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Banerjee S, Buhrlage SJ, Huang HT, Deng X, Zhou W, Wang J, Traynor R, Prescott AR, Alessi DR, Gray NS. Biochem J 457 215-225 (2014)
  4. Cushing's syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity. Walker C, Wang Y, Olivieri C, Karamafrooz A, Casby J, Bathon K, Calebiro D, Gao J, Bernlohr DA, Taylor SS, Veglia G. Sci Adv 5 eaaw9298 (2019)
  5. Design and synthesis of rho kinase inhibitors (III). Iwakubo M, Takami A, Okada Y, Kawata T, Tagami Y, Sato M, Sugiyama T, Fukushima K, Taya S, Amano M, Kaibuchi K, Iijima H. Bioorg Med Chem 15 1022-1033 (2007)
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  16. Diamondoid Amino Acid-Based Peptide Kinase A Inhibitor Analogues. Müller J, Kirschner RA, Berndt JP, Wulsdorf T, Metz A, Hrdina R, Schreiner PR, Geyer A, Klebe G. ChemMedChem 14 663-672 (2019)


Related citations provided by authors (1)

  1. Protein kinase A in complex with Rho-kinase inhibitors Y-27632, Fasudil, and H-1152P: structural basis of selectivity.. Breitenlechner C, Gassel M, Hidaka H, Kinzel V, Huber R, Engh RA, Bossemeyer D Structure 11 1595-607 (2003)

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