Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors.

作者: Eric J. Baude , Michael D. Uhler , Susan S. Dignam , Erwin M. Reimann

DOI: 10.1016/S0021-9258(17)32426-2

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摘要: Abstract The protein kinase inhibitors (PKIs) are potent of the catalytic (C) subunit cAMP-dependent kinase. In this study, interaction between Phe10 PKI and C residues Tyr235 Phe239 was investigated using site-directed mutagenesis. Previous peptide studies as well crystal structure suggested that these may play a key role in C-PKI binding. codons for were changed singly combination to serine codons. mutated alpha proteins overexpressed Escherichia coli. purified Y235S, F239S, Y235S/F239S did not exhibit any differences their Km(app) substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) or Vmax(app), with respect wild-type alpha. All mutants displayed less than 2-fold changes ATP. isoform increased IC50 values Y235S (71-fold), F239S (150-fold), (1800-fold). Similarly, beta 1 showed against proteins, 9.4-, 11-, 44-fold, respectively. addition, F10 codon altered an alanine codon, mutation decreased its ability inhibit activity, but affect Y235S/F239S. serines, however, alter type II R phosphotransferase activity. These results suggest Y235 F239 important specific inhibition by both mutations at would be useful vivo analysis interactions.

参考文章(40)
S.R. Olsen, M.D. Uhler, Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase. Journal of Biological Chemistry. ,vol. 266, pp. 11158- 11162 ,(1991) , 10.1016/S0021-9258(18)99142-8
B E Kemp, D J Graves, E Benjamini, E G Krebs, Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. Journal of Biological Chemistry. ,vol. 252, pp. 4888- 4894 ,(1977) , 10.1016/S0021-9258(17)40137-2
M J Zoller, J Kuret, S Cameron, L Levin, K E Johnson, Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1. Journal of Biological Chemistry. ,vol. 263, pp. 9142- 9148 ,(1988) , 10.1016/S0021-9258(19)76518-1
L. Misconi, S. M. Van Patten, Heung-Chin Cheng, B. E. Kemp, R. B. Pearson, D. A. Walsh, A. J. Smith, A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. Journal of Biological Chemistry. ,vol. 261, pp. 989- 992 ,(1986) , 10.1016/S0021-9258(17)36041-6
Y.J. Buechler, F.W. Herberg, S.S. Taylor, Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95. Journal of Biological Chemistry. ,vol. 268, pp. 16495- 16503 ,(1993) , 10.1016/S0021-9258(19)85447-9
D.A. Fantozzi, A.T. Harootunian, W. Wen, S.S. Taylor, J.R. Feramisco, R.Y. Tsien, J.L. Meinkoth, Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus. Journal of Biological Chemistry. ,vol. 269, pp. 2676- 2686 ,(1994) , 10.1016/S0021-9258(17)41997-1