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.