Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors.
The protein kinase inhibitors (PKIs) are potent inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase. In this study, the interaction between Phe10 of PKI and the C subunit residues Tyr235 and Phe239 was investigated using site-directed mutagenesis. Previous peptide studies as well as the crystal structure suggested that these residues may play a key role in C-PKI binding. The C subunit codons for Tyr235 and Phe239 were changed singly and in combination to serine codons. The mutated C alpha proteins were overexpressed in Escherichia coli. The purified C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins did not exhibit any differences in their Km(app) for the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) or Vmax(app), with respect to wild-type C alpha. All of the C subunit mutants displayed less than 2-fold changes in their Km(app) for ATP. The PKI alpha isoform displayed increased IC50 values for C alpha Y235S (71-fold), C alpha F239S (150-fold), and C alpha Y235S/F239S (1800-fold). Similarly, the PKI beta 1 protein showed increased IC50 values against the C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins, 9.4-, 11-, and 44-fold, respectively. In addition, the PKI alpha F10 codon was altered to an alanine codon, and this mutation decreased its ability to inhibit C alpha kinase activity, but did not affect its ability to inhibit C alpha Y235S/F239S. The mutation of Tyr235 and Phe239 to serines, however, did not alter the ability of the type II R subunit to inhibit phosphotransferase activity. These results suggest that C alpha Y235 and C alpha F239 are important for specific inhibition by both PKI alpha and PKI beta but not the type II R subunit and that mutations at these residues would be useful for in vivo analysis of C-PKI interactions.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
E J
EJ
Baude
S S
SS
Dignam
E M
EM
Reimann
M D
MD
Uhler
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003062:Codon; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000378:metabolism; D014867:Water
D003062:Codon; D014867:Water; D014443:Tyrosine; D010649:Phenylalanine; D004247:DNA; D010770:Phosphotransferases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases