Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor.
Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.
Baude
E J
EJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Dignam
S S
SS
Olsen
S R
SR
Reimann
E M
EM
Uhler
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D014644:Genetic Variation; D005971:Glutamates; D018698:Glutamic Acid; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002352:Carrier Proteins; D017931:DNA Primers; D005971:Glutamates; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011994:Recombinant Proteins; C015482:protein kinase modulator; D018698:Glutamic Acid; C037642:kemptide; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases