GLUTAMIC ACID-203 OF THE CAMP-DEPENDENT PROTEIN-KINASE CATALYTIC SUBUNIT PARTICIPATES IN THE INHIBITION BY 2 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 Calpha isoform (CalphaE203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKIalphaR15) (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 CalphaE203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the CalphaE203 codon of the murine Calpha cDNA to alanine and glutamine codons. Initially, the CalphaE203 mutant proteins were expressed and purified from Escherichia coli. CalphaE203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent K(m) for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent K(m) for ATP. The V(max(app)) was not affected by the mutation of CalphaE203. The mutation of CalphaE203 compromised the ability of PKIalpha(5-24), PKIalpha, and PKIbeta to inhibit phosphotransferase activity. PKIalpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with CalphaE203. The PKIalphaR15A mutant was reduced in its inhibition of Calpha. Preliminary studies of the expression of these Calpha mutants in COS cells gave similar results. These results suggest that the CalphaE203 mutants may be useful in assessing the role of PKI in vivo.