IN-VIVO ACTIVATION OF RECOMBINANT CAPK CATALYTIC SUBUNIT ACTIVE-SITE MUTANTS BY COEXPRESSION OF THE WILD-TYPE ENZYME, EVIDENCE FOR INTERMOLECULAR COTRANSLATIONAL PHOSPHORYLATION

The catalytic subunit of cAMP dependent protein kinase (cAPK) carries two stable autophosphorylated residues, One of them, Thr(197), resides in the so-called protein kinase activation segment, and needs to be p hosphorylated for full activity and protein kinase inhibitor binding o f the enzyme, While wild-type recombinant mammalian C-subunit, express ed in E. coli, can fully autoactivate itself by phosphorylation at Thr (197), many active site mutants lack this autophosphorylation activity , so that the primary effects of the mutations become obscured, Two ac tive site mutants of bovine C-subunit, defective in protein kinase inh ibitor peptide binding, were activated by wild-type enzyme in vivo, bu t could not be activated in vitro, demonstrating intermolecular and pr esumably cotranslational autophosphorylation. The results may delineat e strategies for the expression and mutagenesis of other protein kinas es with requirements for activation segment phosphorylation.