BACTERIAL EXPRESSION OF CHINESE-HAMSTER REGULATORY TYPE-I AND CATALYTIC SUBUNITS OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE AND MUTATIONAL ANALYSIS OF THE TYPE-I REGULATORY SUBUNIT

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned a nd expressed in a strain of BL21(DE3) Escherichia coli lacking adenyla te cyclase [BL21(DE3)/Delta cya]. RI expressed in this bacterial syste m free of cyclic AMP is soluble and can reconstitute functional PKA. R ecombinant CHO C alpha is predominantly insoluble with some active sol uble protein. C beta is entirely insoluble and inactive. Soluble recom binant RI and soluble recombinant C alpha can associate in vitro and b e activated by cyclic AMP. Six site-directed mutations of RI were gene rated to study the interaction of cyclic AMP with RI and RI-C alpha su bunit interactions. Four cyclic AMP-binding-site point mutants were ge nerated [W261R (tryptophan to arginine at position 261), a novel mutat ion in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N-3-[H-3]cyclic AMP binding as well as 5-fold reduced affinity for [H-3]cyclic AMP, with threefold increased EC(50) values for cyclic AMP activation of kinase activity from reconstitute d mutant holoenzymes. The mutation at V376G did not alter cyclic AMP b inding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not b ind cyclic AMP but can inhibit C alpha subunit activity. A novel mutat ion outside the cyclic AMP-binding regions of RI (V89A) weakened the i nteraction with C alpha indicated by a 7-fold lower EC(50) for mutant holoenzyme activation by cyclic AMP.