CHARACTERIZATION OF THE ISOLATED CAMP-BINDING B-DOMAIN OF CAMP-DEPENDENT PROTEIN-KINASE

A 14.4-kDa cAMP-binding fragment was generated during bacterial expres sion and purification of recombinant bovine cAMP-dependent protein kin ase type I alpha regulatory subunit (RI alpha). The full-length RI alp ha from which the fragment was derived contained a point mutation allo wing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fr agment was Ser-252, indicating that it encompassed the entire predicte d B domain. Although the [H-3]cAMP and [H-3]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact R I alpha, the [H-3]cAMP exchange rate was comparable to that of the B d omain of full-length RI alpha containing an unoccupied A domain. A pla smid encoding only the isolated B domain was overexpressed in Escheric hia coli, and a monomeric form of the B domain was purified that had i dentical properties to the proteolytically generated fragment, indicat ing that all of the elements for the high-affinity cAMP-binding B doma in are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could b e reverted to the monomer by incubation in 2-mercaptoethanol, Dimeriza tion caused an approximate fivefold increase in the rate of cyclic nuc leotide exchange relative to the monomer. The results show that an iso lated cAMP-binding domain can function independently of any other doma in structures of the R subunit.