High affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II-B. Cloning, characterization, and expression of cDNAs for rat brain P150.

Cyclic AMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in the central nervous system, tightly associated with cerebral cortex membranes, and avidly complexed by the bovine brain calmodulin-binding protein designated P75 (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9840-9846). Complexes of RII-B and P75 polypeptides can be purified to near homogeneity from either membrane or cytosolic fractions of brain homogenates, suggesting that the binding protein plays a role in determining the central nervous system-specific properties of protein kinase II-B. To investigate the properties of a prototypic, nonabundant, RII-B-binding protein, we have cloned and characterized cDNAs for rat brain P150, a homolog of bovine brain P75. cDNAs were retrieved from a lambda gt11 expression library using 32P-labeled RII-B as a functional probe. cDNA inserts (800 and 1100 base pairs) subcloned into expression plasmids directed the production of partial P150 polypeptides in Escherichia coli that bind RII-B. Sequence analyses disclosed that P150 is a previously uncharacterized protein that contains multiple octapeptide repeats as well as unique sequences. Antibodies directed against 15-residue peptides corresponding to either repeated or unique sequences bound the polypeptides expressed in E. coli and a 150-kDa protein in rat brain membranes and cytosol. Moreover, the immunoprecipitated 150-kDa protein exhibited high affinity RII-B-binding activity. Finally, 3' deletion analysis demonstrated that a 15-amino acid segment of P150 is essential for binding with RII-B.