High affinity binding proteins for the regulatory subunit of cAMP-dependent protein kinase: Cloning, characterization and expression of P150 and P75

Abstract

cAMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system (CNS) via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in brain, tightly associated with cerebral cortex membranes and avidly complexed by the bovine brain calmodulin-binding protein designated P75. 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 subcellular localization and/or other CNS-specific properties of protein kinase II-B. In general, a single high-affinity RII-B binding protein is expressed in the brains of mammals, but the size of the protein varies (e.g., cow: 75 kDa; rat: 150 kDa). To investigate these non-abundant and CNS-enriched RII-B binding proteins, cDNAs for bovine brain P75 and rat brain P150, the homologue of P75, have been cloned and characterized. The cDNAs were retrieved from bovine and rat lambda gt11 expression libraries using {dollar}\sp{lcub}32{rcub}{dollar}P-RII-B as a functional probe. cDNA inserts subcloned into pIN-IA2 or pET-3b expression plasmids directed the production of partial P75 and P150 polypeptides in E. coli that exhibited RII-B binding activity. P47, the expressed product of the largest P75 cDNA isolated, was purified to homogeneity from E. coli transformed with the recombinant pET-3b subclone. On Western blots, a monoclonal antibody specific for P75 bound P47. An antiserum subsequently raised against P47 precipitated 75 kDa and 150 kDa proteins from bovine and rat brain cytosols respectively; both of the precipitated proteins possessed RII-B binding activity. Sequence analyses disclosed that P75 and P150 are previously uncharacterized proteins that include extensive regions of sequence identity. A unique feature of P150 is the presence of 36 copies of an octapeptide. P150 cDNAs hybridize with 3 rat mRNAs (7.3, 5.0, 4.2 kb) that are present in brain and lung, but not other tissues. These mRNAs are not detected in fetal brain, but are expressed during the period of post-natal synaptogenesis and in adult rats. The P75 cDNA hybridizes to a 6 kb bovine brain mRNA. Finally, 3{dollar}\sp\prime{dollar}- deletion analysis demonstrated that the C-terminal 15-25 amino acids of P150 or P75 are essential for binding with RII-B.