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.
Bregman
D B
DB
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
Bhattacharyya
N
N
Rubin
C S
CS
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002540:Cerebral Cortex / Q000032:analysis; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D013552:Swine; D014169:Transformation, Bacterial
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C415703:Akap5 protein, rat; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; C015482:protein kinase modulator; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases