High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins.
Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods. Complexes of RII and its binding proteins were isolated on a cAMP analog-Sepharose affinity column, eluted from the column, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Alternatively, nitrocellulose blots made from polyacrylamide gels containing samples of tissue extracts or affinity column eluates were treated with sequential overlays of RII, monospecific antibody, and radioiodinated protein A. In bovine cerebrum, specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized by the two methods. The 300-kDa and 68-kDa proteins were identified as microtubule-associated protein 2 (300 kDa) and a protein of lower molecular weight (68 kDa) that copurifies with it. The additional major binding protein of 80 kDa requires further characterization. In addition, several binding proteins distinct from those observed in bovine cerebrum were found in bovine heart. Many of the RII binding proteins from brain and heart served to differing extents as substrates for the purified catalytic subunit of cAMP-dependent protein kinase. One hypothesis of the significance of the protein kinase regulatory subunit interaction with cellular binding proteins is that this may control the protein kinase holoenzyme localization and, thereby, define the substrate targets most accessible for phosphorylation by the activated protein kinase catalytic subunit. Alternatively, RII binding to a variety of cellular proteins might regulate their function--i.e., RII could be a regulator for multiple proteins in addition to the catalytic subunit of the cAMP-dependent protein kinase.
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000201:enzymology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003101:Collodion; D000242:Cyclic AMP / Q000494:pharmacology*; D007158:Immunologic Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; C015482:protein kinase modulator; D003101:Collodion; D000242:Cyclic AMP; D011494:Protein Kinases