Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.
1. A simple purification procedure for microtubule proteins is described, which involves a single assembly step in vitro in the absence of glycerol, followed by centrifugation through sucrose. 2. The preparation contains 80% tubulin (mol.wt. 54000), 15-20% of a 280000-mol.wt. protein and several other minor components of intermediate molecular weight after polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. 3. In the presence of [gamma-32P]ATP, [32P]phosphate was incorporated into the 280000-mol.wt. component reaching half-maximal incorporation at 1-2 min, but no phosphorylation of tubulin was detected. Cyclic AMP (Km 0.8 micrometer) increased both the initial rate and the extent of incorporation of [32P]phosphate into this component. 4. About half of the endogenous protein kinase activity did not require cyclic AMP and was not inhibited by a heat-stable inhibitor protein from muscle. The remainder of the activity was cyclic AMP-dependent and sensitive to the inhibitor protein. A regulatory subunit was not dissociable from microtubules assembled in vitro in the presence of saturating concentrations of cyclic AMP. 5. The endogenous substrate and the endogenous protein kinase activity could be partially resolved chromatography on phosphocellulose. 6. The data show that cyclic AMP can moduate the activity of an endogenous protein kinase(s) with unusual properties and which phosphorylates a prominent microtubule-associated protein.
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009124:Muscle Proteins / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D009124:Muscle Proteins; D047428:Protein Kinase Inhibitors; D014404:Tubulin; D000242:Cyclic AMP; D011494:Protein Kinases