Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase

Abstract

A simple purification procedure for microtubule proteins was described, which involved a single assembly step in vitro in the absence of glycerol, followed by centrifugation through sucrose. The preparation contained 80% tubulin (MW 54,000), 15-20% of a 280,000 MW protein and several other minor components of intermediate MW after polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. In the presence of [.gamma.-32P]ATP, [32P]phosphate was incorporated into the 280,000 MW component reaching half-maximal incorporation at 1-2 min, but no phosphorylation of tubulin was detected. Cyclic[c]AMP (Km 0.8 .mu.M) increased both the initial rate and the extent of incorporation of [32P]phosphate into this component. About half of the endogenous protein kinase activity did not require cAMP and was not inhibited by a heat-stable inhibitor protein from muscle. The remainder of the activity was cAMP-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 cAMP. The endogenous substrate and the endogenous protein kinase activity could be partially resolved by chromatography on phosphocellulose. Apparently cAMP can modulate the activity of an endogenous protein kinase(s) with unusual properties and which phosphorylates a prominent microtubule-associated protein.