Incubation of clathrin-coated vesicles with Mg2+-[γ-32P]ATP results in the autophosphorylation of a 50-kDa polypeptide (pp50). We describe here a second protein kinase that is associated with calf brain and liver coated vesicles. This kinase, which phosphorylates casein and phosvitin but not histone and protamine using either ATP or GTP, co-fractionates with coated vesicles as assayed by gel filtration, electrophoresis, and sedimentation. The enzyme can be extracted with 0.5 M Tris-HCl or 1 M NaCl, and can be separated from the pp50 kinase as well as the other major coat proteins. We identified this enzyme as casein kinase II based on physical and catalytic properties and by comparative studies with casein kinase II isolated from brain cytosol. It has a Stokes radius of 4.5 nm, a catalytic moiety of ~ 45 kDa, and labels a polypeptide of 26 kDa when the pure enzyme is assayed for autophosphorylation. Its activity is inhibited by heparin and not affected by cAMP, phospholipids, or calmodulin. This protein kinase preferentially phosphorylates clathrin β-light chain. The phosphorylation is markedly stimulated by polylysine and inhibited by heparin. Isolated β-light chain as well as β-light chain in triskelions or in intact coated vesicles is phosphorylated. All of the phosphate (0.86 mol of P(i)/mol of clathrin β-light chain) is incorporated into phosphoserine.
|Journal||Journal of Biological Chemistry|
|State||Published - 1 Dec 1986|