Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II.
Incubation of clathrin-coated vesicles with Mg2+-[gamma-32P]ATP results in the autophosphorylation of a 50-kDa polypeptide (pp50) (Pauloin, A., Bernier, I., and Jollès, P. (1982) Nature 298, 574-576). 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 approximately 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 beta-light chain. The phosphorylation is markedly stimulated by polylysine and inhibited by heparin. Isolated beta-light chain as well as beta-light chain in triskelions or in intact coated vesicles is phosphorylated. All of the phosphate (0.86 mol of Pi/mol of clathrin beta-light chain) is incorporated into phosphoserine.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure; D047388:Casein Kinases; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D046911:Macromolecular Substances; D008970:Molecular Weight; D011107:Polylysine / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002966:Clathrin; D006657:Histones; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; D011107:Polylysine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D047388:Casein Kinases; D005033:Ethylmaleimide