Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.
A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrin-associated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl S-1000 column chromatography, and all buffers utilized in these procedures contained a mixture of proteolysis inhibitors to maintain CAP2 kinase activity. Incubation of vesicles with [gamma-32P]ATP in the presence of 7 microM polylysine resulted in an overall increase in the incorporation of phosphate. NaDodSO4/PAGE revealed that the principal recipient of this additional phosphate was CAP2 (Mr 33,000), the faster-migrating component of the clathrin coat-associated proteins, whereas CAP1 (Mr 36,000) was not phosphorylated. A number of other proteins, in the Mr 140,000 and 100,000 regions, were phosphorylated to a lesser extent. Polyarginine and polyethylenimine also supported CAP2 phosphorylation, but arginine and lysine were ineffective. The phosphorylated protein was identified as CAP2 because addition of exogenous CAPs resulted in increased incorporation of label into Mr 33,000 polypeptides and because heat treatment of labeled vesicles followed by ultracentrifugation resulted in recovery of labeled Mr 33,000 protein in the supernatant. Phosphorylation of CAP2 may play a regulatory role in clathrin coat/coated vesicle functions.
Schook
W J
WJ
Puszkin
S
S
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D006657:Histones / Q000378:metabolism; D008970:Molecular Weight; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002966:Clathrin; D006657:Histones; D010750:Phosphoproteins; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers; D011494:Protein Kinases