Identification of a protein kinase as an intrinsic component of rat liver coated vesicles

Abstract

Purified rat liver coated vesicles phosphorylate 2 peptides, MW 53,000 and MW 51,000, in the presence of [.gamma.-32P]ATP. Incorporation of phosphate into these peptides is not stimulated by cAMP, Ca2+ or Ca2+ plus calmodulin and occurs principally on a threonine residue. Mild conditions that result in removal of coat proteins from coated vesicles remove most of the protein kinase activity, suggesting the enzyme(s) is (are) not an integral membrane protein. Photolabeling of coated vesicles with 8-azido-[.alpha.-32P]ATP results in specific labeling of only the MW 53,000 and MW 51,000 peptides. Preincubation with 10 mM N-ethylmaleimide inhibits kinase activity and concomitantly reduces photolabeling of the 2 peptides. Thus, the data are consistent with the hypothesis that protein kinase activity resides with these 2 coated vesicle proteins and that they are catalyzing an autophosphorylation reaction.