THE 50 KDA PROTEIN SUBUNIT OF ASSEMBLY POLYPEPTIDE (AP) AP-2 ADAPTER FROM CLATHRIN-COATED VESICLES IS PHOSPHORYLATED ON THREONINE-156 BY AP-1 AND A SOLUBLE AP50 KINASE WHICH COPURIFIES WITH THE ASSEMBLY POLYPEPTIDES

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity asso ciated with AP. We have undertaken an analysis of the amino acid seque nce around the AP50 phosphorylation site. After phosphorylation in vit ro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: e-Thr-Ser-Gln-Val-Th r-Gly-Gln-Ile-Gly-Trp-Arg162, displayed two threonine residues. Analy sis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflectin g the presence of a single phosphorylation site in AP50. AP phosphoryl ated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to a utophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co -purified with APs but resolved from the latter by hydroxyapatite-colu mn exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gelfiltration data.