The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies 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 associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro 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: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. 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-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.
Pauloin
A
A
INRA, Laboratoire de Biologie Cellulaire et Moléculaire, Centre de Recherche de Jouy, Jouy-en-Josas, France.
Thurieau
C
C
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000032:analysis; D016755:Proto-Oncogene Proteins c-jun / Q000302:isolation & purification / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D016755:Proto-Oncogene Proteins c-jun; C438885:adaptor protein complex 2, mu 1 subunit; D010769:Phosphothreonine; D013912:Threonine; D000255:Adenosine Triphosphate