Phosphorylation of the cytoplasmic domain of the bovine cation-independent mannose 6-phosphate receptor. Serines 2421 and 2492 are the targets of a casein kinase II associated to the Golgi-derived HAI adaptor complex.
A kinase activity of purified bovine brain clathrin-coated vesicles phosphorylates the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) with high efficiency (Km approximately 50-100 nM). The kinase copurifies in gel filtration, adsorption on hydroxylapatite, and ion exchange chromatography with the HAI assembly proteins which are part of the coat of Golgi-derived clathrin-coated vesicles. The kinase is associated to the 47-kDa subunit of the complex and exhibits properties similar to a casein kinase II: it uses either ATP or GTP as substrate and its activity is stimulated by poly-L-lysine and inhibited by heparin. Using different domains of the CI-MPR as potential substrates, we show that the phosphorylation is restricted to its cytoplasmic domain. Inhibition studies using synthetic peptides and two-dimensional mapping of the tryptic phosphopeptides indicate that this posttranslational modification occurs on serines 2421 and 2492 of the full-length bovine CI-MPR precursor, residues which are located in typical casein-kinase II recognition sequences. Labeling of Madin-Darby bovine kidney cells followed by immunoprecipitation of the CI-MPR and analysis of the corresponding tryptic phosphopeptides shows that the same serines are phosphorylated in vivo.
Méresse
S
S
Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Ludwig
T
T
Frank
R
R
Hoflack
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D047388:Casein Kinases; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D006056:Golgi Apparatus / Q000378:metabolism*; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D013379:Substrate Specificity
D008360:Mannosephosphates; D010455:Peptides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases