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

J Biol Chem. 1990 Nov 5;265(31):18833-42.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / enzymology*
  • Casein Kinases
  • Cattle
  • Coated Pits, Cell-Membrane / enzymology*
  • Golgi Apparatus / metabolism*
  • Kinetics
  • Mannosephosphates / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Peptides / chemical synthesis
  • Phosphorylation
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Receptor, IGF Type 2
  • Receptors, Cell Surface / isolation & purification
  • Receptors, Cell Surface / metabolism*
  • Serine*
  • Substrate Specificity

Substances

  • Mannosephosphates
  • Peptides
  • Receptor, IGF Type 2
  • Receptors, Cell Surface
  • Serine
  • Protein Kinases
  • Casein Kinases