Reconstitution of the B cell antigen receptor signaling components in COS cells

J Biol Chem. 1995 Nov 10;270(45):27072-8. doi: 10.1074/jbc.270.45.27072.

Abstract

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • Enzyme Precursors / genetics
  • Enzyme Precursors / metabolism
  • Gene Expression
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Peptide Mapping
  • Phosphorylation
  • Protein Sorting Signals / genetics
  • Protein Sorting Signals / metabolism*
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism
  • Receptors, Antigen, B-Cell / genetics
  • Receptors, Antigen, B-Cell / metabolism*
  • Receptors, IgE / genetics
  • Receptors, IgE / metabolism
  • Receptors, Platelet-Derived Growth Factor / genetics
  • Receptors, Platelet-Derived Growth Factor / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Syk Kinase
  • Transfection
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism

Substances

  • Enzyme Precursors
  • Intracellular Signaling Peptides and Proteins
  • Protein Sorting Signals
  • Receptors, Antigen, B-Cell
  • Receptors, IgE
  • Recombinant Fusion Proteins
  • protein-tyrosine kinase p55(blk)
  • Protein-Tyrosine Kinases
  • Receptors, Platelet-Derived Growth Factor
  • SYK protein, human
  • Syk Kinase
  • src-Family Kinases