Reconstitution of the B Cell Antigen Receptor Signaling Components in COS Cells

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 Igα or Igβ. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with co-transfected Igα and Igβ chimeras in vivo. Association between Blk and the Igα and Igβ cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Igα and Igβ cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Igα and Igβ chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Igα chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Igα. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.