Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.