NOVEL COMPLEX, P40 42, IS CONSTITUTIVELY ASSOCIATED WITH THE B-CELL ANTIGEN RECEPTOR AND PHOSPHORYLATED UPON RECEPTOR STIMULATION/

Stimulation of the B cell Ag receptor (BCR), a multimeric complex cont aining heterodimers of Ig-alpha and Ig-beta, initiates a cascade of ty rosine phosphorylation that results in cellular activation. One of the earliest substrates phosphorylated is Ig-alpha beta, and it appears t hat kinase activation emanates from this structure with the most proxi mal kinases themselves, and some of their immediate substrates, associ ating with the heterodimer. To identify other molecules that may be in volved in proximal BCR signaling, we examined the substrates that were tyrosine phosphorylated following stimulation with either anti-IgG Ab s or pervanadate in the murine B cell lymphoma A20 IIA1.6 and in resti ng splenic B cells. Immunoblotting with anti-phosphotyrosine Abs revea led that a doublet of 40 and 42 kDa was phosphorylated within 1 min of stimulation with either agonist. The phosphorylation of p40/42 in A20 cells induced by anti-Ige was rapid and transient, peaking at 2 min a fter stimulation and becoming almost undetectable after 10 min. Furthe rmore, at least 25% of phosphorylated p40/42 co-immunoprecipitated wit h Ig-alpha beta, but none precipitated with MHC II, CD40, Fc gamma RII , Fyn, HS-1, or Syk, suggesting that this protein complex specifically associates with the Ig-alpha beta heterodimer, p40/42 did not react w ith Abs to Ig-alpha, Ig-beta, mitogen-activated protein kinase, or Lnk . Furthermore, and in contrast to Ig-alpha beta, p40/42 was highly aci dic and not part of a disulfide-linked complex. Finally, p40/42 was de monstrated to be a glycosylated surface protein that was constitutivel y associated with Ig-alpha beta. These results suggest that p40/42 is a novel constituent of the resting B cell Ag receptor complex.