In RBL-2H3 rat tumor mast cells, cross-linking the high-affinity IgE receptor Fc epsilon RI causes tyrosine phosphorylation of multiple proteins. These phosphoproteins include phospholipase C gamma 1, the beta and gamma subunits of the Fc epsilon RI, the Src family protein-tyrosine kinase Lyn, and a 72-kDa protein that coimmunoprecipitates from lysates of antigen-stimulated cells with antibody to the receptor beta subunit. We now present evidence that the 72-kDa Fc epsilon RI-associated protein is the protein-tyrosine kinase PTK72 that forms part of the antigen receptor complex in B lymphocytes. The identification is based on immunoreactivity with anti-PTK72 antiserum, chromatographic profiles on the affinity resin heparin/agarose, and one-dimensional phosphopeptide mapping studies. Enzymatic activity of the kinase is increased in anti-PTK72 immune complexes prepared from lysates of antigen-activated RBL-2H3 cells. The 72-kDa protein-tyrosine kinase is the principal substrate for in vitro tyrosine phosphorylation in anti-phosphotyrosine immunoprecipitates of RBL-2H3 cells. The discovery that RBL-2H3 mast cells share a receptor-activated protein-tyrosine kinase, PTK72, with B lymphocytes provides additional support for the existence of common signaling pathways initiated by multichain immune recognition receptors.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cγ1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.