STIMULATION OF THE HIGH-AFFINITY IGE RECEPTOR RESULTS IN THE TYROSINEPHOSPHORYLATION OF A 60 KD PROTEIN WHICH IS ASSOCIATED WITH THE PROTEIN-TYROSINE KINASE, CSK

The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction thr ough several Src kinase-associated receptors. Because the Src-family k inase Lyn plays a pivotal role in Fc epsilon RI-mediated cellular acti vation, we examined whether Csk is involved in Fc epsilon RI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein ref erred to as 'p60') that associates with the SH2 domain of Csk after st imulation of the Fc epsilon RI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggre gated; disaggregation of the receptors resulted in rapid dephosphoryla tion of p60. The phosphorylation of p60 was only detected after activa tion by IgE and antigen and not by stimulation with PMA and/or ionomyc in. Phosphorylated p60 was associated entirely with the membrane fract ion of the cells. A considerable fraction of Csk was associated with t he membrane in both unstimulated and stimulated cells, this fraction d id not change upon activation. p60 coprecipitated with Csk from both u nstimulated and Fc epsilon RI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon Fc epsilon RI stimulation. p60 did not react with antib odies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62(dok). Our data demonstrate that C sk associates with a membrane-anchored protein complex that is directl y involved in Fc epsilon RI signal transduction. (C) 1998 Elsevier Sci ence Ltd. All rights reserved.