Stimulation of the high-affinity IgE receptor results in the tyrosine phosphorylation 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 through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in Fc epsilon RI-mediated cellular activation, 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 referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the Fc epsilon RI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated 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 antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62(dok). Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in Fc epsilon RI signal transduction. (C) 1998 Elsevier Science Ltd. All rights reserved.