Characterization of the Tec tyrosine kinase signaling network in the murine myocardium

The Tec family of non-receptor tyrosine kinases (Tec, Btk, Bmx, Emt and Txk) have well established roles in cell growth and differentiation. These proteins are known to be activated by growth factors and cytokines and are critically involved in T-cell activation. Recent studies have suggested a role for Bmx in ischemic injury to mouse heart and skeletal muscle, however the actions of the Tec family in cardiac signal transduction remain largely unknown. In the present study, we conducted comprehensive analyses of Tec family expression in whole heart and isolated cardiac myocytes under normal and ischemic conditions. Immunoblotting for Tec kinases revealed expression of Bmx and Tec in both whole heart and myocyte lysates. Interestingly, we reproducibly observe two distinct bands when blotting for Tec in myocyte lysates and only one band in whole heart lysate, suggesting the existence of two Tec isoforms in myocytes. We have optimized immunoprecipitations for isolation of Tec-interacting proteins (including preclearing with IgG control to reduce nonspecific contamination) and are using this approach to evaluate the Tec signaling network in cardiac cells. These Tec-associated proteins have been electrophoresed, trypsinized and are being identified by LC/MS/MS. These studies are an important first step to understanding the mechanistic role of Tec family kinases in myocardial ischemia. Supported by AHA & NIH/NHLBI.