The purpose of this research proposal is to elucidate the biological functions of two Tec subfamily tyrosine kinases, Btk and Emt, in mast cells and their roles in the signal transduction pathway triggered by crosslinking of the high-affinity IgE receptor (FceRI). 1) Since btk and emt null mutant mice are available, extensive characterization of activation events will be performed on the mast cells derived from these mice to gain insight on the functions of Btk and Emt in mast cell biology. 2) Defects found in primary cultured mast cells derived from these mice will be corrected by the recently established retroviral gene transfer technique. Preliminary characterization of btk mutated mast cells has revealed multiple defects: defective degranulation, defective cytokine production/secretion, and prolonged cell survival upon growth factor withdrawal. Further defective JNK/SAPK activation upon FceRI cross-linking was correlated with longer cell survival of btk mutated cells in medium without IL-3. 3) Since preliminary data suggest the presence of a Btk-dependent, but not SEK1-dependent, pathway for JNK/SAPK activation induced by FceRI cross-linking, this possibility will be investigated in more depth. As a mechanism for defective cytokine production/secretion, btk mutations seem to result in defective transcription of several cytokine genes. Therefore, 4) they will attempt to identify Btk-dependent transcription factor(s) regulating the expression of these cytokine genes. Furthermore, in order to understand the biochemical mechanisms of Btk (and Emt) functions, 5) Btk-associated proteins will be identified. Since the SH2 domain was shown to be indispensable for the restoration of cytokine production in btk-reconstituted btk null mast cells, SH2-binding proteins and a 120kDa protein, whose association with Btk was identified by immune complex kinase assays on anti-Btk immunoprecipitates, will be identified. 6) Regulatory mechanisms of Btk (and Emt) kinase activity will also be investigated. Relations with lipid second messengers, Src family PTKs, and ERK will be examined. Particularly, interactions of Btk with Src PTKs and ERK might suggest positive and negative, respectively, feedback mechanisms for FceRI induced mast cell activation.
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