T lymphocyte development in the thymus is dependent on interactions with the T cell antigen receptor (TCR). Early in development, thymocytes which fail to rearrange a functional TCR beta chain are arrested at the CD4-8- stage. Subsequently, CD4+8+ thymocytes, which are the first cells in the thymus to express a complete TCR alphabeta/CD3 complex on their cell surface, undergo two selection processes: positive selection and clonal deletion. Together these two selection processes create a TCR repertoire which is self-MHC-restricted and self-tolerant. In an effort to understand the signal transduction pathways underlying these thymic selection events, a PCR based molecular screen for novel tyrosine kinases expressed in the thymus was initiated. One of the genes identified in the screen, called TSK for T cell specific kinase, is the subject of this proposal. The expression pattern of TSK (also called ITK or EMT by other workers) is highly suggestive of a role in T cell development. Analysis of RNA isolated from a wide range of mouse tissues and cell lines indicates that TSK is expressed only in T cells. In addition the levels of TSK mRNA are approximately tenfold higher in the thymus than in resting peripheral T cells. TSK is expressed throughout fetal thymic ontogeny, starting as early as fetal day 14, but is not expressed in bone marrow stem cells. TSK, BTK, and a third protein, TECA, define a new family of tyrosine kinases. Mutations in the BTK gene have been shown to be responsible for the human immunodeficiency disease, X-linked agammaglobulinemia (XLA), and for the xid deficiency in mice. Besides a causative role in immune deficiency diseases, tyrosine kinases are involved in the decisions leading to proliferation as opposed to differentiation of developing cells in many lineages, and have been implicated in a variety of cancers. The elucidation of signal transduction pathways in T lymphocytes will also promote our understanding of the factors leading to autoimmune diseases. To examine the role of TSK in T cell development, biochemical and genetic approaches will be taken. Preliminary results indicating an interaction between the TSK-SH2 domain and Syk in activated thymocytes will be extended and applied to the analysis of TSK Zap-70 complexes. Whether TSK itself is tyrosine phosphorylated and/or activated in thymocytes stimulated by a number of surface receptors including the TCR/CD3 complex will be investigated. In addition, the function of TSK in vivo will be studied by analyzing transgenic mice overexpressing wild type or kinase-inactive TSK. A TSK-deficient mouse line is being generated by gene targeting in embryonal stem cells. The T cell-restricted expression pattern of TSK, together with its high level of expression in the thymus, strongly suggest a role for the TSK kinase in a signaling pathway unique to cells of the T lymphocyte lineage.
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