| Signal transduction in T lymphocytes has been the focus of intensive investigation over the last decade and a half. During this period, the critical role of tyrosine kinases in the transmission of signals initiated at the cell surface has become clear. Tyrosine kinases of the Src and Syk families have been shown to play essential early roles in T cell activation. More recently, a third family of tyrosine kinases has been identified in hematopoietic cells, the Tec family.;Genetic evidence from mice lacking the Tec family kinase Itk indicates that this molecule plays a role in signal transduction downstream of the T cell antigen receptor. These mice have reduced CD4+ T cell numbers in the periphery as a result of impaired positive selection in the thymus. Furthermore, these peripheral T cells are functionally defective, producing very little IL-2 in response to T cell receptor engagement. The cells' inability to maintain elevated intracellular calcium levels following stimulation is thought to be responsible for this defect in cytokine production.;In work presented in this thesis, I have investigated the role of Itk in T cell signal transduction, using a combination of biochemical and genetic approaches. Using an insect cell expression system, I identified sites of trans- and autophosphorylation in Itk, and characterized the effects of these phosphorylation events on the catalytic activity of Itk. I also used this system to demonstrate that Itk can directly phosphorylate PLC-gamma, an important regulator of calcium mobilization in lymphocytes. To study the biochemical mechanisms by which Itk carries out its role in T cells, I developed a system in which Itk-deficient murine cells can be expanded in culture, and induced to express exogenous proteins through retroviral transduction. In this way, I investigated the effects of replacing endogenous Itk with a series of mutant versions, and determined that multiple protein domains, including the PH, SH3, and SH2 domains, play essential roles in Itk's potentiation of inducible IL-2 production.;In combination with work by other groups, the data presented here supports a multistep model of Itk activation. Following TCR ligation, Itk translocates to the site of engagement by binding newly formed lipid moieties in the membrane. Itk then becomes associated through SLP-76 with the LAT-nucleated signaling complex forming at the T cell/APC contact site. Itk becomes catalytically active through phosphorylation by Lck, and then synergizes with ZAP-70 to phosphorylate and activate PLC-gamma. PLC-gamma generates the second messengers IP3 and DAG, which in turn trigger calcium mobilization and PKC dependent signaling pathways. In this way, Itk serves as an important link between TCR engagement and the activation of downstream pathways required for cytokine production and immune function. |
