PLECKSTRIN HOMOLOGY DOMAINS AND T CELL ACTIVATION

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DESCRIPTION In T cells, Phosphatidylinositol 3-kinase (PI 3-K) has been implicated in T cell activation-dependent cell adhesion mediated by T cell receptor (TCR)/CD3, CD2, CD7, and CD28 stimulation. Recent studies in non-T cell lines have suggested that the lipid products generated by PI 3-K can recruit pleckstrin homology (PH) domain containing -proteins to cell domains where upon they become activated. We have found that the PH domain containing protein Itk is involved in CD3 and CD28 activation induced increase in beta1 integrin-mediated adhesion. Itk, a Tec kinase family member, requires both PI 3-K and 1ck for activation. I propose to test the following hypothesis: Signaling through PH domain containing proteins requires PI 3-K activity for recruitment to specific microdomains with in the cell membrane. Located within these microdomains are other signaling molecules (such as 1ck) capable of activating or enhancing the activation of the PH domain containing protein. Having demonstrated that Itk is involved in activation induced T cell adhesion further studies examining the over-expression of constitutively active PI 3-K and lck will be used to assess their role in Itk dependent T cell adhesion. Since membrane targeting is thought to be essential for Itk kinase activity, confocal microscopy and Western Blotting will be used to determine the sub-cellular location of Itk in T cell during activation. The effect of membrane association of Itk kinase activity will also be assessed. These studies will contribute important information to the role of PI 3-K and PH domain containing proteins during antigen presentation and during CTL interaction with target cells.