PLECKSTRIN HOMOLOGY DOMAINS AND T CELL ACTIVATION

在T细胞中，磷脂酰肌醇3-激酶（PI 3-K）与T细胞受体（TCR）/CD 3、CD 2、CD 7和CD 28刺激介导的T细胞活化依赖性细胞粘附有关。最近在非T细胞系中的研究表明，由PI 3-K产生的脂质产物可以将含有普列克底物蛋白同源（PH）结构域的蛋白募集到细胞结构域，在细胞结构域中，它们被激活。我们已经发现，PH结构域包含蛋白Itk参与CD 3和CD 28活化诱导的β 1整合素介导的粘附增加。Itk是Tec激酶家族成员，需要PI 3-K和1ck两者来激活。我建议测试以下假设：通过PH结构域包含蛋白质的信号传导需要PI 3-K活性，以募集到细胞膜中的特定微结构域。位于这些微结构域内的是能够激活或增强含有PH结构域的蛋白质的激活的其他信号分子（例如1ck）。已经证明Itk参与活化诱导的T细胞粘附，进一步的研究检查组成型活性PI 3-K和lck的过表达将用于评估它们在Itk依赖性T细胞粘附中的作用。由于膜靶向被认为是必不可少的Itk激酶活性，共聚焦显微镜和蛋白质印迹将被用来确定Itk在T细胞活化过程中的亚细胞定位。还将评估Itk激酶活性的膜结合的影响。这些研究将为PI 3-K和PH结构域蛋白在抗原提呈和CTL与靶细胞相互作用中的作用提供重要信息。

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.