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)/CD3、CD2、CD7和CD28刺激介导的T细胞活化依赖性细胞粘附有关。最近对非t细胞系的研究表明，PI 3-K产生的脂质产物可以将含有-蛋白的pleckstrin同源（PH）结构域的蛋白招募到细胞结构域，在那里它们被激活。我们发现含有蛋白Itk的PH结构域参与了CD3和CD28激活诱导的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才能激活。我建议测试以下假设：通过含有PH结构域的蛋白质的信号传导需要PI 3-K活性才能招募到细胞膜中的特定微结构域。位于这些微结构域内的其他信号分子（如1ck）能够激活或增强含有PH结构域的蛋白质的激活。在证明Itk参与激活诱导的T细胞粘附后，进一步的研究将检测组成型活性PI 3-K和lack的过表达，以评估它们在Itk依赖性T细胞粘附中的作用。由于膜靶向被认为对Itk激酶活性至关重要，因此将使用共聚焦显微镜和Western Blotting来确定Itk在T细胞活化过程中的亚细胞位置。膜结合对Itk激酶活性的影响也将被评估。这些研究将为pi3 - k和PH结构域蛋白在抗原呈递和CTL与靶细胞相互作用过程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.