REGULATION OF INOSITOL LIPIDS BY THE T CELL MOLECULE CD5

CD5，一种淋巴细胞特异性细胞表面分子，可以提供 允许抗原受体所需的辅助信号 (CD3/Ti)介导的静息外周T细胞活化 淋巴细胞。CD3/Ti的信号转导涉及 膜聚磷酰肌醇(PPI)的水解性 产生肌醇磷酸盐(INSPs)和二酰基甘油。我们 观察到CD5的微扰显著增加了CD3/Ti- 中介生成的INSP，并允许以其他方式短暂 督察的反应将持续。在没有其他刺激的情况下， CD5的扰动不会改变InSP水平，但会 对磷脂酰肌醇磷酸(PIP)和 二磷酸(PIP2)。这些观察表明CD5可能 增加PI激酶或PIP激酶的活性， 从而提高PPI水平，增大CD3/Ti- 诱导产生Insps。为了检验这一假设，其影响 单独和联合刺激CD5和CD3/Ti， 对PI、PIP、PIP2和二酰甘油的质量进行检测。 利用T细胞制备的膜，我们将确定 CD5的刺激是否促进32P的掺入 从(~(32)P)ATP转化为PIP和PIP2以及鸟嘌呤是否 核苷酸可以调节这一过程。CD5的可能性 在物理上与PI/PIP激酶相关，而CD5可以 功能上与PI/PIP激酶的相互作用将使用 免疫抑制的CD5和部分纯化的PI和PIP激酶 准备工作。通过将CD5基因导入非淋巴系细胞 基因，我们将确定CD5是否能调节PPI水平 非淋巴样细胞与CD5是否能促进INSP生成 通过CD3/Ti以外的受体。因为细胞质 CD5的结构域包含三个酪氨酸残基，我们将 确定CD5的酪氨酸磷酸化是否在 T细胞活化。如果是这样的话，苯丙氨酸将取代 酪氨酸，以及这种替代对能力的影响 将确定CD5对PPI的调节作用。的总体目标 这些研究旨在通过以下途径来了解分子机制 哪个CD5调节PIP和PIP2，并提供对 T细胞中肌醇磷脂代谢的调节 激活。

CD5, a lymphocyte-specific cell-surface molecule, can provide the accessory signals necessary to permit antigen receptor (CD3/Ti)-mediated activation of resting peripheral T lymphocytes. Signal transduction by CD3/Ti involves the hydrolysis of membrane polyphosphoinositides (PPI) with the generation of inositol phosphates (InsPs) and diacylglycerol. We observe that perturbation of CD5 substantially augments CD3/Ti- mediated generations of InsPs and allows an otherwise transient InsP response to be sustained. In the absence of other stimuli, perturbation of CD5 does not change InsP levels but does have selective effects on phosphatidylinositol phosphate (PIP) and bisphosphate (PIP2). These observations suggest that CD5 may increase the activity of either the PI kinase, or the PIP kinase, thereby increasing the levels of PPI and augmenting CD3/Ti- induced generation of InsPs. To test this hypothesis, the effects of stimulating CD5 and CD3/Ti, individually and in combination, on the mass of PI, PIP, PIP2, and diacylglycerol will be examined. Using membranes prepared from T cells, we will determine whether stimulation of CD5 enhances the incorporation of 32p from (32p) ATP into PIP and PIP2 and whether guanine nucleotides can regulate this process. The possibilities that CD5 is physically associated with a PI/PIP kinase and that CD5 can functionally interact with PI/PIP kinases will be examined using immunoprecitated CD5 and partially purified PI and PIP kinase preparations. By transfecting non-lymphoid cells with the CD5 gene, we will determine whether CD5 can regulate PPI levels in non-lymphoid cells and whether CD5 can augment InsP generation by receptors other than CD3/Ti. Because the the cytoplasmic domain of CD5 contains three tyrosine residues, we will determine whether tyrosine phosphorylation of CD5 occurs during T cell activation. If so, phenylalanine will be substituted for tyrosine, and the consequences of this substitution on the ability of CD5 to regulate PPI will be determined. The general aims of these studies are to understand the molecular mechanisms by which CD5 regulates PIP and PIP2 and to provide insights into the regulation of inositol phospholipid metabolism during T cell activation.