TYROSINE AND C-KINASE ACTIVITY AND PI TURNOVER IN T-CELL

T淋巴细胞可通过CD2和CD3受体被激活 通过使用特定的M.Ab和有丝分裂凝集素。许多人 需要第二个使者为后续的活动搭建舞台 通过分泌IL-2和表达IL-2实现自分泌增殖 它的受体。长期目标是理解 磷脂代谢、C-激酶活性与细胞的重要性 CA2沿着这些激活途径的观点来定义 疾病反应调整的目标区域。一个 对这些机制的理解也可能解释 某些免疫缺陷的病理生理学基础。 因此，特定的目的是使用结核分枝杆菌抗体和有丝分裂凝集素 明确C-激酶和CA2i.t.o的细胞生物学作用.I1-2 受体表达，底物磷酸化，受体 C-fos、c-myc、IL-2、 γ-干扰素和IL-2受体。这些研究还将着眼于 C-激酶激活之前的一些过程， 即PI周转、IP3、释放、Ca~2通量和受体 聚合。这可能揭示出效力的不同。 例如在凝集素之间生成第二信使 和抗T_3 M抗体。化学调节剂/药物对药物的影响 这些过程也将被审查。信号转导作用 将研究与受体相关的G结合蛋白 它们刺激腺苷环化酶的潜力 磷脂酶C活性。体内试验的重要性 细胞的自磷酸化、易位和蛋白水解性激活 将解决C-激酶的问题。最后，重要的相互作用 带有酪氨酸激酶的C-激酶，pp56Tck，将用 了解膜之间可能的联系的观点 磷脂代谢和有丝分裂事件。 为了进行这些研究，采用了以下方法 已经发展起来：蛋白质磷酸化研究， 特定磷酸化底物的免疫沉淀，SDS- PAGE和放射自显影，磷酸氨基酸分析，薄层层析 磷脂萃取物，离子交换层析测定 IP3释放和胞质斑点杂交分析。完好无损 细胞反应通过流式细胞术、~3H-TD摄取、 IL-2分泌和Quin-2荧光。

T-lymphocytes can be activated via the CD2 and CD3 receptors through the use of specific M.Ab and mitogenic lectins. A host of second messengers are required to set the stage for subsequent autocrine proliferation through secretion of IL-2 and expression of its receptor. The long term objective is an understanding of the importance of phospholipid metabolism, C-kinase activity and cell CA+2 along these activation pathways with the view to define target areas for response modification in disease. An understanding of these mechanisms may also explain the pathophysiological basic of certain immuno-deficiencies. The specific aims are therefore to use M.Ab and mitogenic lectins to define the cell biological role of C-kinase and CA+2 i.t.o. I1-2 receptor expression, substrate phos-phorylation, receptor modulation and expression of mRNA for c-fos, c-myc, IL-2, gamma-IFN, and the IL-2 receptor. These studies will also look at some of the processes which precedes the activation of C-kinase, namely PI turnover, IP3, release, CA+2 flux and receptor aggregation. This may reveal differences in the potency by which second messengers are generated, for instance between lectins and anti-T3 M.Ab. The influence of chemical modulators/drugs on these processes will also be examined. The signal transducing role of receptor related G-binding proteins will be investigated in terms of their potential for stimulating both adenylate cyclase and phospholipase C activities. The importance of in vivo autophosphorylation, translocation and proteolytic activation of C-kinase will be addressed. Finally, the important interaction of C-kinase with the tyrosine kinase, pp56 Tck, will be studied with a view to understanding the possible link between membrane phospholipid metabolism and mitogenic events. In order to perform these studies, the following methodology has been developed: protein phosphorylation studies, immunoprecipitation of specific phosphorylated substrates, SDS- PAGE and autoradiography, phospho-amino acid analysis, TLC of phospholipid extracts, ion exchange chromatography to measure IP3 release and cytoplasmic dot-blot analysis for mRNA. Intact cell responses are monitored by flow cytometry, 3H-Td uptake, IL-2 secretion and Quin 2 fluorescence.