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

T 细胞中的酪氨酸和 C 激酶活性以及 PI 周转

• 批准号: 3299792
• 负责人: Andre Elias Nel
• 金额: $ 9.56万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299792
• 关键词: T lymphocyte; adenylate cyclase; calcium; complementary DNA; flow cytometry; gel electrophoresis; guanine nucleotide binding protein; human tissue; immunoprecipitation; inositol phosphates; interleukin 2; laboratory rabbit; lectin; leukocyte activation /transformation; membrane lipids; monoclonal antibody; phosphatidylinositols; phospholipase C; phosphorylation; protein kinase C; protein tyrosine kinase; receptor

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.

T淋巴细胞可通过CD 2和CD 3受体活化 通过使用特异性M.Ab和促有丝分裂凝集素。 一系列 第二信使需要为随后的 通过分泌IL-2和表达 它的受体。 长期目标是了解 磷脂代谢、C-激酶活性与细胞 CA+2沿着这些激活途径与视图来定义 疾病反应调整的目标区域。 一个 对这些机制的理解也可以解释 某些免疫缺陷的病理生理基础。 因此，具体目标是使用M.Ab和促有丝分裂凝集素 以确定C-激酶和CA+2 i.t.o. I1-2 受体表达，底物磷酸化，受体 调节和表达c-fos、c-myc、IL-2、 γ-IFN和IL-2受体。 这些研究还将着眼于 一些先于C激酶活化的过程， 即PI周转、IP 3、释放、CA+2通量和受体 聚合来 这可能揭示了效力的差异， 第二信使产生，例如在凝集素之间 和抗T3 M. Ab。 化学调节剂/药物对 这些过程也将得到审查。 信号转导作用 受体相关的G-结合蛋白将在 它们刺激腺苷酸环化酶 和磷脂酶C活性。 In vivo的重要性 自身磷酸化，易位和蛋白水解激活 将讨论C激酶。 最后，重要的互动 C-激酶和酪氨酸激酶pp 56 Tck将用 以了解膜之间的可能联系 磷脂代谢和促有丝分裂事件。 为了进行这些研究， 已经发展了：蛋白质磷酸化研究， 特异性磷酸化底物的免疫沉淀 PAGE和放射自显影，磷酸化氨基酸分析， 磷脂提取物，离子交换色谱法测定 IP 3释放和mRNA的细胞质斑点印迹分析。 完整 通过流式细胞术，3 H-Td摄取， IL-2分泌和Quin 2荧光。