T CELL TYROSINE KINASE AND PHOSPHATE-ACCEPTOR PROTEIN

T 细胞酪氨酸激酶和磷酸受体蛋白

• 批准号: 3438626
• 负责人: F P INMAN
• 金额: $ 6.25万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438626
• 关键词: affinity chromatography; gel filtration chromatography; high performance liquid chromatography; human tissue; ion exchange chromatography; molecular weight; phosphoproteins; phosphorylation; protein structure; protein tyrosine kinase

Protein phosphorylation occurs in T lymphocytes which are activated to proliferate after MHC-restricted antigen recognition or interaction of IL-2 with its receptor. The various reactions which take place and ultimately instruct the cell to enter S phase are essentially unknown. Under certain conditions, T cell activation causes heightened proteins tyrosine kinase activity, and it is probable that tyrosine kinases are involved in the initiation, and, perhaps, in the propagation of the signal to proliferate. Loss of control of signal transmission may be a factor in development of neoplasms of lymphocytes or other diseases related to hypoplasia or hyperplasia of lymphoid tissue. Our long-term goal is to sort out the reactions which culminate in initiation of DNA synthesis in lymphocytes. In preliminary work we found that particulate fractions of murine EL-4 T cells contained 60kd and 64kd proteins heavily phosphorylated on tyrosine residues. For comparative purposes, we will examine human CEM T cells for similar proteins. It is likely that the 60kd protein is a tyrosine kinase; the 64kd protein may be a tyrosine kinase or the substrate for one. The proposed work also deals with isolation, characterization and comparison of the 60kd phosphorylated proteins derived from both cell lines. We propose to isolate the proteins using combinations of affinity and ion-exchange chromatography, chromatofocusing, and gel filtration, and to compare them with respect to molecular weights, activities as tyrosine kinase, and similarity of their proteolytic cleavage maps. We also intend to isolate the 64kd proteins using appropriate methods selected from those mentioned above, and to compare them according to their potential tyrosine kinase activities, molecular weight and peptide maps. Finally, we plan to identify the major substrate of the isolated 32P-60 kd proteins in the particulate fractions using autoradiography, and to determine if the isolated 64kd proteins are, themselves, substrates which can accept the 32P directly from the 32P-60 kd proteins or from 32P- ATP catalyzed by the 60kd proteins.

蛋白质磷酸化发生在T淋巴细胞中， 在MHC限制性抗原识别后活化增殖 或IL-2与其受体的相互作用。 的各种反应 最终指导细胞进入S期 基本上是未知的。 在一定条件下，T细胞 激活引起蛋白质酪氨酸激酶活性升高，和 酪氨酸激酶可能参与了起始， 也许，在信号的传播中也是如此。 损失 信号传输的控制可能是发展的一个因素 淋巴细胞肿瘤或其他与 淋巴组织发育不全或增生。 我们的长期目标 就是找出最终导致DNA起始的反应 在淋巴细胞中合成。 在初步工作中，我们发现， 小鼠EL-4 T细胞的颗粒级分含有60 kd， 64 kd蛋白酪氨酸残基高度磷酸化。 为 为了比较的目的，我们将检查人CEM T细胞， 相似的蛋白质 60 kd蛋白可能是一种酪氨酸蛋白， 激酶; 64 kd蛋白可以是酪氨酸激酶或底物 一个是 拟议的工作也涉及隔离， 60 kd磷酸化的 来自两种细胞系的蛋白质。 我们建议隔离 使用亲和性和离子交换的组合的蛋白质 色谱法、色谱聚焦和凝胶过滤，以及 比较它们的分子量，活性， 酪氨酸激酶，以及它们的蛋白水解裂解图谱的相似性。 我们还打算用合适的方法分离64 kd蛋白质， 从上述方法中选择，并比较 根据它们潜在的酪氨酸激酶活性， 分子量和肽图谱。 最后，我们计划确定 分离的32 P-60 kd蛋白的主要底物是 使用放射自显影法测定微粒分数，并确定 分离的64 kD蛋白本身是底物， 直接从32 P-60 kd蛋白质或从32 P-60 kd蛋白质接受32 P， 由60 kd蛋白催化的ATP。