T CELL TYROSINE KINASE AND PHOSPHATE-ACCEPTOR PROTEIN

蛋白质磷酸化发生在T淋巴细胞中，这是 MHC限制性抗原识别后被激活为增殖 或IL-2与其受体的相互作用。各种反应 它们的发生并最终指示细胞进入S阶段 基本上是未知的。在一定条件下，T细胞 激活会导致蛋白质酪氨酸激酶活性升高，并且 酪氨酸激酶很可能参与了这一起始过程， 而且，也许，在信号的传播中扩散。损失 信号传输的控制可能是发展中的一个因素 淋巴细胞肿瘤或与以下相关的其他疾病 淋巴组织发育不良或增生。我们的长期目标 就是找出最终引发DNA的反应 淋巴细胞的合成。在前期工作中，我们发现 小鼠EL-4T细胞的颗粒组分含有60kd和 64kd的蛋白质在酪氨酸残基上高度磷酸化。为 比较目的，我们将检测人类CEM T细胞 相似的蛋白质。60kd的蛋白质很可能是一种酪氨酸 64kd的蛋白可能是酪氨酸酶或底物。 这是其中之一。拟议的工作还涉及隔离， 60kd磷酸化产物的表征与比较 来自两个细胞系的蛋白质。我们建议分离出 结合亲和和离子交换的蛋白质 层析、层析聚焦和凝胶过滤， 比较它们的相对分子质量、活性和 酪氨酸激酶，以及它们的蛋白分解裂解图谱的相似性。 我们还打算用适当的方法分离64kd的蛋白。 从上述方法中选择方法，并进行比较 根据它们潜在的酪氨酸激酶活性， 分子量和多肽图谱。最后，我们计划确定 分离的32P-60kd蛋白的主要底物 使用放射自显影，并确定是否 分离的64kd蛋白质本身就是底物，可以 直接从32P-60kd蛋白质或从32P-60kd蛋白质接受32P ATP由60kd的蛋白质催化。

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.