CD45 REGULATION OF T CELL TYROSINE KINASES

T 细胞酪氨酸激酶的 CD45 调节

• 批准号: 2330425
• 负责人: CHRISTOPHER M BURNS
• 金额: $ 11.34万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-15 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330425
• 关键词: CD antigens; T cell receptor; T lymphocyte; biological signal transduction; enzyme activity; laboratory mouse; leukocyte activation /transformation; phosphorylation; protein sequence; protein tyrosine kinase; protein tyrosine phosphatase; site directed mutagenesis; tissue /cell culture; transfection

Recognition of antigen by the T cell antigen receptor complex (TCR) leads to the activation of protein tyrosine kinases (PTKs), the generation of second messengers from the hydrolysis of phosphatidylinositol (PI), and ultimately an appropriate T cell immune response. Recent studies have demonstrated that the transmembrane tyrosine phosphatase (PTP) CD45 is essential for T cell activation. In CD45-negative (CD45-) mutants of various T cell lines, the TCR is uncoupled from PI hydrolysis and PTK activation. In these CD45- T cells, the kinase activity of the src family PTKs lck and fyn is altered, implying that one critical aspect of the role of CD45 in T cell activation is the modulation of lck and fyn activity. Our central hypothesis is that CD45 regulates the activity of lck, fyn, and a third PTK, ZAP-70, and that this regulation is purposefully altered during T cell activation to allow signal transduction to occur. We propose a model in which CD45 normally maintains lck, fyn, and ZAP-70 in a relatively inactive state through the control of phosphorylation of critical regulatory tyrosine residues. The goal of the proposed studies is to explore the validity of this model and, in so doing, elucidate the precise molecular nature of the regulation of lck, fyn, and ZAP-70 by CD45. First, the effect of T cell activation on the catalytic activity and specific phosphorylation of lck, fyn, and ZAP-70 will be characterized using in vitro kinase assays and highly specific phosphopeptide mapping. These studies will be done in normal murine splenic T cells and a variety of T cell lines and clones to allow the selection of a model system that most closely approximates the normal T cell. We will then compare the identified changes in activity and phosphorylation of lck, fyn, and ZAP-70 seen after activation to those seen in situations where CD45 activity is known to be diminished, namely pharmacologic inhibition of CD45 and cD45 mutant T cell lines or clones. The focus will then shift to the effects of TCR-mediated activation on CD45 itself, in terms of its phosphatase activity and phosphorylation. A temporal correlation between the post- activation changes in CD45 and those in lck, fyn, and ZAP-70 can then be evaluated. In this regard, we present new data in this proposal that CD45 activity is decreased after activation through the TCR. Finally, we will determine the effects of the identified CD45-regulated phosphorylations of lck on lck enzymatic activity and the ability of lck to transduce an activation signal after TCR engagement, using a site-directed mutagenesis and transfection approach in an lck-deficient T cell line. Unraveling the molecular mechanisms of the interaction between CD45 and these critical T cell PTKs will aid our understanding of how an immune response is generated and maintained. Furthermore, our results may suggest specific strategies to interrupt aberrant immune responses at a very proximal site, potentially through specific targeting of these PTKs or CD45 itself.

T细胞抗原受体复合物（TCR）导致的抗原识别 蛋白酪氨酸激酶（PTK）的活化， 来自磷脂酰肌醇（PI）水解的第二信使，和 最终产生适当的T细胞免疫应答。最近的研究 证明跨膜酪氨酸磷酸酶（PTP）CD 45是 对T细胞活化至关重要。在CD 45阴性（CD 45-）突变体中， 在各种T细胞系中，TCR与PI水解和PTK解偶联， activation.在这些CD 45- T细胞中，src家族的激酶活性 PTKs lck和fyn发生了改变，这意味着 CD 45在T细胞活化中的作用是对lck和fyn活性的调节。 我们的中心假设是CD 45调节lck，fyn， 以及第三种PTK，ZAP-70，并且这种调节被故意改变， 在T细胞活化过程中允许信号转导发生。我们提出 一个模型，其中CD 45正常维持lck，fyn和ZAP-70在一个细胞内， 相对不活跃的状态，通过磷酸化的控制， 关键的调节酪氨酸残基。拟议研究的目标是 探讨这一模式的有效性，并在这样做，阐明 LCK、Fyn和ZAP-70调节的精确分子性质， CD45。首先，研究了T细胞活化对催化活性的影响， 将表征lck、fyn和ZAP-70的特异性磷酸化 使用体外激酶测定和高度特异性磷酸肽作图。 这些研究将在正常鼠脾T细胞和各种 的T细胞系和克隆，以允许选择模型系统， 最接近正常的T细胞。然后我们将比较 确定了lck、fyn和ZAP-70的活性和磷酸化的变化 在活化后观察到的那些与在CD 45活性被激活的情况下观察到的那些相比， 已知减少，即对CD 45和CD 45的药理学抑制 突变T细胞系或克隆。然后，焦点将转移到以下方面的影响： 就其磷酸酶而言，TCR介导的对CD 45本身的活化 活性和磷酸化。后- CD 45和lck、fyn和ZAP-70中的活化变化， 评估。在这方面，我们在本提案中提出了新的数据，即CD 45 在通过TCR活化后活性降低。最后我们将 确定鉴定的CD 45调节的磷酸化的影响， lck对lck酶活性及lck抗胰蛋白酶能力的影响 TCR接合后的激活信号，使用定点诱变 以及在lck缺陷型T细胞系中的转染方法。Unraveling the CD 45与这些关键T细胞相互作用的分子机制 细胞PTKs将有助于我们理解免疫反应是如何 生成和维护。此外，我们的研究结果可能表明， 在非常接近的位点中断异常免疫应答的策略， 可能通过特异性靶向这些PTK或CD 45本身。