Negative regulation of TCR-associated PTKs

超出所提供的空间。酪氨酸磷酸化是向细胞传递生长信号的关键机制。在T淋巴细胞中，T细胞抗原受体（TCR）和生长因子白介素-2 （IL-2）受体都使用多种已知的蛋白酪氨酸激酶（PTKs）进行信号传导。相比之下，对蛋白酪氨酸磷酸酶（PTPases）知之甚少，PTPases通过去磷酸化ptk及其底物来中和这些ptk。本研究的重点是PEP PTPase和Csk PTK，它们形成一个复合物，负调控早期TCR信号转导事件。1)首先，我们将通过PEP敲除小鼠与TCR转基因动物杂交，生成PEP-/-、抗原特异性、CD4+和CD8+ T细胞并进行分析。我们还将通过一种新的体细胞基因缺失技术产生Jurkat T白血病细胞系的PEP-/-克隆。我们将确定在没有PEP的情况下TCR信号的改变程度，以及Lck和Fyn是否过度活跃。作为对照，我们将使用IL-2R系统来测试不依赖于Src家族激酶的ptk，如Jakl和Jak3，对PEP的丧失不敏感。2)其次，我们将使用pep - 1 - T细胞来深入了解PEP-Csk复合物对脂筏或两种相互作用蛋白PAG/Cbp和G3BP的结构-功能关系、特异性和亚细胞靶向性。3)第三，我们将继续研究我们的发现，即PKA在体外和完整T细胞中磷酸化Csk的丝氨酸364($364)，从而激活Csk 3- 5倍。这种激活导致Lck的抑制增加和TCR信号的减少。我们将研究PEP是否也在$35处直接磷酸化并受PKA调控。PEP调控的分子机制也将被研究。4)第四，我们将进行大量的结构研究，尝试解决单个结构域及其复合物的晶体结构，最终希望了解整个酶的结构，甚至它们的复合物。这些研究对于理解PEP的调控和pka介导的364位点的Csk磷酸化的影响尤为重要。总的来说，这些研究将从全动物生理相关性水平到原子结构和调控水平详细描述PEP和Csk的功能和结构。这些问题对于我们理解T细胞活化和免疫反应的分子机制非常重要。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Tyrosine phosphorylation is a key mechanism for transmitting growth signals into cells. In T lymphocytes, both the T cell antigen receptor (TCR) and the receptor for the growth factor interleukin-2 (IL-2) use multiple well known protein tyrosine kinases (PTKs) for signaling. In contrast, very little is known about the protein tyrosine phosphatases (PTPases) that counteract these PTKs by dephosphorylating them and their substrates. This proposal focuses on the PEP PTPase and the Csk PTK, which form a complex that negatively regulates early TCR signaling events. 1) First, we will generate and analyze PEP-/-, antigen-specific, CD4+ and CD8+ T cells by crossing PEP knock-out mice with TCR transgenic animals. We will also generate PEP-/- clones of the Jurkat T leukemia cell line by a novel somatic gene deletion technology. We will determine to what extent TCR signaling is altered in the absence of PEP and if Lck and Fyn are hyperactive. As a control, we will use the IL-2R system to test the notion that PTKs that do not depend on Src family kinases, such as Jakl and Jak3, are insensitive to loss of PEP. 2) Second, we will use the PEP-l- T cells to gain some insight into the structure-function relationships, specificity, and subcellular targeting of the PEP-Csk complex to lipid rafts or two interacting proteins, PAG/Cbp and G3BP. 3) Third, we will continue to study our finding that PKA phosphorylates Csk at serine 364 ($364) in vitro as well as in intact T cells and thereby activates Csk 3- to 5-fold. This activation results in increased suppression of Lck and reduced TCR signaling. We will investigate whether PEP is also directly phosphorylated at $35 and regulated by PKA. The molecular mechanism by which PEP is regulated will also be examined. 4) Fourth, we will undertake a number of structural studies and will attempt to solve the crystal structure of individual domains and their complexes, ultimately hoping to understand the structure of the whole enzymes and even their complex. These studies are particularly important ifor understanding the regulation of PEP and the impact of PKA-mediated phosphorylation of Csk at $364. Overall, these studies will provide a detailed characterization of the function and structure of PEP and Csk from the level of physiological relevance in whole animals to atomic structure and regulation. These issues are important for our understanding of the molecular machinery of T cell activation and the immune response. PERFORMANCE SITE ========================================Section End===========================================