Negative regulation of TCR-associated PTKs

超出所提供的空间。酪氨酸磷酸化是将生长信号传递到细胞中的关键机制。在T淋巴细胞中，T细胞抗原受体（TCR）和生长因子白细胞介素-2（IL-2）受体都使用多种众所周知的蛋白质酪氨酸激酶（PTK）进行信号传导。相反，很少有人知道的蛋白酪氨酸磷酸酶（PTPases），抵消这些PTKs通过去磷酸化他们和他们的底物。该提案关注PEP PTK和Csk PTK，它们形成负调节早期TCR信号传导事件的复合物。1)首先，我们将通过将PEP敲除小鼠与TCR转基因动物杂交来产生和分析PEP-/-、抗原特异性、CD 4+和CD 8 + T细胞。我们还将通过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信号传导改变的程度以及Lck和Fyn是否过度活跃。作为对照，我们将使用IL-2 R系统来测试不依赖于Src家族激酶（例如Jakl和Jak 3）的PTK对PEP损失不敏感的概念。2)其次，我们将使用PEP-1- T细胞来获得对PEP-Csk复合物的结构-功能关系、特异性和亚细胞靶向脂筏或两种相互作用蛋白PAG/Cbp和G3 BP的一些了解。3)第三，我们将继续研究我们的发现，PKA磷酸化Csk在丝氨酸364（$364）在体外以及在完整的T细胞，从而激活Csk 3至5倍。这种激活导致Lck的抑制增加和TCR信号传导减少。我们将研究PEP是否也在35美元直接磷酸化并受PKA调节。还将研究PEP调节的分子机制。4)第四，我们将进行一些结构研究，并试图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，甚至是它们的复合物。这些研究对于理解PEP的调节和PKA介导的Csk磷酸化在$364的影响特别重要。总体而言，这些研究将提供PEP和Csk的功能和结构的详细表征，从整个动物的生理相关性水平到原子结构和调节。这些问题对于我们理解T细胞活化和免疫应答的分子机制非常重要。性能现场=

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===========================================