Negative regulation of TCR-associated PTKs

描述(申请人提供)：酪氨酸磷酸化是将生长信号传递到细胞内的关键机制。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(PTK)进行信号传递。相比之下，人们对蛋白酪氨酸磷酸酶(PTPase)知之甚少，这些蛋白酪氨酸磷酸酶通过使这些PTK及其底物去磷酸化来抵消它们。这一建议侧重于PEP PTPase和CSK PTK，它们形成了一个复合体，对早期的TCR信号事件进行负面调节。1)首先，我们将通过将PEP基因敲除小鼠与TCR转基因动物杂交，产生并分析PEP-/-、抗原特异性、CD4+和CD8+T细胞。我们还将通过一种新的体细胞基因缺失技术来产生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-2R系统来测试不依赖于Src家族激酶的PTK，如Jak L和Jak 3，对PEP丢失不敏感的概念。2)其次，我们将利用PEP-L-T细胞来深入了解PEP-CSK复合体对脂筏或两种相互作用的蛋白质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，在体外和在完整的T细胞中，PKA使丝氨酸364位(364美元)的CSK磷酸化，从而激活CSK 3-5倍。这种激活导致对Lck的抑制增加和TCR信号的减少。我们将调查PEP是否也在S35被直接磷酸化，并受到PKA的调控。PEP被调控的分子机制也将被研究。4)第四，我们将进行一些结构研究，试图解决单个结构域及其复合体的晶体结构，最终希望了解整个酶的结构，甚至它们的复合体。这些研究对于理解PEP的调节和PKA介导的CSK在S364的磷酸化的影响尤为重要。总之，这些研究将从整体动物的生理相关性水平到原子结构和调节提供详细的PEP和CSK的功能和结构的表征。这些问题对于我们理解T细胞激活和免疫反应的分子机制是重要的。

DESCRIPTION (provided by applicant): 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 Jak l and Jak 3, 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 S35 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 for understanding the regulation of PEP and the impact of PKA-mediated phosphorylation of Csk at S364.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.