Role of Tyrosine Phosphorylation of TbRI

描述（由申请人提供）：转化生长因子-β（TGF-β）在生长和发育中起许多作用，并且异常的TGF-β信号传导有助于各种疾病的发病机制和进展，包括癌症向转移和纤维化的起始和进展。TGF-β信号传导诱导上皮细胞获得侵袭性间充质特征，这一过程称为上皮-间充质转化（EMT）。由TGF-β信号传导驱动的EMT现在越来越被视为导致癌的侵袭行为并促成纤维化的关键分化事件。 TGF-β通过细胞表面的两种I型和两种II型跨膜受体激酶（TbRI和TbRII）的复合物发出信号，其中TGF-β诱导TbRII受体磷酸化并激活TbRI受体。这些反过来又通过直接丝氨酸磷酸化激活Smad，Smad信号传导导致基因表达的变化。TGF-β还激活非Smad途径，包括PI 3 K-Akt-mTOR和Erk-MAP激酶途径，这是由受体酪氨酸激酶诱导的关键信号传导途径，并且在疾病中经常被错误调节。最近，TbRI被证明是磷酸化的酪氨酸，这可能使它激活这些非Smad途径。此外，TGF-b受体也在酪氨酸上磷酸化，因此作为双特异性激酶发挥作用，使它们在信号激活中发挥更大的作用，而不仅仅是激活Smad信号。TbRI的酪氨酸磷酸化在TGF-β诱导的Smad和非Smad信号通路的激活中的作用尚未被探索。 我的项目的总体目标是确定TbRI中磷酸化酪氨酸在响应TGF-β的非Smad通路的激活中以及在上皮细胞对TGF-β的EMT响应中的作用。在目的1中，我建议确定TbRI中的酪氨酸磷酸化位点，并使用TbRI突变体，将特定的酪氨酸磷酸化位点与它们在TGF-β激活的非Smad信号传导和受体的内体区室化中的作用相关联。在目的2中，我的目的是确定TbRI酪氨酸残基在细胞对TGF-β的反应，并在TGF-β诱导的EMT的重要性。TbRI突变对TGF-b的EMT应答的可能后果将通过评价对小鼠模型中皮下肿瘤形成和转移性肺定植的影响而在体内扩展。

DESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-b) plays many roles in growth and development, and aberrant TGF-b signaling contributes to the pathogenesis and progression of various diseases, including the initiation and progression of cancers toward metastasis, and fibrosis. TGF-b signaling induces epithelial cells to acquire invasive, mesenchymal characteristics, a process known as epithelial-mesenchymal transition (EMT). EMT, driven by TGF-b signaling, is now increasingly seen as a key differentiation event leading to the invasive behavior of carcinomas, and contributing to fibrosis. TGF-b signals through a complex of two type I and two type II transmembrane receptor kinases (TbRI and TbRII) at the cell surface, in which TGF-b induces the TbRII receptors to phosphorylate and activate the TbRI receptors. These in turn activate Smads through direct Ser phosphorylation, and Smad signaling leads to changes in gene expression. TGF-b also activates non-Smad pathways including the PI3K-Akt-mTOR and Erk- MAP kinase pathways, key signaling pathways that are induced by receptor tyrosine kinases and are often misregulated in disease. Recently, TbRI was shown to be phosphorylated on tyrosines, which may allow it to activate these non-Smad pathways. In addition, TGF-b receptors also phosphorylate on tyrosines, and thus function as dual specificity kinases, giving them a bigger role in signal activation than just activating Smad signaling. The roles of tyrosine phosphorylation of TbRI in the activation of TGF-b-induced Smad and non- Smad signaling pathways have not been explored. The overall goal of my project is to define the roles of the phosphorylated tyrosines in TbRI in the activation of non-Smad pathways in response to TGF-b, and in the EMT response of epithelial cells to TGF-b. In Aim 1, I propose to identify the tyrosine phosphorylation sites in TbRI, and, using TbRI mutants, to correlate specific tyrosine phosphorylation sites with their roles in TGF-b-activated non-Smad signaling, and endosomal compartmentalization of the receptors. In Aim 2, I aim to determine the importance of TbRI tyrosine residues in the cellular response to TGF-b, and in TGF-b-induced EMT. Possible consequences of TbRI mutations on the EMT response to TGF-b will be extended in vivo, through an evaluation of the effects on subcutaneous tumor formation and metastatic lung colonization in a mouse model.