Role of Tyrosine Phosphorylation of TbRI

描述（申请人提供）：转化生长因子- β (TGF-b)在生长发育过程中发挥着多种作用，TGF-b信号异常参与多种疾病的发病和进展，包括癌症的发生和转移、纤维化等。TGF-b信号诱导上皮细胞获得侵袭性间充质特征，这一过程被称为上皮-间充质转化（EMT）。由TGF-b信号驱动的EMT，现在越来越多地被视为导致癌症侵袭性行为的关键分化事件，并有助于纤维化。TGF-b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）复合物进行信号传递，其中TGF-b诱导TbRII受体磷酸化并激活TbRI受体。这些反过来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-b还激活非smad通路，包括PI3K-Akt-mTOR和Erk- MAP激酶通路，这是由受体酪氨酸激酶诱导的关键信号通路，在疾病中经常被错误调节。最近，TbRI在酪氨酸上被磷酸化，这可能允许它激活这些非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比仅仅激活Smad信号更大的作用。酪氨酸磷酸化TbRI在tgf -b诱导的Smad和非Smad信号通路激活中的作用尚未被探索。我项目的总体目标是定义TbRI中磷酸化酪氨酸在响应TGF-b激活非smad途径以及上皮细胞对TGF-b的EMT反应中的作用。在Aim 1中，我建议鉴定TbRI中的酪氨酸磷酸化位点，并使用TbRI突变体，将特定酪氨酸磷酸化位点与其在tgf -b激活的非smad信号传导和受体内体区室化中的作用联系起来。在Aim 2中，我的目标是确定TbRI酪氨酸残基在细胞对TGF-b的反应以及TGF-b诱导的EMT中的重要性。通过在小鼠模型中评估对皮下肿瘤形成和转移性肺定植的影响，TbRI突变对EMT对TGF-b反应的可能影响将在体内得到扩展。

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.