Role of TFII-I in signal transduction by growth factors

TFII-I - i最近被发现是导致c-fos启动子的信号转导途径的重要组成部分。虽然TFII-I - i最初被认为是一种可以结合启动子启动元件的因子，但现在很清楚它在信号转导中也起直接作用。在淋巴细胞中，已发现它与细胞质BTK酪氨酸激酶相关，并被其磷酸化。TFII-I的半合子性与人类神经发育障碍威廉姆斯-伯伦综合征（WBS）密切相关。我们已经证明TFII-I与c-fos启动子的上游调控元件结合，并与血清反应因子（SRF）和STAT家族蛋白形成体内复合物。过表达TFII-I可增强c-fos启动子激活，显性阴性TFII-I可抑制c-fos启动子激活。我们发现TFII-I的酪氨酸磷酸化受到血清生长因子的调控，JAK2激酶在这一调控中起重要作用。此外，我们发现TFII-I也受RhoA和MAP激酶途径的调控，并通过其D结构域直接与ERK和p38结合。这些发现表明，TFII-I是血清/RhoA/SRF通路中直接早期基因表达的关键转录介质。在本研究中，我们将研究TFII-I在细胞核信号转导中的作用。我们将确定蛋白质中的关键磷酸化位点，并确定调节TFII-I在丝氨酸、苏氨酸和酪氨酸上磷酸化的途径。然后，我们将确定这些磷酸化位点在TFII-I活性中的作用，包括DNA结合、蛋白质结合、反激活和核易位。此外，我们将在TFII-I及其相关因子中产生突变，使这些相互作用分离，并使我们能够评估TFII-I与血清反应因子和MAP激酶相互作用在其功能中的作用。我们还将评估TFII-I在RhoA- SRF通路中作为信号中间体的可能作用。这些研究结果将有助于阐明和理解一个可能参与人类疾病的重要的新的信号转导分子。具体目标1：绘制TFII-I磷酸化位点，分析磷酸化在TFII-I功能中的作用。特异性目的2：确定特异性蛋白相互作用在TFII-I功能中的作用。特异性目的3：确定MAP激酶和RhoA信号通路在TFII-I调节中的作用。特异性目的4：确定JAK激酶对TFII-I的调控机制。特异性目的5：确定内源性c-fos基因的诱导是否需要TFII-I。

TFII-I has recently emerged as an important component of the signal transduction pathways that lead to the c-fos promoter. Though TFII-I was initially identified as a factor that can bind to promoter initiator elements, it is now clear that it also plays a direct role in signal transduction. In lymphocytes, it has been found to be associated with the cytoplasmic BTK tyrosine kinase and is phosphorylated by it. Hemizygousity for TFII-I is closely linked to the neurodevelopmental disorder Williams-Beuren syndrome (WBS) in humans. We have shown that TFII-I binds to upstream regulatory elements of the c-fos promoter and forms in vivo complexes with the serum response factor (SRF) and the STAT family proteins. Overexpression of TFII-I enhances c-fos promoter activation and dominant negative TFII-I inhibits c-fos promoter activation. We have found that the tyrosine phosphorylation of TFII-I is regulated by serum growth factors and that the JAK2 kinase is important for this regulation. In addition, we have found that TFII-I is also regulated by the RhoA and MAP kinase pathways and associates directly with ERK and p38 through its D domain. These findings suggest that TFII-I is a key transcriptional mediator in the serum/RhoA/SRF pathway for immediate early gene expression. In this proposal, we will investigate the role of TFII-I in signal transduction to the nucleus. We will identify critical phosphorylation sites in the protein and will determine the pathways which regulate phosphorylation of TFII-I on both serine threonine and tyrosine. We will then determine the role of these phosphorylation sites in TFII-I activity including DNA binding, protein binding, transactivation, and nuclear translocation. Moreover, we will generate mutations in TFII-I and its associated factors that will disassociate these interactions and allow us to assess the role of TFII-I interactions with serum response factor and MAP kinases in its function. We will also evaluate the possible role of TFII-I as a signaling intermediate in the RhoA- SRF pathway. The results of these studies will help to elucidate and understand an important new signal transduction molecule which may be involved in human disease. Spec. Aims Specific Aim number 1: To map the Phosphorylation sites in TFII-I and analyze the role of Phosphorylation in TFII-I function. Specific Aim number 2: To determine the role of specific protein interactions in TFII-I function. Specific Aim number 3: To determine the role of MAP kinase and RhoA signaling pathways in TFII-I regulation. Specific Aim number 4: To determine the mechanism of regulation of TFII-I by of JAK kinases. Specific Aim number 5: To determine whether TFII-I is required for the induction of endogenous c-fos gene.