TBC1D3: A HOMINOID-SPECIFIC GENE THAT REGULATES GROWTH FACTOR RECEPTOR SIGNALING

描述(申请人提供)：人类和类人族特有基因的分子细胞生物学在基础研究中实际上是一个隐蔽的领域。了解人类特有基因的分子细胞生物学将为理解人类生物学和疾病开辟新的研究前景。TBC1D3是一个广泛表达的类人族特异性基因，最近的研究表明，TBC1D3是一种强大的EGF和IGF-1受体通路刺激剂。因此，TBC1D3的表达促进了细胞的增殖，并增强了RAS对生长因子的反应。TBC1D3含有一个TBC结构域，它与小GTPase Rab5相互作用，Rab5是内吞作用的关键调节因子。我们推测，TBC1D3可能既是生长因子受体调节因子，又是Rab5效应器。这项建议侧重于定义由TBC1D3启动并导致细胞增殖的生化机制。在具体目标1中，我们将进行结构-功能研究，以确定TBC1D3中参与细胞定位和RAS激活的结构域。为了了解TBC1D3与RAS途径的联系，我们将确定TBC1D3上的丝氨酸/苏氨酸和酪氨酸-磷酸化位点，并验证与TBC1D3相互作用的蛋白Grb2和14-3-3在RAS激活中的作用。在特定的目标2中，我们将量化TBC1D3和GTP酶的Rab5亚家族之间的相互作用。通过使用已知TBC结构域的三维结构对TBC1D3的TBC结构域进行建模，我们将确定TBC1D3：Rab5界面的关键残基，并测试它们在Rab5结合中的作用。为了确认和扩展这些初步的结构研究，将解决TBC结构域的结构问题。在特定的目标3中，我们假设TBC1D3通过调节IRS1的磷酸化和通过TBC1D3与E3泛素连接酶Cul7的相互作用来刺激细胞的信号/增殖。我们将表征TBC1D3对IRS1磷酸化和周转的影响。为了了解Cul7在TBC1D3功能中的作用，我们将确定介导TBC1D3-Cul7相互作用的结构域，并利用缺失突变体，确定TBC1D3-Cul7复合体的功能。TBC1D3的细胞和分子生物学可能代表了一种模型，可以理解人类特有基因的进化，这些基因的功能是调节和调节进化上保守的细胞信号通路。公共卫生相关性：TBC1D3是类人族特有的基因，调节生长因子受体信号转导。研究表明，TBC1D3是一种癌基因，在某些增殖性疾病中过度表达。尽管人类特定基因与人类状况相关，并在人类特定疾病中发挥潜在作用，但尚未得到广泛研究。本工作拟从细胞和分子水平阐明TBC1D3的作用机制。TbC1D3可以作为一个模型来发展对人类特有基因在调节复杂信号通路中的功能的理解。

DESCRIPTION (provided by applicant): The molecular cell biology of human- and hominoid-specific genes is virtually a terra incognita in basic research. Understanding the molecular cell biology of human-specific genes will open a new vista of research of wide import to understanding human biology and disease. TBC1D3 is a widely expressed hominoid-specific gene, and recent work indicates that TBC1D3 is a powerful stimulant of the EGF and IGF-1 receptor pathways. Thus, expression of TBC1D3 promotes cellular proliferation and enhances the activation of Ras in response to growth factors. TBC1D3 contains a TBC domain that interacts with the small GTPase Rab5, a key regulator of endocytosis. We speculate that TBC1D3 may be both a growth factor receptor regulator and a Rab5 effector. This proposal focuses upon defining the biochemical mechanisms that are set in motion by TBC1D3 and result in cell proliferation. In Specific Aim 1, we will carry out a structure-function study to determine domains in TBC1D3 involved in cell localization and in Ras activation. To understand the linkage between TBC1D3 and the Ras pathway, we will identify serine/threonine and tyrosine-phosphorylation sites on TBC1D3 and validate the role TBC1D3-interacting proteins, Grb2 and 14-3-3, in Ras activation. In Specific Aim 2, we will quantify the interaction between TBC1D3 and the Rab5 subfamily of GTPases. By modeling the TBC domain of TBC1D3 using 3-dimensional structures of known TBC domains, we will identify key residues at the TBC1D3:Rab5 interface and test their role in Rab5 binding. To confirm and extend these initial structural studies, the structure of the TBC domain will be solved. In Specific Aim 3, we hypothesize that TBC1D3 stimulates cell signaling/proliferation by modulating IRS1 phosphorylation and via the interaction between TBC1D3 and the E3 ubiquitin ligase Cul7. We will characterize the effect of TBC1D3 on IRS1 phosphorylation and turnover. To understand the role of Cul7 in TBC1D3 function, we will determine the domains that mediate TBC1D3-Cul7 interaction and using deletion mutants, determine the function of the TBC1D3-Cul7 complex. The cell and molecular biology of TBC1D3 may represent a model to understand the evolution of human-specific genes that function to modulate and regulate evolutionarily conserved cell signaling pathways. PUBLIC HEALTH RELEVANCE: TBC1D3 is a hominoid-specific gene that regulates growth factor receptor signaling. TBC1D3 has been shown to be an oncogene and to be over-expressed in certain proliferative disorders. Human specific genes have not been extensively studied in spite of their relevance to the human condition and their potential role in human specific diseases. This work proposes to elucidate the mechanism of action of TBC1D3 at the cellular and molecular level. TBC1D3 may serve as a model for developing an understanding of the function of human-specific genes in regulating complex signaling pathways.