Molecular Mechanisms of TGF-beta Signaling Pathway

通过其膜结合I型受体的作用，tgf - β引发了广泛的细胞反应，以环境依赖的方式调节细胞增殖、分化和凋亡。许多这些信号反应是由Smad蛋白介导的。因此，控制Smad活性对于tgf - β及其相关因子的正确信号传导至关重要。我们发现除了两个c端丝氨酸残基外，tgf - β还诱导Smad3连接体区域的三个位点的磷酸化。这些连接位点也可以被MAPK和CDKs磷酸化，以响应生长因子刺激或致癌Ras激活。此外，Smad3也会受到smurf2介导的单泛素化，通过阻断Smad4的复合物形成抑制其活性。我们发现连接子T179的磷酸化是Smad3与Smurf2相互作用并经历Smurf2介导的泛素化所必需的。因此，Smad3连接子磷酸化会降低Smad复合物的形成和转录活性。在许多类型的癌细胞中，Smad3连接位点被组成性磷酸化。我们目前正在研究Smad3连接子磷酸化的变化是否有助于tgf - β从肿瘤抑制因子转变为转移启动因子。虽然smad参与了tgf - β的大部分活动，但活化的tgf - β受体也通过其他细胞内信号通路转导信号。在过去的几年里，我的团队投入了大量的精力来破译tgf - β受体激活独立于smad的MAP激酶的具体机制，并阐明这种独立于smad的tgf - β信号传导的生物学意义。为了实现这些目标，我们发现TRAF6对于JNK和p38的smad非依赖性激活是特别需要的。目前，我们正在扩大这一发现，以揭示tgf - β信号传导的其他机制和途径。我们采用了靶向蛋白质组学方法来鉴定tgf - β I型受体复合物的其他相关蛋白。我们还采用了全球磷酸化蛋白质组学方法，使用SILAC识别与tgf - β信号相关的差异磷酸化蛋白。这两种方法相辅相成，这些努力的结果将产生tgf - β信号网络的定量磷酸化蛋白质组学谱。我们希望发现在tgf - β信号传导的早期阶段相互作用和/或磷酸化的新蛋白。候选蛋白的进一步表征将导致阐明可能解释smad非依赖性tgf - β信号反应的其他机制，并推进我们对tgf - β诱导多种生物反应能力的理解。

Through the action of its membrane bound type I receptor, TGF-beta elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis in the context-dependent manner. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. We found that TGF-beta induces phosphorylation at three sites in the linker region of Smad3 in addition to the two C-terminal serine residues. These linker sites can also be phosphorylated by MAPK and CDKs in response to growth factor stimulation or oncogenic Ras activation. In addition, Smad3 is also subjected to Smurf2-mediated mono-ubiquitination that inhibits its activity through blocking complex formation with Smad4. We found that phosphorylation of the linker T179 is required for Smad3 to interact with Smurf2 and undergo Smurf2-mediated ubiquitination. Therefore, Smad3 linker phosphorylation decreases Smad complex formation and transcriptional activity. In many types of cancer cells, the Smad3 linker sites are constitutively phosphorylated. We are currently investigating if changes in the linker phosphorylation of Smad3 contribute to TGF-beta switching from a tumor suppressor to a metastasis promoter. Although Smads are involved in the most actions of the TGF-beta, activated TGF-beta receptors also transduce signals through other intracellular signaling pathways. For the past several years, my group has devoted considerable effort in deciphering the specific mechanism by which TGF-beta receptors activate MAP kinases independent of Smads, and elucidating the biological significance of this Smad-independent TGF-beta signaling. Toward these goals, we found that TRAF6 is specifically required for the Smad-independent activation of JNK and p38. Currently, we are in the midst of expanding this finding to uncover additional mechanisms and pathways that function in TGF-beta signaling. We have taken a targeted proteomics approach to identify additional associated proteins of the TGF-beta type I receptor complex. We have also taken a global phosphoproteomics approach to identify differentially phosphorylated proteins associated with TGF-beta signaling using SILAC. These two approaches complement each other, and the outcome of these efforts will generate a quantitative phosphoproteomic profile of TGF-beta signaling network. We hope to uncover novel proteins that interact and/or are phosphorylated at the early stages of TGF-beta signaling. Further characterization of the candidate proteins should lead to elucidation of additional mechanisms that may account for Smad-independent TGF-beta signaling responses and advance our understanding of the ability of TGF-beta to induce a plethora of diverse biological responses.