Molecular Mechanisms of TGF-beta Signaling Pathway

通过其膜结合 I 型受体的作用，TGF-β 引发广泛的细胞反应，以环境依赖性方式调节细胞增殖、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。除了两个 C 端丝氨酸残基外，TGF-β 还可诱导 SMAD3 连接区的三个位点发生磷酸化。这些连接位点还可以响应生长因子刺激或致癌 Ras 激活而被 MAPK 和 CDK 磷酸化。此外，SMAD3 还受到 SMURF2 介导的单泛素化作用，通过阻断与 SMAD4 的复合物形成来抑制其活性。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 连接位点被组成型磷酸化。我们发现，Smad3 连接体磷酸化的变化有助于 TGF-β 从肿瘤抑制因子转变为转移促进因子。在寻找通过连接区中苏氨酸 179 (T179) 磷酸化来调节 SMAD3 的蛋白质时，我们鉴定了一种 RNA 结合蛋白聚（RC）结合蛋白 1（PCBP1，也称为 hnRNP E1），并发现通过与 PCBP1 合作，SMAD3 被带到癌症干细胞标记基因 CD44 的前 mRNA 上，以调节其选择性剪接。我们还将 Smad3 的这一作用扩展到控制 TAK1 的选择性剪接，TAK​​1 具有全长和缩短的亚型。我们发现短 TAK1 亚型是介导 TGF-β 诱导的 EMT 和 NF-kB 信号传导所必需的，并赋予耐药性，而全长 TAK1 支持 TGF-β 诱导细胞凋亡。我们的数据表明，阻断 TGF-β 诱导的 TAK1 选择性剪接可能被证明是对抗癌症治疗药物耐药性的可行策略。除了 CD44 和 TAK1 之外，我们的全球 RNA 测序研究还揭示了大量癌症基因，其剪接模式因 SMAD3-PCBP1 相互作用而改变，有利于肿瘤进展。这些发现让我们提出，通过受体激活的 SMAD3 和 PCBP1 的协同作用来调节选择性剪接是推动 TGF-β 成为肿瘤促进剂的关键机制。尽管 SMAD 参与 TGF-β 的大部分作用，但激活的 TGF-β 受体也通过其他细胞内信号传导途径转导信号。在过去的几年里，我的团队投入了大量的精力来破译TGF-β受体独立于Smads激活MAP激酶的具体机制，并阐明这种不依赖于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 独立激活是特别需要的。为了揭示在 TGF-β 信号传导中发挥作用的其他机制和途径，我们采用有针对性的定量蛋白质组学方法来鉴定 TGFbRI 复合物的其他相关蛋白。通过这种方法，我们发现了几种在 TGF-β 信号传导早期相互作用和/或被磷酸化的蛋白激酶。我们表明，JAK1 是一种 TGF-β 受体相互作用蛋白，并且 JAK1 以不依赖于 Smad 和依赖于 Smad 的方式激活 STAT3，以响应肝细胞中的 TGF-β。我们证明STAT3需要与Smad3配合才能诱导肝星状细胞的纤维化反应。 其他候选蛋白的进一步表征应有助于阐明其他机制，这些机制可能解释不依赖于 SMAD 的 TGF-β 信号传导反应，并加深我们对 TGF-β 诱导多种生物反应能力的理解。 通过合作协议，我们还表征了两种氧甾醇化合物抑制非小细胞肺癌 (NSCLC) 中 TGF-β、Shh 和 Wnt 信号传导的潜在机制。

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. 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 showed that changes in the linker phosphorylation of Smad3 contribute to TGF-beta switching from a tumor suppressor to a metastasis promoter. In searching for proteins that confer regulation of the SMAD3 via phosphorylation of threonine 179 (T179) in the linker region, we identified an RNA-binding protein poly(RC) binding protein 1 (PCBP1, also known as hnRNP E1), and discovered that by partnering with PCBP1, SMAD3 is brought onto the pre-mRNA of a cancer stem cell marker gene CD44 to regulate its alternative splicing. We also extended this role of Smad3 to controlling alternative splicing of TAK1, which is made in both a full length and a shortened isoforms. We showed that the short TAK1 isoform is required for mediating TGF-beta-induced EMT and NF-kB signaling and confers drug resistance, whereas the full length TAK1 supports TGF-beta induction of apoptosis. Our data suggest that blocking TGF-beta-induced alternative splicing of TAK1 may prove to be a viable strategy to combat resistance to cancer therapeutic drugs. In addition to CD44 and TAK1, our global RNA-seq study revealed a plethora of cancers genes whose splicing patterns are altered by the SMAD3-PCBP1 interaction in favor of tumor progression. These findings let us to propose that regulation of alternative splicing by the concerted action of receptor-activated SMAD3 and PCBP1 is a key mechanism that propels TGF-beta to a tumor 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. In order to uncover additional mechanisms and pathways that function in TGF-beta signaling, we took a targeted quantitative proteomics approach to identify additional associated proteins of the TGFbRI complex. Through this approach, we uncovered several protein kinases that interact and/or are phosphorylated at the early stages of TGF-beta signaling. We showed that JAK1 is a TGF-beta receptor interaction protein and JAK1 activates STAT3 in both Smad-independent and dependent manners in response to TGF-beta in hepatic cells. We demonstrated that STAT3 is required to cooperate with Smad3 to induce fibrotic response in hepatic stellate cells. Further characterization of other 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. Through a collaboration agreement, we also characterize underlying mechanism of two oxysterol compounds in the inhibition of TGF-beta, Shh and Wnt signaling in non-small cell lung cancer (NSCLC).