Molecular Mechanisms of TGF-beta Signaling Pathway

通过其膜结合型I型受体的作用，TGF-β可广泛地促进肿瘤细胞的增殖。 调节细胞增殖、分化和凋亡的细胞反应 context-dependent方式。这些信号应答中的许多由SMAD蛋白介导。作为 因此，控制SMAD活性对于TGF-β及其相关蛋白的适当信号传导至关重要。 因素TGF-β诱导SMAD 3连接区三个位点磷酸化， 除了两个C-末端丝氨酸残基。这些连接位点也可以被磷酸化 通过MAPK和CDKs对生长因子刺激或致癌Ras激活的反应。在 此外，SMAD 3也受到SMURF 2介导的单泛素化，其抑制SMAD 3的表达。 通过阻断与SMAD 4的复合物形成来实现活性。我们发现， SMAD 3与SMURF 2相互作用并经历SMURF 2介导的 泛素化因此，SMAD 3接头磷酸化减少SMAD复合物形成， 转录活性在许多类型的癌细胞中，SMAD 3接头位点是 组成型磷酸化。我们发现Smad 3连接子磷酸化的变化 有助于TGF-β从肿瘤抑制因子转换为转移促进因子。在 寻找通过苏氨酸磷酸化调节SMAD 3的蛋白质 179（T179）的连接区，我们确定了RNA结合蛋白聚（RC）结合蛋白 1（PCBP 1，也称为hnRNP E1），并发现通过与PCBP 1合作，SMAD 3是 在癌症干细胞标记基因CD 44的前体mRNA上， 拼接我们还将Smad 3的这种作用扩展到控制TAK 1的选择性剪接， 其以全长和缩短的同种型制备。我们发现短的TAK 1 同种型是介导TGF-β诱导的EMT和NF-κ B信号传导所必需的，并赋予药物 TAK 1全长支持TGF-β诱导细胞凋亡。我们的数据 这表明阻断TGF-β诱导的TAK 1的选择性剪接可能被证明是一种可行的 对抗癌症治疗药物耐药性的战略。除了CD 44和TAK 1，我们的 全球RNA-seq研究揭示了大量剪接模式改变的癌症基因 通过SMAD 3-PCBP 1相互作用促进肿瘤进展。这些发现让我们提出 通过受体激活的SMAD 3的协同作用调节可变剪接， PCBP 1是推动TGF-β成为肿瘤促进剂的关键机制。虽然SMAD 参与TGF-β的大多数作用，激活的TGF-β受体也参与TGF-β的大多数作用。 通过其他细胞内信号传导途径。在过去的几年里，我的团队 已经投入了相当大的努力来破译TGF-β的具体机制， 受体激活不依赖于Smads的MAP激酶，并阐明了 这种SMAD非依赖性TGF-β信号传导的重要性。为了实现这些目标，我们发现， TRAF 6是SMAD非依赖性激活JNK和p38所必需的。为了 为了揭示在TGF-β信号传导中起作用的其他机制和途径，我们采取了一种 有针对性的定量蛋白质组学方法，以确定其他相关的蛋白质的 TGFbRI复合物。通过这种方法，我们发现了几种蛋白激酶， 和/或在TGF-β信号传导的早期被磷酸化。我们发现JAK 1是一个 TGF-β受体相互作用蛋白和JAK 1在Smad非依赖性和非依赖性细胞中激活STAT 3。 肝细胞对TGF-β的依赖性反应。我们证明了STAT 3是 需要与Smad 3合作以诱导肝星状细胞中的纤维化反应。 对其他候选蛋白质的进一步表征应导致对其他蛋白质的阐明。 可能解释SMAD非依赖性TGF-β信号传导反应的机制，并促进我们的研究。 了解TGF-β诱导多种生物反应的能力。 通过合作协议，我们还表征了两种氧甾醇的潜在机制 抑制非小细胞肺癌中TGF-β、Shh和Wnt信号传导的化合物 （NSCLC）。

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).