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。tgf - β诱导SMAD3连接体区域的三个位点磷酸化，除了两个c端丝氨酸残基。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还会受到smurf2介导的单泛素化作用，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。我们发现Smad3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启动因子。在寻找通过苏氨酸179 （T179）磷酸化来调节SMAD3的蛋白过程中，我们发现了一种rna结合蛋白poly(RC) binding protein 1 (PCBP1，也称为hnRNP E1)，并发现通过与PCBP1合作，SMAD3被带到了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，TAK1以全长和缩短同种异构体的形式出现。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亚型则支持tgf - β诱导的细胞凋亡。我们的数据表明，阻断tgf - β诱导的TAK1的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，我们的全球RNA-seq研究揭示了大量的癌症基因，其剪接模式被SMAD3-PCBP1相互作用改变，从而有利于肿瘤的进展。这些发现让我们提出，通过受体激活的SMAD3和PCBP1的协同作用调节选择性剪接是推动tgf - β成为肿瘤启动子的关键机制。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是JNK和p38不依赖smad激活所必需的。为了揭示在tgf - β信号传导中起作用的其他机制和途径，我们采用了靶向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，我们发现了几种蛋白激酶在tgf - β信号传导的早期阶段相互作用和/或被磷酸化。我们发现JAK1是一种tgf - β受体相互作用蛋白，在肝细胞中，JAK1以smad独立和依赖的方式激活STAT3，以响应tgf - β。我们证明了STAT3需要与Smad3合作才能诱导肝星状细胞的纤维化反应。对其他候选蛋白的进一步表征将导致阐明可能解释不依赖smad的tgf - β信号反应的其他机制，并促进我们对tgf - β诱导多种生物反应的能力的理解。通过合作协议，我们还描述了两种氧甾醇化合物在非小细胞肺癌（NSCLC）中抑制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).