Molecular Mechanisms of TGF-beta Signaling Pathway

通过其膜结合I型受体的作用，TGF-β以环境依赖性方式引发广泛的细胞应答，所述细胞应答调节细胞增殖、分化和凋亡。这些信号应答中的许多由SMAD蛋白介导。因此，控制SMAD活性对于TGF-β及其相关因子的适当信号传导至关重要。TGF-β在SMAD 3的连接区中除了两个C-末端丝氨酸残基之外的三个位点处诱导磷酸化。这些连接位点也可以被MAPK和CDK磷酸化，以响应生长因子刺激或致癌Ras激活。此外，SMAD 3还经历SMURF 2介导的单泛素化，其通过阻断与SMAD 4的复合物形成来抑制其活性。我们发现SMAD 3与SMURF 2相互作用并经历SMURF 2介导的泛素化需要连接子T179的磷酸化。因此，SMAD 3接头磷酸化降低SMAD复合物形成和转录活性。在许多类型的癌细胞中，SMAD 3接头位点是组成性磷酸化的。我们发现，Smad 3的连接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。在寻找通过连接区中苏氨酸179（T179）的磷酸化来调节SMAD 3的蛋白质时，我们鉴定了RNA结合蛋白poly（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被带到癌症干细胞标记基因CD 44的前mRNA上以调节其选择性剪接。我们还将Smad 3的这种作用扩展到控制TAK 1的选择性剪接，TAK 1以全长和缩短的同种型形式产生。我们发现短的TAK 1亚型是介导TGF-β诱导的EMT和NF-κ B信号传导所必需的，并赋予耐药性，而全长TAK 1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TAK 1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CD 44和TAK 1之外，我们的全球RNA-seq研究还揭示了许多癌症基因，其剪接模式被SMAD 3-PCBP 1相互作用改变，有利于肿瘤进展。这些发现让我们提出，通过受体激活的SMAD 3和PCBP 1的协同作用调节可变剪接是推动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，但活化的TGF-β受体也通过其他细胞内信号传导途径抑制信号。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MAP激酶的特定机制，并阐明这种SMAD独立的TGF-β信号传导的生物学意义。为了实现这些目标，我们发现TRAF 6是SMAD非依赖性激活JNK和p38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TGF β RI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信号传导的早期阶段相互作用和/或磷酸化的蛋白激酶。我们表明JAK 1是一种TGF-β受体相互作用蛋白，JAK 1以Smad非依赖性和依赖性方式激活STAT 3以响应肝细胞中的TGF-β。我们证明了STAT 3需要与Smad 3合作来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应导致阐明可能解释SMAD非依赖性TGF-β信号传导应答的其他机制，并促进我们对TGF-β诱导过多多样生物应答的能力的理解。

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. 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.