Molecular Mechanisms of TGF-beta Signaling Pathway

TGF-β信号通路的分子机制

• 批准号: 10014367
• 负责人: YING E Zhang
• 金额: $ 94.8万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014367
• 关键词: Activins; Alternative Splicing; Apoptosis; Binding Proteins; Biological; Bone Morphogenetic Proteins; C-terminal; CD44 gene; Cell Growth Processes; Cell Proliferation; Cell physiology; Cell surface; Complex; Data; Development; Differentiation and Growth; Disease; Drug resistance; Epigenetic Process; Family; Family member; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Goals; Growth Factor; Hepatic Stellate Cell; Heterogeneous-Nuclear Ribonucleoproteins; Human; Induction of Apoptosis; JAK1 gene; Lead; Length; Liver Fibrosis; MADH3 gene; MADH4 gene; MAP Kinase Gene; MAP3K7 gene; MAPK14 gene; MAPK8 gene; Malignant Neoplasms; Mediating; Membrane; Mitogen-Activated Protein Kinases; Molecular; Monoubiquitination; NF-kappa B; Neoplasm Metastasis; Oncogenes; Oncogenic; Pathway interactions; Pattern; Peptides; Pharmaceutical Preparations; Phase; Phosphorylation; Protein Biosynthesis; Protein Isoforms; Protein Kinase; Proteins; Proteomics; RNA Splicing; RNA-Binding Proteins; Receptor Serine/Threonine Kinase; Receptor Signaling; Regulation; Reporting; Research; Resistance; Role; STAT3 gene; Serine; Signal Pathway; Signal Transduction; Site; Smad Proteins; TRAF6 gene; Therapeutic; Threonine; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor Promoters; Tumor Suppressor Proteins; Ubiquitination; cancer cell; cancer stem cell; cancer type; combat; mRNA Precursor; member; promoter; receptor; response; transcriptome; transcriptome sequencing; tumor progression; tumorigenesis

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. In addition to CD44, 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. We recently 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. Out 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. 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 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. Among them, we showed that JAK1 is a constitutive TGFbRI binding protein and is absolutely required for phosphorylation of STATs in a SMADs-independent manner within minutes of TGF-beta stimulation. Following the activation of SMAD, TGF-beta also induces a second phase of STAT phosphorylation that requires SMADs, de novo protein synthesis, and contribution from JAK1. Our global gene expression profiling indicate that the non-SMAD JAK1/STATs pathway is essential for the expression of a subset of TGF-beta target genes in hepatic stellate cells, and the cooperation between JAK1-STAT3 and SMADs pathways is critical to the roles of TGF-beta in liver fibrosis. 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.

通过其膜结合的I型受体的作用，转化生长因子-β诱导了广泛的细胞反应，以上下文依赖的方式调节细胞的增殖、分化和凋亡。这些信号反应中有许多是由SMAD蛋白介导的。因此，控制SMAD活性对于转化生长因子-β及其相关因子的正确信号转导至关重要。除了两个C-末端丝氨酸残基外，转化生长因子-β还能诱导Smad3连接区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还受到SMURF2介导的单一泛素化作用的影响，该作用通过阻止Smad4形成复合体来抑制其活性。我们发现连接蛋白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连接蛋白的磷酸化降低了Smad3复合体的形成和转录活性。在许多类型的癌细胞中，SMAD3连接位点是结构性磷酸化的。我们发现，Smad3连接蛋白磷酸化的变化有助于转化生长因子-β从肿瘤抑制因子转变为肿瘤转移促进剂。在寻找通过连接区苏氨酸179(T179)的磷酸化来调节Smad3的蛋白质时，我们鉴定了一个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。除了CD44，我们的全球RNA-SEQ研究揭示了过多的癌症基因，其剪接模式被SMAD3-PCBP1相互作用改变，有利于肿瘤的进展。这些发现使我们提出，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转化生长因子-β成为肿瘤促进剂的关键机制。我们最近将Smad3的这一作用扩展到控制TAK1的选择性剪接，TAK1的剪接既有全长的，也有缩短的异构体。我们发现，短的TAK1亚型是介导转化生长因子-β诱导的EMT和核因子-kB信号所必需的，并导致耐药，而全长的TAK1则支持转化生长因子-β诱导的细胞凋亡。我们的数据表明，阻断转化生长因子-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。尽管Smads参与了转化生长因子-β的大部分活动，但激活的转化生长因子-β受体也通过其他细胞内信号通路传递信号。在过去的几年里，我的团队致力于破译转化生长因子-β受体激活不依赖于Smads的MAP激酶的具体机制，并阐明这种不依赖于SMAD的转化生长因子-β信号的生物学意义。为了达到这些目标，我们发现TRAF6是SMAD非依赖性激活JNK和p38所必需的。为了揭示在转化生长因子-β信号转导中发挥作用的其他机制和途径，我们采用了有针对性的蛋白质组学方法来鉴定TGFbRI复合体的其他相关蛋白。通过这种方法，我们发现了几个在转化生长因子-β信号传递的早期阶段相互作用和/或被磷酸化的蛋白激酶。其中，我们证明了JAK1是一种结构性的TGFbRI结合蛋白，在转化生长因子-β刺激的几分钟内，JAK1是STATS以Smads非依赖性方式磷酸化所必需的。在SMAD激活后，转化生长因子-β还诱导第二阶段的STAT磷酸化，这需要Smads、从头合成蛋白质和JAK1的贡献。我们的全球基因表达谱表明，非SMAD JAK1/STATS通路是肝星状细胞表达一系列转化生长因子-β靶基因所必需的，JAK1-STAT3和Smads通路之间的协同作用对于转化生长因子-β在肝纤维化中的作用至关重要。对其他候选蛋白的进一步鉴定将有助于阐明可能解释SMAD非依赖性转化生长因子-β信号反应的其他机制，并促进我们对转化生长因子-β诱导过多不同生物反应能力的理解。