Molecular Mechanisms of TGF-beta Signaling Pathway

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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.