Transforming growth factor-β (TGFβ) signaling regulates cell proliferation, differentiation, and development. The binding of TGFβ to TGFβ receptor 2 (TGFBRII) induces the interaction between TGFβ receptor 1 (TGFBRI) and TGFBRII, leading to the phosphorylation and activation of transcriptional regulators SMAD2 and SMAD3. Using an siRNA screen of the human kinome and a live-cell reporter for TGFBR activity, we identified BUB1 (budding uninhibited by benzimidazoles-1), a Ser/Thr kinase, as an essential mediator of TGFβ signaling. BUB1 interacted with TGFBRI in response to stimulation with TGFβ and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2/3 and their interaction with SMAD4, SMAD-dependent transcription, and TGFβ-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Non-canonical signaling cascades of the TGFβ pathway mediated by the kinases AKT and p38 MAPK also mediated by BUB1, suggesting an upstream positioning for BUB1 in the TGFβ pathway. Although the substrate for BUB1 was elusive, its function in promoting TGFβ signaling was dependent on its kinase function: A small-molecule inhibitor of BUB1 kinase (2OH-BNPP1) and a kinase-deficient mutant of BUB1 abrogated TGFβ signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings provide evidence for a role of BUB1 as a kinase in mediating TGFβ-dependent signaling beyond its established function in cell-cycle regulation and chromosome cohesion.

The transforming growth factor-beta (TGF-β) family of cytokines regulates many processes such as immune suppression, angiogenesis, wound healing and epithelial to mesenchymal transition (EMT. Early in tumorigenesis, when epithelial cells retain exquisite growth sensitivity to this ligand, TGF-β signaling elicits a tumor suppressing activity. However, transformed cells become refractory to TGF-β-mediated growth inhibition and acquire a phenotype wherein the intracellular signaling circuitry of the cells is altered, leading to tumorigenic and metastatic effects in response to TGF-β exposure. Although TGF-β activating pathways have been studied, the molecular participants are poorly defined. Here we identify budding uninhibited by benzimidazoles-1 (Bub1) as an integral component of canonical and non-canonical TGF-β signaling pathways, where Bub1 is required for TGFBRI-TGFBRII complex formation and activation. Bub1-depleted cells exhibited reductions in TGF-β dependent Smad2/3 phosphorylation, recruitment of Smad2/3 to the TGFBRI-II complex, PI3K/Akt and p38MAPK activation, Smad binding element driven promoter activity (SBE4-Luc), and invasion and migration. Furthermore, a targeted small molecule inhibitor of Bub1 kinase activity (2OH-BNPP1), as well as an inactive kinase mutant of Bub1, abrogated ligand mediated TGF-β signaling and phenotypic response. These studies demonstrate a role for the Bub1 kinase in mediating TGF-β dependent signaling beyond its established function in cell-cycle regulation and chromosome cohesion and uncover the underlying basis for the pleiotropic cellular response commonly observed upon activation of the pathway. Citation Format: Shyam Nyati, Katrina Schinske-Sebolt, Sethuramasundaram Pitchiaya, Katerina Chekhovskiy, Areeb Chator, Nauman Chaudhry, Joseph Dosch, Marcian E. Van Dort, Varambally, Kumar-Sinha, Nyati, Ray, Walter, Sooryanarayana Varambally, Chandan Kumar-Sinha, Mukesh K. Nyati, Dipankar Ray, Nils G. Walter, Hongtao Yu, Brian D. Ross, Alnawaz Rehemtulla. Bub1 is a key regulator of TGF-β signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1137. doi:10.1158/1538-7445.AM2014-1137