It is well established that the level and duration of R-Smad activity has critical effects on TGF-β
-induced transcriptional responses.27
It is therefore logical that pathways that regulate the dephosphorylation inactivating R-Smads would have a significant impact on TGF-β
and BMP signaling. Although multiple Smad phosphatases have been identified,28
it has been unclear how the activity and expression of these phosphatases are regulated. In this study, we have shown that Bat3 promotes Smad1/5/8 dephosphorylation and thereby influences BMP signaling. In addition, we have started to unravel the molecular mechanisms underlying this regulation.
The regulation of R-Smad dephosphorylation has been difficult to dissect, partly because of the diverse and dynamic functions of these phosphatases, and partly because of our poor understanding of the tissue-specific mechanisms that control these enzymes.29
Among the known Smad phosphatases, the SCPs have been reported to differentially regulate Smad2/3 and Smad1/5/8.7, 8, 26
SCP1 and 2 clearly dephosphorylate the linker regions of Smad2 and 3, thereby enhancing TGF-β
signaling. However, controversy exists over whether SCP-mediated dephosphorylation of sites within the linker and C-terminal regions of Smad1 can inactivate this molecule, thereby suppressing BMP signaling.7, 8, 26
Our study has partially resolved this issue by demonstrating that SCP2 can indeed dephosphorylate the C-terminal phosphorylation sites in Smad1 and Smad5, while showing weaker activity for Smad8 (Supplementary Figure 2
). It is currently unknown whether SCPs require additional factors and/or modifications to initiate Smad1 dephosphorylation, or whether SCP-mediated Smad1 dephosphorylation is tissue or species specific. Nevertheless, our data indicate that Smad1–SCP2 interaction is enhanced by Bat3, and that this series of events regulates BMP signaling ().
Bat3 has been reported to act as a regulator of molecular chaperone proteins,30
and also contains a ubiquitin-like (UBL) domain. Therefore, like other UBL-containing proteins,31
Bat3 may suppress or enhance protein stability, protein–protein interactions, or both. Although Bat3 target proteins have yet to be fully delineated, our biochemical analyses indicate that SCP2 is a Bat3-interacting protein. Interestingly, Bat3 enhances Smad1–SCP2 interaction only in the presence of constitutively active ALK6 (). It has been previously shown that ALK5–Bat3 interaction accelerates the translocation of Bat3 into the nucleus.22
However, in the context of BMP signaling, we were unable to detect any interaction between Bat3 and ALK2, 3, or 6 (Supplementary Figure 4
). In addition, Bat3 is located predominantly in the nucleus in the cell lines used in our study, obscuring any increase in levels of total Bat3 protein or nuclear Bat3 protein that might have been induced by BMP treatment (, Supplementary Figure 1
and data not shown). Finally, although our data strongly suggest that Bat3 enhances the interaction between SCP and phosphorylated Smad1, it remains to be elucidated whether additional post-translational modifications (e.g., ubiquitination or acetylation) of Smad1, SCP2, and/or Bat3 are required for physiological regulation of BMP signaling.
In their detailed analyses of the developmental defects of Bat3-deficient mice, Desmots et al.15
reported that these mutants showed impaired branching of the terminal bronchiolar alveoli in the lung as well as abnormal renal branching in the kidney, two processes for which BMP signaling is known to be crucial.32, 33, 34
These in vivo
findings raised the possibility that dysregulated BMP signaling might be responsible for the defects in their Bat3-deficient mice. Our demonstration in cell lines that Bat3 inactivation sustained BMP signaling and enhances osteoblast differentiation support a role for Bat3 in development. However, further investigation of tissue-specific Bat3 knockout mice will be required to confirm any such in vivo
functions for Bat3.
In conclusion, our work demonstrates that Bat3 positively regulates dephosphorylation of Smad1/5/8 and thus functions as a negative regulator of BMP signaling. As BMPs enhance bone generation, modification of the BMP–Bat3 signaling may improve efficacy of bone replacement and implant. In addition, because dysregulation of the TGF-β
and BMP signaling pathways has been observed in numerous human disorders, including cancer,32, 35, 36
it would be interesting to determine whether mutations of Bat3 are associated with disease progression.