Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling

Abstract

Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1–Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1–Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses. Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1–Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1–Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses. Bone morphogenetic proteins (BMPs) are extracellular ligands that carry out their function through the activation of cell surface BMP type I and type II receptors, which subsequently activate the R-Smads, Smad1 and Smad5, via C-terminal phosphorylation. The activated R-Smads then form complexes with the Co-Smad, Smad4, and translocate into the nucleus to regulate transcription (1Chaikuad A. Bullock A.N. Structural basis of intracellular TGF-beta signaling: Receptors and Smads.Cold Spring Harb. Perspect. Biol. 2016; 8a022111Crossref PubMed Scopus (57) Google Scholar, 2Macias M.J. Martin-Malpartida P. Massague J. Structural determinants of Smad function in TGF-beta signaling.Trends Biochem. Sci. 2015; 40: 296-308Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Among BMP target genes is Smad6, which encodes an inhibitory Smad that restrains the BMP response at multiple levels (3Miyazawa K. Miyazono K. Regulation of TGF-beta family signaling by inhibitory Smads.Cold Spring Harb. Perspect. Biol. 2017; 9a022095Crossref PubMed Scopus (262) Google Scholar). At the membrane level, Smad6 inhibits BMP-induced Smad1/5 activation through competition for association with the BMP type I receptor (4Goto K. Kamiya Y. Imamura T. Miyazono K. Miyazawa K. Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors.J. Biol. Chem. 2007; 282: 20603-20611Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 5Xu J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). At the cytosolic level, Smad6 competes with Smad4 for Smad1/5 binding, therefore disrupting Smad1/5 and Smad4 complex formation (6Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (583) Google Scholar). At the nuclear level, Smad6 recruits transcription corepressors to suppress BMP-driven transcription (7Bai S. Shi X. Yang X. Cao X. Smad6 as a transcriptional corepressor.J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 8Lin X. Liang Y.Y. Sun B. Liang M. Shi Y. Brunicardi F.C. Shi Y. Feng X.H. Smad6 recruits transcription corepressor CtBP to repress bone morphogenetic protein-induced transcription.Mol. Cell. Biol. 2003; 23: 9081-9093Crossref PubMed Scopus (82) Google Scholar). As their name implies, the BMPs control bone development, repair, and regeneration (9Katagiri T. Watabe T. Bone morphogenetic proteins.Cold Spring Harb. Perspect. Biol. 2016; 8a021899Crossref PubMed Scopus (259) Google Scholar). 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Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The of protein arginine methylation is by a family of protein arginine which signaling transcriptional and S. The functions and of arginine Rev. Biol. PubMed Scopus Google Scholar, Y. arginine and Rev. Cancer. 2013; PubMed Scopus Google Scholar). roles in processes including cell and Y. arginine and Rev. Cancer. 2013; PubMed Scopus Google Scholar, T.J. Jin J. T. arginine the Rev. Biol. 12: PubMed Scopus Google Scholar, arginine methylation in and Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Among the protein arginine methyltransferase 1 is for more of arginine methylation in cells arginine methylation in and Cell. Full Text Full Text PDF PubMed Scopus Google and to signaling by and J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, M. N. Y. S. K. S. S. L. Regulation of signaling through arginine methylation by Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Zhu K. M. Arginine methylation of Full Text Full Text PDF PubMed Scopus Google Scholar, K. H. Y. K. K. K. H. Y. A. Arginine methylation of transcription inhibits their phosphorylation by Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that methylated Smad6 at arginine and that methylation inhibitory function at the cell surface receptor J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). that Smad6 is also methylated on arginine 81 However, not at the membrane, and functional not the we show that BMP Smad6 methylation at R81 in the to interaction with Smad1 and of Smad1–Smad4 in of transcription and inhibition of BMP that Smad6 is methylated at and R81 J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). At the membrane level, Smad6 methylation at BMP signaling R81 methylation the function of Smad6 R81 we the of Smad6 with Smad6 using J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). BMP4-induced methylation of R81 methylation specifically in the cytosolic of Smad6 R81 of treatment, by of cytosolic Smad6 methylation methylation of and R81 in the nuclear on BMP signaling with our J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google methylation in the membrane but is the methyltransferase that catalyzes methylation of Smad6 J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). therefore also methylated R81 of using in methylation we that methylated Smad6 at R81 in the of control methylation not the R81 methylation-deficient mutant R81A is for R81 methylation in we expression using which Smad6 R81 methylation in cells suggest that R81 methylation in the cytosolic and that is the for Smad6 R81 Smad6 to with Smad1 and nuclear (6Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (583) Google Scholar). R81 methylation regulates Smad6 to Smad1, we the R81 methylation-deficient Smad6 we using the which in the to a nuclear J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The R81A mutant a J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google and therefore in Smad6 to Smad1, we WT R81A and methylated in by with in the of Smad6 as the the of and WT Smad6 to Smad1 that cells and on WT Smad6 with Smad1 WT Smad6 R81A Smad6 and The of Smad6 in the the results the WT Smad6 and WT Smad6 in the of the on the and results that Smad6 R81 methylation is for Smad1 the role of R81 methylation in Smad6 interaction with Smad1 in with WT R81A and with for Smad6 complexes and the of Smad1 in complexes by WT Smad6 with Smad1 in a Smad6 which is in R81 to Furthermore, Smad6 that with Smad1 in cells R81 methylated that Smad6 interaction with Smad1 in in which Smad1 phosphorylated that Smad6 activated Smad1, the C-terminal phosphorylated form (1Chaikuad A. Bullock A.N. Structural basis of intracellular TGF-beta signaling: Receptors and Smads.Cold Spring Harb. Perspect. Biol. 2016; 8a022111Crossref PubMed Scopus (57) Google Scholar, 2Macias M.J. Martin-Malpartida P. Massague J. Structural determinants of Smad function in TGF-beta signaling.Trends Biochem. Sci. 2015; 40: 296-308Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). by phosphorylated and Smad1 to an in to that in cells the BMP type I and type II Smad1, we the Smad1 with in The levels of Smad1 phosphorylation by the then Smad1 to As methylated Smad6 to with phosphorylated Smad1 but not with Smad1 that Smad6 phosphorylated the of BMP-induced R81 methylation and Smad1 phosphorylation and that BMP-induced Smad1 phosphorylation the of Smad6 R81 methylation R81 methylation is for the inhibition of BMP4-induced Smad1 nuclear translocation. cells with Smad6 WT the R81A mutant the with for 1 and then to with and on The the nuclear Smad1 in cells WT Smad6 with cells cells mutant Smad6 for of cells Smad6 the R81A as control with for and to with are in The of Smad1 in to cells using cells Smad6 Smad6 as control with for the of and to The cytosolic and nuclear by using the and as for in and using and Smad6 but not the R81 BMP-responsive cells Smad6 Smad6 as control with for and expression of and by at Smad6 at R81, which facilitates interaction that competes with Smad1–Smad4 complex formation to BMP bone morphogenetic protein arginine methyltransferase R81, arginine Smad6 interaction with Smad1 via C-terminal (6Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (583) Google Scholar). on for interaction the and of Smad6 (6Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (583) Google Scholar, A. Y. T. T. Imamura T. Miyazono K. The of is essential for inhibition of Biol. PubMed Scopus Google we R81 methylation in the regulates interaction the and of Smad6 to Smad6 interaction with Smad6 the and the C-terminal in and the interaction by The WT with with (6Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (583) Google Scholar, A. Y. T. T. Imamura T. Miyazono K. The of is essential for inhibition of Biol. PubMed Scopus Google Scholar). we interaction and the R81A mutant that the the WT and the R81A mutant on R81 and not on the arginine residue, we the interaction and in cells control cells with R81A WT in control However, WT and R81A to in cells results that Smad6 R81 methylation at the the interaction and of a R81 methylation of Smad6 the interaction and facilitates Smad6 association with the Smad6 R81 interaction with Smad1 inhibited Smad1 to the Co-Smad, we a signaling in cells by the active BMP type receptor S. T. M. N. N. K. H. N. T. active BMP type I the in PubMed Scopus Google Smad1 and Smad4, with WT mutant The Smad1 activation and Smad1–Smad4 complex formation WT Smad6 disrupted Smad1–Smad4 complex formation the Smad6 R81A mutant not that methylation of Smad6 at R81 is for to Smad1–Smad4 complex also in the Smad6 which WT Smad6 but is at Smad6 also to Smad1–Smad4 complex formation suggest a role for Smad6 R81 methylation in disrupting Smad1–Smad4 complex we WT and mutant Smad6 for their to the formation of Smad1–Smad4 complexes in complexes of cells WT R81A mutant Smad6, and the of phosphorylated Smad1 in the complex by with the WT Smad6, but not the R81A mutant inhibited the of with Smad4 Smad6 R81 methylation is for to complex Smad1–Smad4 complex formation is for Smad1 nuclear X.H. Derynck R. and in signaling through Rev. Dev. Biol. PubMed Scopus Google we the role of Smad6 R81 methylation in Smad1 cells with Smad6 the R81A mutant the with and the of Smad1 on of Smad1 in WT cells in and Smad1 in of cells in the and in cells with the R81A the mutant Smad6 in and also the of Smad6 on BMP4-induced Smad1 nuclear in cell levels of Smad6 R81A as of the nuclear and that the of Smad1 and that by the of Smad6 WT and not R81A and The with a approach the cell levels of R81A with and the levels of Smad1 in cytosolic and nuclear Smad6 WT the BMP4-induced in nuclear Smad1, Smad6 R81A to and Smad1 in the of cells WT but not the R81A mutant Smad6 and with the inhibition of BMP4-induced Smad1 nuclear WT Smad6, but not the R81A antagonized BMP-mediated of the Smad1 target genes and and R81 methylation of Smad6, which is BMP is for inhibition of BMP-induced Smad1 nuclear and transcriptional Taken together, we a Smad6 R81 methylation the interaction and and facilitates Smad6 to phosphorylated active Smad1, which inhibits Smad1–Smad4 complex formation and transcription into the of Smad6 R81 we role in the of osteogenic of cells with osteogenic BMPs differentiation the T. A. M. N. T. A. T. Bone morphogenetic the differentiation of into the Biol. PubMed Scopus Google Scholar). cell to the of Smad6 WT the methylation-deficient R81A mutant to the effects of The WT and R81A mutant Smad6 at levels as the methylated on R81 WT Smad6 inhibited the expression of the osteogenic and but the R81A mutant not repress osteogenic Furthermore, for that Smad6 but not the R81A inhibited osteogenic differentiation and suggest that loss of R81 methylation the inhibitory of Smad6 in the of BMP-induced osteogenic R81, methylation of not in the inhibitory of Smad6 in BMP-induced signaling J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). At the biochemical level, R81 methylation-deficient mutant methylation T. J. N. Y. Feng J. Y. Liu J. S. Jin J. G. Y. J. Smad6 methylation activation and 2018; PubMed Scopus Google methylation-deficient mutant R81 methylation J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google that and R81 the functional of and R81 methylation in BMP-induced osteogenic cells with to osteogenic differentiation T. A. J. B. differentiation in of Cell. Biochem. PubMed Scopus Google Scholar, Li M. B. to for of and Cell. 2017; PubMed Scopus Google Scholar). the of Smad6 WT with the methylation-deficient R81A to the effects of Here, effects on R81 methylation in biochemical J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The WT and mutant Smad6 at levels WT Smad6 inhibited the expression of the osteogenic and and with a role for R81A methylation in Smad6 the R81A mutant not repress osteogenic the mutant inhibitory effects and Taken together, suggest that and R81 play methylation the inhibitory function of Smad6, R81 methylation is for the inhibitory of Smad6 in the of BMP-induced osteogenic osteogenic differentiation, BMP signaling inhibits the in (9Katagiri T. Watabe T. Bone morphogenetic proteins.Cold Spring Harb. Perspect. Biol. 2016; 8a021899Crossref PubMed Scopus (259) Google we Smad6 cells T. J. N. Y. Feng J. Y. Liu J. S. Jin J. G. Y. J. Smad6 methylation activation and 2018; PubMed Scopus Google which are to BMP and the cells and the expression of Smad6 WT in the Smad6 we the Smad6 R81A and cell invasion through Smad6 WT and mutant cell the R81A mutant Smad6 to suggest that R81 methylation is is for the of into the of BMP response to BMP Smad6 is methylated at R81, and facilitates interaction with Smad1, which in Smad1–Smad4 complex inhibits Smad1 to the and The Smad6 R81 methylation is therefore to play a role in to The that BMP signaling in the of osteogenic differentiation and cell invasion. The methylation of Smad6 R81 by also Smad6 on the J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, M. S. Ten Dijke P. M. of Smad6 by protein arginine PubMed Scopus Google Scholar). that Smad6 methylation at the cell surface BMP treatment, Smad1 phosphorylation and activation J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). that methylation Smad6 BMP type I as an of BMP signaling to Smad1 activation J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). the R81 methylation of Smad6 with a by to the Smad6 methylation Smad1 phosphorylation and at the cell surface J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google the Smad6 R81 methylation to Smad1 phosphorylation and the of active Smad1 in the and in the nucleus as also show that Smad6 to phosphorylated Smad1, that Smad6 active Smad1 to BMP Smad6 methylation at the membrane and R81 methylation at the is for control of BMP signaling by of Smad6 R81 the with the R81 methylation as a for the interaction Smad6 and differentiation response which for differentiation response protein in an Smad6 in the to Factor repress and tissue T. J. N. Y. Feng J. Y. Liu J. S. Jin J. G. Y. J. Smad6 methylation activation and 2018; PubMed Scopus Google Scholar). The that R81 methylation also plays roles in Smad6-mediated inhibition of osteogenic differentiation, as as Smad6-mediated of a function recently to Smad6 with in multiple types of M. S. Ten Dijke P. 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Full Text Full Text PDF PubMed Scopus Google Scholar). specifically R81 methylation by Smad6 and BMP-induced in a development, tissue repair, and a role for arginine methylation in Smad6-mediated of BMP signaling that Smad6 to BMP signaling and Smad6 as with methyltransferase Smad6 the of for by of BMP and their cell in with cells Smad6 WT as control into the using The then with into cells to the cell and then by at cells by the for in the of The cells to and levels of Smad6 WT the R81A mutant protein in for of cells and at 1 and to The for are in For cells with control using with a for in a tissue 1 to cells in the in the and the cell with in the in a tissue for the cells with the the and cells on the surface of the membrane in with and with a cells in with by with for 1 with in in and with with a with by and in and and nuclear by at for at in the to and the the and by at for at The the with and in the nuclear and the nuclear by at for at in the and The cell for and at for The to and the proteins then to which and with at and then with at using and by as J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Bruckner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google and and and and and with in for and with with with at with and the for 1 at then and with with and using a as T. J. N. Y. Feng J. Y. Liu J. S. Jin J. G. Y. J. Smad6 methylation activation and 2018; PubMed Scopus Google Scholar). cells in the and the by at for The to the with at protein A for in the and by at for proteins by in the for For protein an by the with a control for by at for to with the to with to 1 of active in the methylation and in the of for at The by protein to and protein methylation by using The cells with and BMP and using by and with the proteins at for by at for The as a and the to the by the with and for in using the in a as the for are in the are the The that of with the of for the J. X. P. H. and J. X. the the and the X. P. T. J. and Y. G. to and B. and H. S. to functional and by including the of to J. and to J. to J. of to T. the of J. and the at the of J. The is the of the and not the of the of

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