Transforming Growth Factor β Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors

Abstract

Transforming growth factor-β (TGFβ) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFβ signaling is mediated via the TβRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFβ can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFβ can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFβ/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFβ induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFβ-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. Transforming growth factor-β (TGFβ) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFβ signaling is mediated via the TβRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFβ can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFβ can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFβ/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFβ induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFβ-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. Transforming growth factor (TGF) 2The abbreviations used are: TGF, transforming growth factor; DM, dorsomorphin; MEF, mouse embryonic fibroblast; ALK, activin receptor-like kinases; caALK, constitutively active ALK; HA, hemagglutinin; GST, glutathione S-transferase; siRNA, small interfering RNA; BMP, bone morphogenic protein. β superfamily members signal through heteromeric complexes of transmembrane serine/threonine kinase receptors to control diverse developmental processes and the pathogenesis of many diseases. The heteromeric receptor complex usually comprises two type II receptors and two type I receptors, and the receptors are classified based on their structural and functional properties. Type II receptors are constitutively active, and they phosphorylate type I receptors on serine/threonine residues in the GS domain in response to ligand binding. Activated type I receptors then phosphorylate specific downstream receptor-activated Smad (R-Smads) proteins in their distal C-terminal SXS motif, activating them to transduce the signal to the nucleus (1Feng X.-H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1507) Google Scholar, 2Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (935) Google Scholar). There are five mammalian type II receptors: TβRII, ActR-II, ActR-IIB, BMPR-II, AMHR-II, and seven type I receptors, which are referred to as activin receptor-like kinases (ALK) 1–7 (1Feng X.-H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1507) Google Scholar, 3Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Crossref PubMed Scopus (645) Google Scholar). Of the ALKs, ALK5 is thought to be specific for TGFβ ligands, and ALK4 and ALK7 are thought to mediate signaling via nodal and activins (2Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (935) Google Scholar). In canonical signaling, the activated ALK4/ALK5/ALK7 phosphorylate downstream R-Smads 2 and 3. The remaining ALKs, ALK1/2/3/6, phosphorylate R-Smads 1, 5, and 8 following specific activation by bone morphogenic proteins (BMP) (2Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (935) Google Scholar). Once activated, R-Smads undergo heterooligomeric complex formation with Smad4, named the common Smad due to its role in all branches of TGFβ superfamily signaling, and this complex accumulates in the nucleus to regulate gene transcription in conjunction with a variety of transcriptional cofactors (1Feng X.-H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1507) Google Scholar, 2Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (935) Google Scholar). The specificity of different ALKs for different Smad proteins is based on the L45 loop and phosphorylated GS motif in the specific type I receptor, and the L3 loop in the MH2 domain of the relevant R-Smad (4Chen Y.G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massague J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (299) Google Scholar, 5Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (207) Google Scholar). The L45 loops of ALK4/5/7 that phosphorylate Smad2/3 are completely conserved, but they differ by four amino acids from the L45 sequences in ALK3 and ALK6, and by seven amino acids from the L45 loops of ALK1 and ALK2 (4Chen Y.G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massague J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (299) Google Scholar, 6Feng X.-H. Derynck R. EMBO J. 1997; 16: 3912-3923Crossref PubMed Scopus (160) Google Scholar). The L45 loop of the ALK interacts directly with the L3 loop in the MH2 domain of the relevant R-Smad (4Chen Y.G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massague J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (299) Google Scholar, 5Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (207) Google Scholar). Although it is generally accepted that TGFβ induces Smad2/3 phosphorylation, several studies have shown that TGFβ can also induce Smad1/5 phosphorylation in endothelial cells (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 8Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (906) Google Scholar). The current model proposes that the type I receptor ALK1 is necessary for this phosphorylation event, in addition to ALK5 activity and the TGFβ transmembrane accessory receptor Endoglin (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 8Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (906) Google Scholar, 9Lebrin F. Goumans M.J. Jonker L. Carvalho R.L. Valdimarsdottir G. Thorikay M. Mummery C. Arthur H.M. ten Dijke P. EMBO J. 2004; 23: 4018-4028Crossref PubMed Scopus (514) Google Scholar). Similarly, in dermal fibroblasts TGFβ can induce Smad1 phosphorylation dependent on both ALK5 and ALK1 activity, although this also appears to require activation of the ERK1/2 pathway for a sustained response (10Pannu J. Nakerakanti S. Smith E. ten Dijke P. Trojanowska M. J. Biol. Chem. 2007; 282: 10405-10413Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). It has recently been shown that TGFβ can also stimulate Smad1/5 phosphorylation in HaCaT keratinocytes dependent on ALK5 activity, and this is also thought to depend on a canonical activator of Smad1, most likely ALK2 (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar). Thus, although there have been several reports that TGFβ can induce Smad1 phosphorylation, revealing cross-talk between the TGFβ ligand and the BMP R-Smads, this phosphorylation event is largely thought to be dependent on the activity of a BMP type I receptor. During our studies we observed TGFβ-induced Smad1 in several cell lines for which this has previously not been reported, including C2C12 mouse mesenchymal stem cells and HepG2 human hepatocellular carcinoma cells. Significantly, we found that in these cell lines TGFβ leads to Smad1 phosphorylation independently of ALK1/2/3/6, exclusively relying on the ALK4/5/7 subset as a type I receptor. This is in line with a study published while our manuscript was in revision, which revealed ALK5-mediated TGFβ-induced Smad1 phosphorylation in mammary epithelial cells (12Liu I.M. Schilling S.H. Knouse K.A. Choy L. Derynck R. Wang X.F. EMBO J. 2009; 28: 88-98Crossref PubMed Scopus (145) Google Scholar). Our data provide firm evidence that TGFβ-induced Smad1 phosphorylation can occur independently of involvement of one of its canonical BMP type I receptors. Cell Culture and Treatment-C2C12 mouse mesenchymal cells, HEK293T human embryonic kidney cells, and COS monkey kidney cells were cultured in Dulbecco’s modified Eagle’s medium, and Smad3 mouse cells E. J. D. M. C. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google were cultured in medium, HepG2 human hepatocellular carcinoma cells and human HaCaT cells were cultured in medium, The Hill C.S. Mol. 2002; PubMed Scopus Google was as a in The was previously C. Nat. Chem. Biol. 2008; PubMed Scopus Google Scholar). cells were with ligand and for to were in with to a of 2 and to a of and and constitutively active ALK5 also for have been previously X.-H. Derynck R. EMBO J. PubMed Scopus Google Scholar). was by constitutively active ALK1 was a from ten HEK293T cells were using HepG2 cells using and C2C12 cells using C2C12 cells were also using the as and of cell cells were in for the of Smad phosphorylation, and was as previously D. J. F. X.-H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was using and Smad were by using Smad SXS motif phosphorylation was by using from proteins were using and protein was using was used to the of Smad1 on TGFβ-induced C2C12 cells were with using the with and cells were cultured in the of TGFβ BMP for to for the of Smad1 on TGFβ-induced C2C12 cells were with as cells were with the which of the activator and L. J. A. J. M. Wang X.F. Massague J. Cell. Full Text PDF PubMed Scopus Google Scholar). a cells were cultured in the of TGFβ for to for as previously D. J. F. X.-H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the of to ALK1 kinase activity, COS cells to were with by ten ten Dijke P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and the constitutively active of BMP type I receptor ALK1 using with was used to control for were with Cell were and activity by the of to activity using the In were largely as previously (12Liu I.M. Schilling S.H. Knouse K.A. Choy L. Derynck R. Wang X.F. EMBO J. 2009; 28: 88-98Crossref PubMed Scopus (145) Google Scholar). In was from HEK293T cells in with inhibitors and and inhibitors and were in and in kinase and was for with from in kinase were by TGFβ Smad1 in a of Cell studies in our we observed that TGFβ induce Smad1 phosphorylation in cell with several reports that it can in endothelial cells (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 8Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (906) Google dermal fibroblasts (10Pannu J. Nakerakanti S. Smith E. ten Dijke P. Trojanowska M. J. Biol. Chem. 2007; 282: 10405-10413Abstract Full Text Full Text PDF PubMed Scopus (171) Google and HaCaT keratinocytes (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar). In to this in we several cell lines in the of TGFβ for in with BMP as a control for Smad1 phosphorylation, and cells for of Smad and shown in 1, we a protein the of in response to both TGFβ and BMP in C2C12 HepG2 and mouse embryonic cells This was using as as which can both and data that TGFβ induce Smad1 phosphorylation, as as the phosphorylation of Smad2 and in the cell lines TGFβ-induced Smad1 the of TGFβ-induced Smad1 phosphorylation as with of TGFβ-induced Smad3 phosphorylation and Smad1 C2C12 cells were with TGFβ a of to and cell were by Smad3 phosphorylation was observed of TGFβ with the the The TGFβ-induced Smad3 phosphorylation to although a for the of the In TGFβ-induced Smad1 phosphorylation was not and was the Thus, TGFβ-induced Smad1 phosphorylation is and a TGFβ-induced Smad3 phosphorylation, which is sustained the Smad1 phosphorylation in C2C12 cells TGFβ-induced Smad1, and was also sustained to a observed a response in TGFβ-induced Smad1 phosphorylation was between and as with TGFβ-induced Smad3 phosphorylation, which from to Smad1 phosphorylation BMP in remaining for the of the in C2C12 cells and TGFβ-induced Smad1 phosphorylation a both TGFβ-induced Smad3 phosphorylation and Smad1 phosphorylation, and is in the of TGFβ to stimulate Smad1 and Smad3 phosphorylation in C2C12 cells. Smad3 phosphorylation was by with their However, Smad1 phosphorylation was not observed with with their Thus, TGFβ-induced Smad1 phosphorylation a of TGFβ TGFβ-induced Smad3 phosphorylation in C2C12 cells. these data due to the different and TGFβ for TGFβ-induced Smad1 phosphorylation, it a role in TGFβ-induced and in cells. of Smad1 in to to that the by and in response to TGFβ to this protein can be by the we E. J. D. M. C. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google to that this protein to a modified of and were cultured in the of TGFβ BMP for to for shown in TGFβ Smad3 phosphorylation, Smad2 phosphorylation, and the Smad1 phosphorylation in as we previously observed for this cell Although TGFβ-induced Smad3 phosphorylation was observed in in line with the of Smad3 Smad2 phosphorylation and the TGFβ-induced protein in response to TGFβ data completely that the in response to TGFβ to a of that this protein is to the TGFβ-induced is in cells of Smad1 protein. we C2C12 cells with Smad1, and cultured cells in the of TGFβ BMP for to for shown in TGFβ was to induce and in cells of in a in Smad1 protein by although and were in cells in response to the we have observed is in C2C12 cells of Smad1 protein Smad1 phosphorylation is also significantly in cells data that TGFβ induces Smad1 phosphorylation in C2C12 cells, as by both our and and that these are most likely TGFβ-induced Smad1 phosphorylation in the cell lines we have Smad1 have on TGFβ-induced C2C12 cells were with Smad1, and cultured for to with the which is used to TGFβ signaling L. J. A. J. M. Wang X.F. Massague J. Cell. Full Text PDF PubMed Scopus Google Scholar). of cell of Smad1 and a of Smad1 phosphorylation in response to TGFβ of cells with TGFβ in a of TGFβ-induced as Significantly, of Smad1 the of TGFβ-induced transcription by as with cells. This in C2C12 cells, Smad1 be in TGFβ-induced transcription in addition to the Smad2/3 TGFβ-induced Smad1 of BMP Type I in Cell reports of TGFβ-induced Smad1 phosphorylation in endothelial cells and dermal fibroblasts a model both the BMP type I receptor and the TGFβ type I receptor are for this (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 8Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (906) Google Scholar, J. Nakerakanti S. Smith E. ten Dijke P. Trojanowska M. J. Biol. Chem. 2007; 282: 10405-10413Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In HaCaT cells it is that and most likely the BMP type I receptor are for TGFβ-induced Smad1 phosphorylation (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar). the ALK of TGFβ-induced Smad1 phosphorylation in the cell lines our we to of the and specific ALK inhibitors Hill C.S. Mol. 2002; PubMed Scopus Google and C. Nat. Chem. Biol. 2008; PubMed Scopus Google Scholar). However, as ALK1 is a for involvement in TGFβ-induced Smad1 phosphorylation, and the of to ALK1 is we to also ALK1 kinase activity. the of and to Smad1 phosphorylation by C2C12 cells were with and cultured in the of for to shown in Smad1 phosphorylation in C2C12 cells This likely activity as TGFβ BMP was to stimulate the activity of receptors. Significantly, Smad1 phosphorylation was completely in cells with can ALK1 activity in C2C12 cells. a in HepG2 cells with and The was to induce phosphorylation of Smad1 and this was also by of cells in can ALK1 kinase activity. also found Smad1 phosphorylation in HEK293T cells the of to induce transcription from gene dependent on BMP R-Smad in the of of shown in transcriptional activity was on with and in 2 activity by the data in is of ALK1 receptor activity, as as the receptor activity C. Nat. Chem. Biol. 2008; PubMed Scopus Google in cell to the involvement of different ALKs in TGFβ-induced Smad1 phosphorylation in the cell lines our we cultured cells for in the of TGFβ BMP in with the and specific ALK Hill C.S. Mol. 2002; PubMed Scopus Google DM, which we can ALK1/2/3/6 in cell lines C. Nat. Chem. Biol. 2008; PubMed Scopus Google C2C12 cells and found that TGFβ Smad3 and Smad1 phosphorylation as we have been to of ALK4/5/7 using in a of TGFβ-induced Smad1, and Smad3 phosphorylation that these TGFβ-induced phosphorylation are dependent on ALK4/5/7 kinase activity. However, in with on Smad1 phosphorylation, that there is on ALK4/5/7 activity for this event of ALK1/2/3/6 by not the TGFβ-induced Smad2 Smad3 phosphorylation, the TGFβ-induced Smad1 phosphorylation that all of these TGFβ-induced phosphorylation are of BMP type I receptor The was to Smad1 phosphorylation in C2C12 cells and that is active the and used in this data that BMP type I receptors not be for TGFβ-induced Smad1 phosphorylation in all cell this is specific to C2C12 cells, also for cells, we a using the specific ALK inhibitors with cell observed that also TGFβ-induced Smad1 phosphorylation in HepG2 cells as as it Smad2 and Smad3 However, as for C2C12 cells, not TGFβ-induced Smad1 phosphorylation although it was to Smad1 phosphorylation In completely TGFβ-induced Smad1 phosphorylation was to TGFβ-induced Smad1 phosphorylation, although it on TGFβ-induced Smad2 and Smad3 phosphorylation in these cells was to Smad1 phosphorylation in HaCaT keratinocytes both and were to completely TGFβ-induced Smad1 phosphorylation and This that ALK1/2/3/6 a role in TGFβ-induced Smad1 phosphorylation in and HaCaT cells. ALK5 ALK5 is thought to be specific for Smad2/3 phosphorylation, we ALK5 also Smad1 HEK293T cells were with and and and Smad SXS phosphorylation was following of observed phosphorylation of Smad1 and the control Smad3 in the of phosphorylation be a of activity as TGFβ BMP was to stimulate the activity of receptors. Thus, this ALK5 have the to phosphorylate Smad1 in addition to the Smad2 and Smad3 in also the of to phosphorylation of in in kinase was from HEK293T cells, and with in the of of revealed phosphorylation of both and on with 2 and that the in phosphorylation observed was due to kinase activity, and not factor in the we a in the of the ALK4/5/7 kinase was to phosphorylation of both and and However, phosphorylation was completely in the of 2 and the data in 8 provide firm evidence that ALK5 is to phosphorylate Smad1, in addition to the ALK5 can directly phosphorylate Smad1, we that these proteins this HEK293T cells were with and and to The control was to in line with its role as a canonical ALK5 phosphorylation was also to these proteins and evidence that Smad1 also be ALK5 phosphorylation In our data that TGFβ-induced Smad1 phosphorylation not require a functional BMP type I receptor, although ALK1/2/3/6 activity is necessary for this to occur in some cell Thus, the of TGFβ-induced Smad1 phosphorylation be largely cell line The receptors for TGFβ superfamily ligands and the specific downstream of these have been and and a canonical for signaling from the cell to the nucleus in response to the ligands has been However, signaling to TGFβ signaling in the cells, that the signaling model is not as as in addition to the canonical Smad2/3 phosphorylation, can a complex with ALK1 in endothelial cells, dependent on ALK5 kinase activity and to in Smad1/5 phosphorylation in response to TGFβ (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 8Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (906) Google Scholar, 9Lebrin F. Goumans M.J. Jonker L. Carvalho R.L. Valdimarsdottir G. Thorikay M. Mummery C. Arthur H.M. ten Dijke P. EMBO J. 2004; 23: 4018-4028Crossref PubMed Scopus (514) Google Scholar). In this activation of canonical TGFβ signaling and of endothelial cells, TGFβ-mediated Smad1/5 phosphorylation and (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). It has been that ALK1 activation in response to TGFβ signaling signaling (7Goumans M.J. Valdimarsdottir G. Itoh S. Lebrin F. Larsson J. Mummery C. Karlsson S. ten Dijke P. Mol. Cell. 2003; 12: 817-828Abstract Full Text Full Text PDF PubMed Scopus (564) Google as of Endoglin that signaling and not signaling F. Goumans M.J. Jonker L. Carvalho R.L. Valdimarsdottir G. Thorikay M. Mummery C. Arthur H.M. ten Dijke P. EMBO J. 2004; 23: 4018-4028Crossref PubMed Scopus (514) Google Scholar). TGFβ-induced Smad1 phosphorylation has also been observed in HaCaT keratinocytes (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar). In with the endothelial cell this signaling is thought to require a BMP type I receptor, in addition to ALK5 kinase activity, with ALK2 the most likely HaCaT cells were found not to ALK1 (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar). this manuscript was in it was also shown that TGFβ can induce Smad1 phosphorylation in a of epithelial cells and fibroblasts via ALK5 and ALK2 ALK3 Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google which a on a BMP type I receptor for TGFβ ligand to have TGFβ-induced Smad1 in different cell lines to by of these Additionally, our data that TGFβ-induced Smad1 phosphorylation can occur independently of BMP type I receptors in some cell lines of cells in the of the ALK DM, which we show can ALK1 kinase activity in addition to its published to was to the of TGFβ-induced Smad1 phosphorylation in C2C12 and HepG2 cells. However, this was completely by the ALK4/5/7 and and This to data in the study by Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google both and TGFβ-induced Smad1/5 phosphorylation in mouse mammary epithelial cells. was to Smad1 phosphorylation in HepG2 and C2C12 cells and as as TGFβ-induced Smad1 phosphorylation in HaCaT cells in with the that HaCaT cells on for this (11Bharathy S. Xie W. Yingling J.M. Reiss M. Cancer Res. 2008; 68: 1656-1666Crossref PubMed Scopus (50) Google Scholar, Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google Scholar). Thus, was active and of ALK1/2/3/6 the in our our manuscript was revision, (12Liu I.M. Schilling S.H. Knouse K.A. Choy L. Derynck R. Wang X.F. EMBO J. 2009; 28: 88-98Crossref PubMed Scopus (145) Google that TGFβ induce Smad1 phosphorylation in the mouse mammary epithelial cell line This phosphorylation event was dependent on ALK5 kinase activity, and the involvement of BMP receptors using Our data from the of ALK inhibitors in the that TGFβ-induced Smad1 phosphorylation is of BMP receptors in some cells. ALK5-mediated phosphorylation of Smad1 was by in kinase in both studies (12Liu I.M. Schilling S.H. Knouse K.A. Choy L. Derynck R. Wang X.F. EMBO J. 2009; 28: 88-98Crossref PubMed Scopus (145) Google Scholar). It is as to ALK5 as this is thought to be for TGFβ-induced signaling, lead to the phosphorylation of Smad1 independently of type I receptors. of type I receptors for the canonical Smad is usually based on their L45 loop and phosphorylated GS motif, and on the L3 loop in the MH2 domain of the Smad (4Chen Y.G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massague J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (299) Google Scholar, 5Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (207) Google Scholar, 6Feng X.-H. Derynck R. EMBO J. 1997; 16: 3912-3923Crossref PubMed Scopus (160) Google Scholar). However, our data that ALK5 can directly Smad1 the constitutively active ALK5 Smad1 phosphorylation in HEK293T cells and phosphorylation in and It is to that most of the studies their on specificity based on functional in cell as and cells. Thus, it is that a factor also be in the of Smad1 by In C2C12 cells, TGFβ-induced Smad1 phosphorylation be completely using on the protein of this our TGFβ-induced However, it be that is also phosphorylated by TGFβ signaling in the cells we and that it is by our Our that TGFβ-induced Smad1 phosphorylation a TGFβ-induced Smad3 phosphorylation in C2C12 and cells, and is significantly in completely with the data recently published for mouse mammary epithelial cells Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google Scholar). Our that a of TGFβ is to induce Smad1 phosphorylation Smad3 phosphorylation in C2C12 cells is also in with this for cells Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google Scholar). Thus, the in ALK for the TGFβ-induced Smad1 phosphorylation between C2C12 and cells, the to be There are several to the TGFβ-induced Smad1 phosphorylation that we TGFβ-induced a role usually activated by BMP is to in with the of the the usually TGFβ-induced a role usually activated by TGFβ sequences is to a of TGFβ and BMP in response to in several different cell were to a canonical gene that to TGFβ not This data showing that TGFβ-induced phosphorylation of Smad1/5 transcription via in epithelial cells Hill C.S. Mol. Cell. Biol. 2008; 28: PubMed Scopus Google Scholar). Additionally, we were to TGFβ-induced in Smad3 cells by of Smad1 not that TGFβ-induced not for in this However, we that of Smad1 significantly the of TGFβ-induced transcription from the in C2C12 cells, Smad1 a role in TGFβ-induced transcription in addition to Smad2/3 It that involvement of Smad1 in TGFβ-induced transcription be to cell lines that are to induce Smad1 phosphorylation in and BMP manner. In we provide firm evidence that TGFβ can Smad1 independently of BMP type I receptor activity in cells. studies are likely to TGFβ-induced Smad1 phosphorylation can occur in cell and this has a as with the TGFβ-induced Smad1 phosphorylation dependent on for ten Dijke for and and Wang for and and and for