Modulation of Smad2-mediated Signaling by Extracellular Signal-regulated Kinase

Abstract

Components of the transforming growth factor-β and mitogen-activated protein kinase pathways interact in controlling cell growth and differentiation. We show that phosphorylation of Smad2, a mediator of the activin/transforming growth factor-β signal, by activated extracellular signal-regulated kinase 1 (ERK1) increases the amount of Smad2 protein and leads to enhanced transcriptional activity. Epidermal growth factor increased phosphorylation of Smad2 in COS7 cells, and Smad2-dependent transcription in a mink lung epithelial cell line, L17, was enhanced by co-transfection of a constitutively active MEK1. In addition, transfection of Smad2 mutants lacking ERK sites resulted in reduced transcription, whereas mutants that mimicked ERK phosphorylation stimulated transcription. The amount of Smad2 protein was increased by transfection with a constitutively active MEK1 and reduced by co-transfection with the ERK phosphatase, HVH2. The elevation of Smad2 protein levels was because of increased half-life and resulted in increased complex formation with Smad4. A site of ERK-dependent phosphorylation on Smad2 was located to Thr8, a site that overlaps with the calmodulin binding region. We show that calmodulin inhibits Smad2 phosphorylation by ERK1, and overexpressing calmodulin, or stimulating calmodulin activity with ionomycin, reduces Smad2 levels. These findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that negative regulation of Smad2 signaling by calmodulin is achieved in part by inhibiting this phosphorylation. Components of the transforming growth factor-β and mitogen-activated protein kinase pathways interact in controlling cell growth and differentiation. We show that phosphorylation of Smad2, a mediator of the activin/transforming growth factor-β signal, by activated extracellular signal-regulated kinase 1 (ERK1) increases the amount of Smad2 protein and leads to enhanced transcriptional activity. Epidermal growth factor increased phosphorylation of Smad2 in COS7 cells, and Smad2-dependent transcription in a mink lung epithelial cell line, L17, was enhanced by co-transfection of a constitutively active MEK1. In addition, transfection of Smad2 mutants lacking ERK sites resulted in reduced transcription, whereas mutants that mimicked ERK phosphorylation stimulated transcription. The amount of Smad2 protein was increased by transfection with a constitutively active MEK1 and reduced by co-transfection with the ERK phosphatase, HVH2. The elevation of Smad2 protein levels was because of increased half-life and resulted in increased complex formation with Smad4. A site of ERK-dependent phosphorylation on Smad2 was located to Thr8, a site that overlaps with the calmodulin binding region. We show that calmodulin inhibits Smad2 phosphorylation by ERK1, and overexpressing calmodulin, or stimulating calmodulin activity with ionomycin, reduces Smad2 levels. These findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that negative regulation of Smad2 signaling by calmodulin is achieved in part by inhibiting this phosphorylation. transforming growth factor-β c-Jun N-terminal kinase epidermal growth factor mitogen-activated protein extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase glutathioneS- transferase hemagglutinin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine l-1-tosylamido-2-phenylethyl chloromethyl ketone high pressure liquid chromatography Activin and transforming growth factor-β (TGF-β)1 are structurally related, multipotent growth and differentiation factors. These peptides are not only among the most potent known cellular growth inhibitors but also regulate other diverse biological processes including early embryonic patterning and cell fate determination (1Zimmerman C.M. Mathews L.S. Muttukrishna S. Ledger W. Inhibins, Activins, Follistatin in Human Reproductive Physiology. Imperial College Press, London2001: 239-278Google Scholar). Signaling by these proteins is initiated by ligand-induced hetero-oligomerization of type I and type II receptor serine kinases. Transphosphorylation of a type I receptor serine kinase, ALK4 for activin and ALK5 for TGF-β, by the type II receptor serine kinase activates the type I receptor, which then phosphorylates the C-terminal Ser residues of Smad2 and -3. At present, no difference has been established between the intracellular signaling pathways of activin and TGF-β. Once phosphorylated, Smad2 and -3 form heteromeric complexes with Smad4, followed by translocation of the complexes into the nucleus. There they modulate target gene transcription either by associating with various DNA binding partners or by inducing ubiquitin-mediated protein degradation (2Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3315) Google Scholar, 3Zhang Y. Derynck R. Trends Cell Biol. 1999; 9: 274-279Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 4Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 5Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar).In addition to Smad activation by activin/TGF-β-dependent phosphorylation (6Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar), other interactions are also likely to regulate Smad signaling. We have revealed previously that calmodulin binds with Smad2 and acts as a Smad modulator (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Scherer and Graff Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have also that calmodulin and extracellular signal-regulated kinase interact in on Smad signaling between the pathway and the kinase pathway is known in various biological a negative by whereas a constitutively mimicked the inducing activity M. Nature. PubMed Scopus Google Scholar). In addition, of epithelial in a M. J. Genes Dev. PubMed Scopus Google Scholar), and signaling by c-Jun N-terminal kinase was for in J. 1999; PubMed Google Scholar). has also been to the growth of Google Scholar). a protein that inhibits the pathway Y. Genes Dev. 2000; 14: Google Scholar), was for in K. J. PubMed Scopus Google was to a for the regulation of Smad signaling by these other intracellular signaling that activated ERK phosphorylates Smad2 and that this in leads to enhanced transcription of a In addition, have located of the ERK phosphorylation sites on site the calmodulin binding and have been to the of calmodulin in of Smad2 findings have revealed that Smad2 is by and that this in enhanced transcription of DNA have also that the enhanced signaling is because of increased levels of Smad2 as a of of the and that this in leads to increased formation of active signaling complexes with Smad4. These suggest that is between the and ERK signaling pathways the of Smad2 activation and that phosphorylation of Smad2 by activated and receptor complexes is for transcriptional have between the and kinase signaling but no has with to the of these activation of kinase cell growth and K. J. PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), whereas signaling and kinase pathways M. Nature. PubMed Scopus Google Scholar, M. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google and in J. 1999; PubMed Google Scholar). In addition, and in and of epithelial M. J. Genes Dev. PubMed Scopus Google Scholar). These suggest that the between signaling and kinase pathway on cellular In the of to growth was not by activation of ERK but the of was in K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar). A for the of ERK on Smad2 signaling revealed in this the between signaling and the kinase pathway and of epithelial is also with the of M. Cell Biol. PubMed Scopus Google that and Smad2 signaling are for epithelial to and that is by elevation of and Smad2 with P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), that Smad2 was in to in revealed that activated Smad2 but not Smad4, and a that by receptor complexes M. Mathews L.S. 2000; PubMed Scopus Google Scholar, M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have also that transfection of constitutively active kinase a of the Smad2 phosphorylation the C-terminal Smad2 phosphorylation by and the these suggest that Smad2 phosphorylation is not only by for activin and but also by the kinase on the of ERK phosphorylation on Smad2-dependent transcription are with findings by P. Genes Dev. 1998; 12: PubMed Scopus Google that activation of receptor by or growth factor activated Smad2-dependent gene In addition, activated kinase 1 enhanced Smad2-dependent gene transcription J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and co-transfection of with and increased of a gene J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google complexes of and S. S. ten Dijke P. S. J. 1998; PubMed Scopus Google Scholar). activin and early in M. Google Scholar, M. Google Scholar). the kinase is with a complex and of and R. 1998; PubMed Scopus Google Scholar), which of the kinase Smad2 in suggest that phosphorylation of Smad2 by activated is for transcription of Smad2-dependent residues and the of Smad2 are in to S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and a Smad2 with for these has been to with receptor complexes and transcription of a was as that the protein in a negative M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar). in and Smad2 transcriptional activity with as that the not have a negative is the M. J. J. Genes Dev. 1999; PubMed Scopus Google have that mutants with Ser or ERK sites in the to the residues in increased of in was to suggest that phosphorylation of by the pathway inhibits a that with as as of P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). M. J. J. Genes Dev. 1999; PubMed Scopus Google the of or growth factor on transcription of a gene P. Genes Dev. 1998; 12: PubMed Scopus Google as to a of transcription by the the has that in Smad2 that either or phosphorylation and transcription, between Smad2 and for the the proteins are and are to has been to transcription of a gene Wrana J.L. L. Mol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is also that between the cell in the for the suggest that Smad2 that has been by has transcriptional activity because has and a of complexes with Smad4. The in complex formation is a because a Smad2 phosphorylation by activated not the of the protein for Smad4. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have that of the pathway the in is known that activation of the kinase pathway protein the half-life of protein increased in to of the pathway R. J. Mol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and phosphorylation of by ERK S. J. Biol. Chem. Full Text PDF PubMed Google was for the of R. Y. K. Genes Dev. 2000; 14: PubMed Scopus Google Scholar).In that calmodulin with Smad2 in a and that the N-terminal of Smad2 is the calmodulin binding (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The show that phosphorylation of Smad2 by is by with Scherer and Graff Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have also that binding of calmodulin to Smad2 inhibits phosphorylation of is that binding of calmodulin the of Smad2 that is the phosphorylation that of calmodulin gene transcription and that of calmodulin activity stimulated transcription (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). also Smad2-dependent in Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). S. Mol. Biol. 2000; PubMed Scopus Google have that of transcriptional activation phosphorylation of Smad2 by kinase II and of In the of calmodulin, or the of Smad2 protein in the of signaling by increased calmodulin activity because of reduced phosphorylation of Smad2 the ERK pathway and by which calmodulin regulates the for regulation of Smad2 signaling by the ERK pathway and by calmodulin findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that calmodulin regulates Smad2 activation by inhibiting this phosphorylation. The of Smad2 a mediator for activin and TGF-β, by these other intracellular signaling pathways of the diverse biological of activin and in and Activin and transforming growth factor-β (TGF-β)1 are structurally related, multipotent growth and differentiation factors. These peptides are not only among the most potent known cellular growth inhibitors but also regulate other diverse biological processes including early embryonic patterning and cell fate determination (1Zimmerman C.M. Mathews L.S. Muttukrishna S. Ledger W. Inhibins, Activins, Follistatin in Human Reproductive Physiology. Imperial College Press, London2001: 239-278Google Scholar). Signaling by these proteins is initiated by ligand-induced hetero-oligomerization of type I and type II receptor serine kinases. Transphosphorylation of a type I receptor serine kinase, ALK4 for activin and ALK5 for TGF-β, by the type II receptor serine kinase activates the type I receptor, which then phosphorylates the C-terminal Ser residues of Smad2 and -3. At present, no difference has been established between the intracellular signaling pathways of activin and TGF-β. Once phosphorylated, Smad2 and -3 form heteromeric complexes with Smad4, followed by translocation of the complexes into the nucleus. There they modulate target gene transcription either by associating with various DNA binding partners or by inducing ubiquitin-mediated protein degradation (2Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3315) Google Scholar, 3Zhang Y. Derynck R. Trends Cell Biol. 1999; 9: 274-279Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 4Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 5Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar). In addition to Smad activation by activin/TGF-β-dependent phosphorylation (6Funaba M. Mathews L.S. Mol. Endocrinol. 2000; 14: 1583-1591Crossref PubMed Google Scholar), other interactions are also likely to regulate Smad signaling. We have revealed previously that calmodulin binds with Smad2 and acts as a Smad modulator (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Scherer and Graff Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have also that calmodulin and extracellular signal-regulated kinase interact in on Smad signaling between the pathway and the kinase pathway is known in various biological a negative by whereas a constitutively mimicked the inducing activity M. Nature. PubMed Scopus Google Scholar). In addition, of epithelial in a M. J. Genes Dev. PubMed Scopus Google Scholar), and signaling by c-Jun N-terminal kinase was for in J. 1999; PubMed Google Scholar). has also been to the growth of Google Scholar). a protein that inhibits the pathway Y. Genes Dev. 2000; 14: Google Scholar), was for in K. J. PubMed Scopus Google Scholar). The was to a for the regulation of Smad signaling by these other intracellular signaling that activated ERK phosphorylates Smad2 and that this in leads to enhanced transcription of a In addition, have located of the ERK phosphorylation sites on site the calmodulin binding and have been to the of calmodulin in of Smad2 findings have revealed that Smad2 is by and that this in enhanced transcription of DNA have also that the enhanced signaling is because of increased levels of Smad2 as a of of the and that this in leads to increased formation of active signaling complexes with Smad4. These suggest that is between the and ERK signaling pathways the of Smad2 activation and that phosphorylation of Smad2 by activated and receptor complexes is for transcriptional have between the and kinase signaling but no has with to the of these activation of kinase cell growth and K. J. PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), whereas signaling and kinase pathways M. Nature. PubMed Scopus Google Scholar, M. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google and in J. 1999; PubMed Google Scholar). In addition, and in and of epithelial M. J. Genes Dev. PubMed Scopus Google Scholar). These suggest that the between signaling and kinase pathway on cellular In the of to growth was not by activation of ERK but the of was in K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar). A for the of ERK on Smad2 signaling revealed in this the between signaling and the kinase pathway and of epithelial is also with the of M. Cell Biol. PubMed Scopus Google that and Smad2 signaling are for epithelial to and that is by elevation of and Smad2 with P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), that Smad2 was in to in revealed that activated Smad2 but not Smad4, and a that by receptor complexes M. Mathews L.S. 2000; PubMed Scopus Google Scholar, M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have also that transfection of constitutively active kinase a of the Smad2 phosphorylation the C-terminal Smad2 phosphorylation by and the these suggest that Smad2 phosphorylation is not only by for activin and but also by the kinase on the of ERK phosphorylation on Smad2-dependent transcription are with findings by P. Genes Dev. 1998; 12: PubMed Scopus Google that activation of receptor by or growth factor activated Smad2-dependent gene In addition, activated kinase 1 enhanced Smad2-dependent gene transcription J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and co-transfection of with and increased of a gene J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google complexes of and S. S. ten Dijke P. S. J. 1998; PubMed Scopus Google Scholar). activin and early in M. Google Scholar, M. Google Scholar). the kinase is with a complex and of and R. 1998; PubMed Scopus Google Scholar), which of the kinase Smad2 in suggest that phosphorylation of Smad2 by activated is for transcription of Smad2-dependent residues and the of Smad2 are in to S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and a Smad2 with for these has been to with receptor complexes and transcription of a was as that the protein in a negative M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar). in and Smad2 transcriptional activity with as that the not have a negative is the M. J. J. Genes Dev. 1999; PubMed Scopus Google have that mutants with Ser or ERK sites in the to the residues in increased of in was to suggest that phosphorylation of by the pathway inhibits a that with as as of P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). M. J. J. Genes Dev. 1999; PubMed Scopus Google the of or growth factor on transcription of a gene P. Genes Dev. 1998; 12: PubMed Scopus Google as to a of transcription by the the has that in Smad2 that either or phosphorylation and transcription, between Smad2 and for the the proteins are and are to has been to transcription of a gene Wrana J.L. L. Mol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is also that between the cell in the for the suggest that Smad2 that has been by has transcriptional activity because has and a of complexes with Smad4. The in complex formation is a because a Smad2 phosphorylation by activated not the of the protein for Smad4. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have that of the pathway the in is known that activation of the kinase pathway protein the half-life of protein increased in to of the pathway R. J. Mol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and phosphorylation of by ERK S. J. Biol. Chem. Full Text PDF PubMed Google was for the of R. Y. K. Genes Dev. 2000; 14: PubMed Scopus Google Scholar).In that calmodulin with Smad2 in a and that the N-terminal of Smad2 is the calmodulin binding (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The show that phosphorylation of Smad2 by is by with Scherer and Graff Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have also that binding of calmodulin to Smad2 inhibits phosphorylation of is that binding of calmodulin the of Smad2 that is the phosphorylation that of calmodulin gene transcription and that of calmodulin activity stimulated transcription (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). also Smad2-dependent in Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). S. Mol. Biol. 2000; PubMed Scopus Google have that of transcriptional activation phosphorylation of Smad2 by kinase II and of In the of calmodulin, or the of Smad2 protein in the of signaling by increased calmodulin activity because of reduced phosphorylation of Smad2 the ERK pathway and by which calmodulin regulates the for regulation of Smad2 signaling by the ERK pathway and by calmodulin findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that calmodulin regulates Smad2 activation by inhibiting this phosphorylation. The of Smad2 a mediator for activin and TGF-β, by these other intracellular signaling pathways of the diverse biological of activin and in and findings have revealed that Smad2 is by and that this in enhanced transcription of DNA have also that the enhanced signaling is because of increased levels of Smad2 as a of of the and that this in leads to increased formation of active signaling complexes with Smad4. These suggest that is between the and ERK signaling pathways the of Smad2 activation and that phosphorylation of Smad2 by activated and receptor complexes is for transcriptional activity. have between the and kinase signaling but no has with to the of these activation of kinase cell growth and K. J. PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), whereas signaling and kinase pathways M. Nature. PubMed Scopus Google Scholar, M. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google and in J. 1999; PubMed Google Scholar). In addition, and in and of epithelial M. J. Genes Dev. PubMed Scopus Google Scholar). These suggest that the between signaling and kinase pathway on cellular In the of to growth was not by activation of ERK but the of was in K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar). A for the of ERK on Smad2 signaling revealed in this the between signaling and the kinase pathway and of epithelial is also with the of M. Cell Biol. PubMed Scopus Google that and Smad2 signaling are for epithelial to and that is by elevation of and Smad2 In with P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, M. J. J. Genes Dev. 1999; PubMed Scopus Google Scholar), that Smad2 was in to in revealed that activated Smad2 but not Smad4, and a that by receptor complexes M. Mathews L.S. 2000; PubMed Scopus Google Scholar, M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have also that transfection of constitutively active kinase a of the Smad2 phosphorylation the C-terminal Smad2 phosphorylation by and the these suggest that Smad2 phosphorylation is not only by for activin and but also by the kinase on the of ERK phosphorylation on Smad2-dependent transcription are with findings by P. Genes Dev. 1998; 12: PubMed Scopus Google that activation of receptor by or growth factor activated Smad2-dependent gene In addition, activated kinase 1 enhanced Smad2-dependent gene transcription J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and co-transfection of with and increased of a gene J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google complexes of and S. S. ten Dijke P. S. J. 1998; PubMed Scopus Google Scholar). activin and early in M. Google Scholar, M. Google Scholar). the kinase is with a complex and of and R. 1998; PubMed Scopus Google Scholar), which of the kinase Smad2 in suggest that phosphorylation of Smad2 by activated is for transcription of Smad2-dependent Ser residues and the of Smad2 are in to S. M. L. Wrana J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. ten Dijke P. C.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and a Smad2 with for these has been to with receptor complexes and transcription of a was as that the protein in a negative M. S. R. L. Wrana J.L. Full Text Full Text PDF PubMed Scopus Google Scholar). in and Smad2 transcriptional activity with as that the not have a negative is the M. J. J. Genes Dev. 1999; PubMed Scopus Google have that mutants with Ser or ERK sites in the to the residues in increased of in was to suggest that phosphorylation of by the pathway inhibits a that with as as of P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, K. M. M. J. Genes Dev. 2000; 14: PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). M. J. J. Genes Dev. 1999; PubMed Scopus Google the of or growth factor on transcription of a gene P. Genes Dev. 1998; 12: PubMed Scopus Google as to a of transcription by the the has that in Smad2 that either or phosphorylation and transcription, between Smad2 and for the the proteins are and are to has been to transcription of a gene Wrana J.L. L. Mol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is also that between the cell in the for the suggest that Smad2 that has been by has transcriptional activity because has and a of complexes with Smad4. The in complex formation is a because a Smad2 phosphorylation by activated not the of the protein for Smad4. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google have that of the pathway the in is known that activation of the kinase pathway protein the half-life of protein increased in to of the pathway R. J. Mol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and phosphorylation of by ERK S. J. Biol. Chem. Full Text PDF PubMed Google was for the of R. Y. K. Genes Dev. 2000; 14: PubMed Scopus Google Scholar). In that calmodulin with Smad2 in a and that the N-terminal of Smad2 is the calmodulin binding (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The show that phosphorylation of Smad2 by is by with Scherer and Graff Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have also that binding of calmodulin to Smad2 inhibits phosphorylation of is that binding of calmodulin the of Smad2 that is the phosphorylation that of calmodulin gene transcription and that of calmodulin activity stimulated transcription (7Zimmerman C.M. Kariapper M.S. Mathews L.S. J. Biol. Chem. 1998; 273: 677-680Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). also Smad2-dependent in Graff J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). S. Mol. Biol. 2000; PubMed Scopus Google have that of transcriptional activation phosphorylation of Smad2 by kinase II and of In the of calmodulin, or the of Smad2 protein in the of signaling by increased calmodulin activity because of reduced phosphorylation of Smad2 the ERK pathway and by which calmodulin regulates signaling. The the for regulation of Smad2 signaling by the ERK pathway and by calmodulin findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that calmodulin regulates Smad2 activation by inhibiting this phosphorylation. The of Smad2 a mediator for activin and TGF-β, by these other intracellular signaling pathways of the diverse biological of activin and in and We and for and cell We are also to and for stimulating