Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling

Xing Ming Shi, Xiangli Yang, Di Chen, Zhijie Chang, Xu Cao

Research output: Contribution to journalArticle

146 Citations (Scopus)

Abstract

Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a 'pull-down' assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.

Original languageEnglish (US)
Pages (from-to)13711-13717
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number19
DOIs
StatePublished - May 7 1999
Externally publishedYes

Fingerprint

Homeodomain Proteins
Bone Morphogenetic Proteins
DNA-Binding Proteins
Osteopontin
Assays
Genes
Bone Morphogenetic Protein Receptors
Phosphorylation
COS Cells
Transcription
Reporter Genes
Immunoprecipitation
Gene expression
Yeast
Transfection
Transcription Factors
Yeasts
Gels
Binding Sites
Gene Expression

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling. / Shi, Xing Ming; Yang, Xiangli; Chen, Di; Chang, Zhijie; Cao, Xu.

In: Journal of Biological Chemistry, Vol. 274, No. 19, 07.05.1999, p. 13711-13717.

Research output: Contribution to journalArticle

Shi, Xing Ming ; Yang, Xiangli ; Chen, Di ; Chang, Zhijie ; Cao, Xu. / Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 19. pp. 13711-13717.
@article{0c8d7f38e3614b39955a38397af00b55,
title = "Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling",
abstract = "Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a 'pull-down' assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.",
author = "Shi, {Xing Ming} and Xiangli Yang and Di Chen and Zhijie Chang and Xu Cao",
year = "1999",
month = "5",
day = "7",
doi = "10.1074/jbc.274.19.13711",
language = "English (US)",
volume = "274",
pages = "13711--13717",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

TY - JOUR

T1 - Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling

AU - Shi, Xing Ming

AU - Yang, Xiangli

AU - Chen, Di

AU - Chang, Zhijie

AU - Cao, Xu

PY - 1999/5/7

Y1 - 1999/5/7

N2 - Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a 'pull-down' assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.

AB - Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a 'pull-down' assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.

UR - http://www.scopus.com/inward/record.url?scp=0033532057&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033532057&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.19.13711

DO - 10.1074/jbc.274.19.13711

M3 - Article

VL - 274

SP - 13711

EP - 13717

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -