Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution.
Smads mediate activin, transforming growth factor beta (TGFbeta), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGFbeta receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smad3 (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2-2E (Ser465, 467Glu) and Smad2-3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2-2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2-2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 +/- 66 nM for wild-type Smad2:Smad4 complexes and 79 +/- 18 nM for Smad2-2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2-2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.
Funaba
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M
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA. funaba@azabu-u.ac.jp
Mathews
L S
LS
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008168:Lung; D008907:Mink; D016296:Mutagenesis; D009154:Mutation; D009414:Nerve Growth Factors; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D009414:Nerve Growth Factors; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; C497785:Smad2 protein, Xenopus; D051901:Smad4 Protein; D015534:Trans-Activators; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D010768:Phosphoserine