Phosphorylation of Smad7 at Ser-249 does not interfere with its inhibitory role in transforming growth factor-beta-dependent signaling but affects Smad7-dependent transcriptional activation.
Smad proteins are major components in the intracellular signaling pathway of transforming growth factor-beta (TGF-beta), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF-beta-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-beta1. Abrogation of the phosphorylation by substitution of Ser-249 with alanine or aspartic acid residues did not affect the ability of Smad7 to inhibit TGF-beta1 and BMP7 signaling. No differences were found in the stability or in the intracellular distribution of Smad7 mutants compared with the wild-type molecule. However, Smad7 fused to the DNA-binding domain of GAL4 induced transcription from a reporter with mutated TATA minimal promoter in a Ser-249-dependent manner. Moreover, a reporter with the SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was also dependent on Ser-249 phosphorylation. The amplitude of effects on transcriptional regulation was dependent on cell type. Our results suggest that phosphorylation of Smad7, unlike phosphorylation of the receptor-regulated Smads, does not regulate TGF-beta signaling but rather affects TGF-beta-independent effects of Smad7 on transcriptional regulation.
Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden.
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Pulaski
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Landström
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Heldin
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Souchelnytskyi
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017930:Genes, Reporter; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D051906:Smad7 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D008024:Ligands; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D001224:Aspartic Acid; D012694:Serine; D004247:DNA; D008156:Luciferases