Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling.
Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.
Department of Growth and Development, University of California, San Francisco, California 94143-0640, USA.
Pierre S W
PS
Lee
Chenbei
C
Chang
Dong
D
Liu
Rik
R
Derynck
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors; D010957:Plasmids / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011233:Precipitin Tests; D050828:Protein Inhibitors of Activated STAT; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009414:Nerve Growth Factors; C485891:PIAS4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT; D018075:RNA, Complementary; C497796:SMAD4 protein, human; D025842:SUMO-1 Protein; D051785:Smad Proteins; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D001120:Arginine; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine