The transcription factor Sox9 is degraded by the ubiquitin-proteasome system and stabilized by a mutation in a ubiquitin-target site.
Sox9 is a transcription factor that is critical for chondrogenesis, testis determination, and development of several other organs in vertebrates. Thus the levels of Sox9 protein and its activity may be tightly regulated. Here we show that inhibitors of the 26S proteasome increase both the levels of Sox9 protein and its transcriptional activity measured with Col2a1 promoter/enhancer construct in RCS cells and C3H10T1/2 cells. Indeed, in intact cells ubiquitination assays indicate that Sox9 is multiply ubiquitinated. The K398A mutation, which was introduced in a potential ubiquitin-binding site, increases the stability of Sox9 protein and its transcriptional activity of Col2a1, Col11a2, and AMH promoter/enhancer constructs without affecting the subcellular localization and the DNA binding efficiency of Sox9. Pulse-chase experiments show that the increased Sox9 levels resulting from treatment with the MG132 proteasome inhibitor or from the K398A mutation produce stabilization of the protein. Our in vitro studies indicate that the ubiquitin-proteasome proteolytic system degrades Sox9 and regulates its transcriptional activity.
Akiyama
Haruhiko
H
Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. hakiyama@mdacc.edu
Kamitani
Tetsu
T
Yang
Xiaohong
X
Kandyil
Roshini
R
Bridgewater
Laura C
LC
Fellous
Marc
M
Mori-Akiyama
Yuko
Y
de Crombrugghe
Benoit
B
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics; D004742:Enhancer Elements, Genetic; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008856:Microscopy, Fluorescence; D009154:Mutation; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D061988:Proteasome Inhibitors; D017434:Protein Structure, Tertiary; D051381:Rats; D055758:SOX9 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
C499177:COL2A1 protein, human; C447413:Col2a1 protein, mouse; D024043:Collagen Type II; D006609:High Mobility Group Proteins; D061988:Proteasome Inhibitors; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease