Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9.
We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xalpha, and -xbeta as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.
Hattori
Takako
T
Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Eberspaecher
Heidi
H
Lu
Jingfang
J
Zhang
Ren
R
Nishida
Tamotsu
T
Kahyo
Tomoaki
T
Yasuda
Hideyo
H
de Crombrugghe
Benoit
B
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D024043:Collagen Type II / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006609:High Mobility Group Proteins / Q000096:biosynthesis*; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism*; D051381:Rats; D055758:SOX9 Transcription Factor; D025842:SUMO-1 Protein / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis*; D014162:Transfection
C499177:COL2A1 protein, human; D024043:Collagen Type II; D018076:DNA, Complementary; D006609:High Mobility Group Proteins; D050828:Protein Inhibitors of Activated STAT; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D025842:SUMO-1 Protein; D014157:Transcription Factors