Hattori Takako, Eberspaecher Heidi, Lu Jingfang, Zhang Ren, Nishida Tamotsu, Kahyo Tomoaki, Yasuda Hideyo, de Crombrugghe Benoit
Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 2006 May 19;281(20):14417-28. doi: 10.1074/jbc.M511330200. Epub 2006 Mar 21.
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.
我们利用基于Gal4的酵母双杂交系统和源自软骨细胞系的cDNA文库，鉴定出PIAS1（活化STAT-1的蛋白抑制剂）、-3、-xalpha和-xbeta为与SOX9相关的多肽。在双杂交、共免疫沉淀和电泳迁移率变动分析中，这些PIAS蛋白被证明可直接与SOX9相互作用。在使用PIAS和SUMO-1（小泛素样修饰物-1）表达载体与COS-7细胞进行共转染实验时，SOX9发生了SUMO化修饰。在SUMO-1、SUMO激活酶和SUMO结合酶存在的情况下，PIAS蛋白在体外也能使SOX9发生SUMO化修饰。在COS-7细胞中，PIAS蛋白刺激了Col2a1启动子-增强子报告基因的SOX9依赖性转录活性。报告基因活性的这种增加与SOX9细胞水平的增加平行。与SUMO表达载体共转染进一步增强了COS-7细胞中这种SOX9依赖性Col2a1报告基因的转录活性，并且在存在SUMO-1突变体或PIAS RING结构域突变体时或通过去SUMO化酶的共表达，这种额外的激活受到抑制。对转染了SOX9的COS-7细胞进行免疫荧光显微镜观察显示，在存在PIAS1和SUMO-1的情况下，SOX9的核内分布变得更加弥散。我们的结果表明，通过控制SOX9的细胞浓度，PIAS蛋白和SUMO化修饰可能是SOX9功能主要调节系统的一部分。
