Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9
Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9
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DOI：
10.1074/jbc.m511330200
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发表时间：
2006-05-19
影响因子：
4.8
通讯作者：
de Crombrugghe, Benoit
中科院分区：
文献类型：
--
作者：
Hattori, Takako;Eberspaecher, Heidi;de Crombrugghe, Benoit
我们已经确定了PIAS 1（激活STAT-1的蛋白质抑制剂），-3，-x α，和-x β作为SOX 9相关的多肽使用Gal 4为基础的酵母双杂交系统和cDNA文库来自软骨细胞系。这些皮亚斯蛋白质被证明直接相互作用与SOX 9在双杂交，共免疫沉淀，和电泳迁移率变动测定。在使用皮亚斯和SUMO-1（小泛素样修饰物-1）表达载体与COS-7细胞的共转染实验中，SOX 9被SUMO化。在SUMO-1、SUMO激活酶和SUMO结合酶存在下，SOX 9也在体外被皮亚斯蛋白类小泛素化。在COS-7细胞中，皮亚斯蛋白刺激SOX 9依赖的Col 2a 1启动子-增强子报告基因的转录活性。这种报告活性的增加被SOX 9的细胞水平的增加所抵消。与SUMO表达载体的共转染进一步增强了这种SOX 9依赖性Col 2a 1报告基因在COS-7细胞中的转录活性，并且这种额外的激活在SUMO-1突变体或皮亚斯RING结构域突变体的存在下或通过去小泛素化酶的共表达而被抑制。免疫荧光显微镜观察显示，在PIAS 1和SUMO-1的存在下，SOX 9在COS-7细胞核内的分布更加弥散。我们的研究结果表明，通过控制SOX 9的细胞浓度，皮亚斯蛋白和类小泛素化可能是SOX 9功能的主要调节系统的一部分。
We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -x alpha, and -x beta 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-7cells 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.