Transcriptional activity of megakaryoblastic leukemia 1 (MKL1) is repressed by SUMO modification
Transcriptional activity of megakaryoblastic leukemia 1 (MKL1) is repressed by SUMO modification
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DOI：
10.1111/j.1365-2443.2005.00880.x
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发表时间：
2005-08-01
期刊：
影响因子：
2.1
通讯作者：
Kuzumaki, N
中科院分区：
文献类型：
--
作者：
Nakagawa, K;Kuzumaki, N
巨核细胞白血病1（MKL 1）最初被鉴定为在巨核细胞白血病中易位的基因。已显示MKL 1作为血清应答因子（SRF）的RhoA调节的转录共激活因子起作用。为了鉴定调节MKL 1功能的蛋白质，我们进行了酵母双杂交筛选，并分离了编码UBC 9的cDNA，UBC 9是小泛素相关修饰物-1（SUMO-1）的E2酶，作为MKL 1结合蛋白。GST pull-down实验发现UBC 9与MKL 1之间存在物理相互作用，并在293 T细胞和体外重组系统中用SUMO-1共价修饰MKL 1。MKL 1类小泛素化通过血清刺激或组成型活性形式RhoA的共表达而增强。突变分析表明499、576和624位赖氨酸残基是SUMO-1的主要受体位点。此外，报告基因分析表明，突变的三个sumoylation位点强烈增强MKL 1的转录活性。SUMO-1通过基因融合共价连接到MKL 1，以互补方式抑制MKL 1依赖性转录。最后，MKL 1类小泛素化位点的突变也增强了SRF依赖的转录，而不影响MKL 1-SRF相互作用。综合结果表明，MKL 1是sumoylated和这种修饰抑制MKL 1的转录活性。
Megakaryoblastic leukemia 1 (MKL1) was originally identified as a gene translocated in megakaryoblastic leukemia. It has been shown that MKL1 functions as a RhoA-regulated transcriptional coactivator of serum response factor (SRF). In order to identify a protein that regulates the function of MKL1, we performed yeast two-hybrid screening and isolated cDNA that encodes UBC9, an E2 enzyme of small ubiquitin-related modifier-1 (SUMO-1), as an MKL1-binding protein. UBC9 was found to physically interact with MKL1 by GST pull-down assay, and MKL1 was covalently modified with SUMO-1 in 293T cells and in vitro reconstitution system. MKL1 sumoylation is enhanced by either serum stimulation or co-expression of constitutively active form of RhoA. Mutational analysis showed that lysine residues at 499, 576, and 624 are the major acceptor sites for SUMO-1. In addition, reporter gene analysis revealed that mutation of the three sumoylation sites strongly enhances the transcriptional activity of MKL1. The covalent attachment of SUMO-1 to MKL1 by gene fusion represses MKL1-dependent transcription in a complementary manner. Finally, mutation of the sumoylation sites of MKL1 also enhances SRF-dependent transcription without affecting MKL1-SRF interaction. The combined results demonstrated that MKL1 is sumoylated and this modification represses transcriptional activity of MKL1.