A RING finger protein praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Needin family protein, Dlxin-1
A RING finger protein praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Needin family protein, Dlxin-1
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DOI：
10.1074/jbc.m109728200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Watanabe, K
中科院分区：
文献类型：
--
作者：
Sasaki, A;Masuda, Y;Watanabe, K
Msx 2和Dlx 5是同源结构域蛋白，在成骨细胞分化中起重要作用，并且其表达由骨形态发生蛋白诱导。最近，我们已经鉴定了一种新的蛋白质，Dlxin-1，其与这些同源结构域蛋白质结合并调节Dlx 5依赖性转录功能（增田，Y.，Sasaki，A.，涩谷，H.，Ueno，N.，Ikeda，K.，Watanabe，K.（2001）J.Biol.Chem.276，5331-5338）。为了阐明Dlxin-1的分子功能，利用Dlxin-1的C-末端necdin同源结构域作为诱饵，通过酵母双杂交筛选，分离了两个密切相关的RING指蛋白Prajal和Neurodap-1。Dlxin-1和Prajal共转染后的Glucose S-转移酶下拉和免疫沉淀/Western印迹分析显示Prajal分别在体外和体内与Dlxin-1的C-末端necdin同源结构域结合。Prajal的过表达引起Dlxin-1蛋白水平的降低，当加入蛋白酶体抑制剂时，这种降低被逆转。在环指中突变的Prajal的过表达抑制了Dlxin-1蛋白的减少，指出了与环指相关的泛素蛋白异肽连接酶（E3）活性的重要性。野生型Prajal，而不是它的环指突变体，促进Dlxin-1在体内的泛素化。最后，Prajal的表达在GAL 4依赖性测定中下调Dlx 5依赖性转录活性。这些结果表明，Prajal通过泛素依赖性降解途径控制Dlxin-1的稳定性来调节同源结构域蛋白Dlx 5的转录功能。
Msx2 and Dlx5 are homeodomain proteins that play an important role in osteoblast differentiation and whose expression is induced by bone morphogenetic proteins. Recently we have identified a novel protein, Dlxin-1, that associates with these homeodomain proteins and regulates Dlx5-dependent transcriptional function (Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., and Watanabe, K. (2001) J. Biol. Chem. 276, 5331-5338). In an attempt to elucidate the molecular function of Dlxin-1, two closely related RING finger proteins, Prajal and Neurodap-1, were isolated by yeast two-hybrid screening using the C-terminal necdin homology domain of Dlxin-1 as bait. Glutathione S-transferase pull-down and immunoprecipitation/Western blotting assays following co-transfection of Dlxin-1 and Prajal revealed that Prajal binds to the C-terminal necdin homology domain of Dlxin-1 in vitro and in vivo, respectively. Overexpression of Prajal caused a decrease in Dlxin-1 protein level, which was reversed when a proteasome inhibitor was added. Overexpression of Prajal with a mutation in the RING finger inhibited the decrease in Dlxin-1 protein, pointing to the importance of ubiquitin-protein isopeptide ligase (E3) activity associated with RING finger. Wild-type Prajal, but not its RING finger mutant, promoted ubiquitination of Dlxin-1 in vivo. Finally, expression of Prajal down-regulated Dlx5-dependent transcriptional activity in a GAL4-dependent assay. These results suggest that Prajal regulates the transcription function of the homeodomain protein Dlx5 by controlling the stability of Dlxin-1 via an ubiquitin-dependent degradation pathway.