Alm-Kristiansen Anne Hege, Norman Ingrid Louise, Matre Vilborg, Gabrielsen Odd Stokke
Department of Molecular Biosciences, University of Oslo, Blindernveien 31, Blindern, Oslo, Norway.
Biochem Biophys Res Commun. 2009 Sep 25;387(3):494-9. doi: 10.1016/j.bbrc.2009.07.053. Epub 2009 Jul 16.
FLASH is a huge multifunctional nuclear protein that has been linked to apoptotic signalling, transcriptional control and Cajal body function. To gain further insight into the functions of the FLASH protein, we performed a yeast two-hybrid screening with FLASH as bait and identified the SUMO-conjugating enzyme Ubc9 as an interaction partner. The main interaction surface for Ubc9 was found in the C-terminal part of FLASH, which is also a target for sumoylation. We identified K1813 as the major sumoylation site in FLASH, being enhanced by the SUMO E3 ligases Pc2 and PIASy. Disruption of this SUMO-conjugation site did not change the speckled subnuclear localization of FLASH, but it caused a reduction in FLASH activity as measured in a Gal4-tethering assay. Interestingly, the SUMO-specific protease SENP1 activated FLASH in the same assay. Overall, our results point to a complex involvement of sumoylation in modulating the function of FLASH.
FLASH是一种巨大的多功能核蛋白，它与凋亡信号传导、转录调控以及 Cajal 体功能相关。为了进一步深入了解 FLASH 蛋白的功能，我们以 FLASH 为诱饵进行了酵母双杂交筛选，并鉴定出 SUMO 缀合酶 Ubc9 为相互作用伴侣。发现 Ubc9 的主要相互作用表面位于 FLASH 的 C 末端部分，该部分也是 SUMO 化的靶点。我们确定 K1813 是 FLASH 中的主要 SUMO 化位点，SUMO E3 连接酶 Pc2 和 PIASy 可增强该位点的 SUMO 化。破坏这个 SUMO 缀合位点并没有改变 FLASH 在核内的斑点状定位，但在 Gal4 系留试验中测量时，它导致了 FLASH 活性的降低。有趣的是，在相同试验中，SUMO 特异性蛋白酶 SENP1 激活了 FLASH。总体而言，我们的结果表明 SUMO 化在调节 FLASH 功能中存在复杂的参与情况。
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