Shimizu Kouhei, Fukushima Hidefumi, Ogura Kohei, Lien Evan C, Nihira Naoe Taira, Zhang Jinfang, North Brian J, Guo Ailan, Nagashima Katsuyuki, Nakagawa Tadashi, Hoshikawa Seira, Watahiki Asami, Okabe Koji, Yamada Aya, Toker Alex, Asara John M, Fukumoto Satoshi, Nakayama Keiichi I, Nakayama Keiko, Inuzuka Hiroyuki, Wei Wenyi
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
Sci Signal. 2017 Jan 3;10(460):eaah4117. doi: 10.1126/scisignal.aah4117.
The SCF E3 ubiquitin ligase complex plays pivotal roles in normal cellular physiology and in pathophysiological conditions. Identification of β-transducin repeat-containing protein (β-TRCP) substrates is therefore critical to understand SCF biology and function. We used a β-TRCP-phosphodegron motif-specific antibody in a β-TRCP substrate screen coupled with tandem mass spectrometry and identified multiple β-TRCP substrates. One of these substrates was Lipin1, an enzyme and suppressor of the family of sterol regulatory element-binding protein (SREBP) transcription factors, which activate genes encoding lipogenic factors. We showed that SCF specifically interacted with and promoted the polyubiquitination of Lipin1 in a manner that required phosphorylation of Lipin1 by mechanistic target of rapamycin 1 (mTORC1) and casein kinase I (CKI). β-TRCP depletion in HepG2 hepatocellular carcinoma cells resulted in increased Lipin1 protein abundance, suppression of SREBP-dependent gene expression, and attenuation of triglyceride synthesis. Moreover, β-TRCP1 knockout mice showed increased Lipin1 protein abundance and were protected from hepatic steatosis induced by a high-fat diet. Together, these data reveal a critical physiological function of β-TRCP in regulating hepatic lipid metabolic homeostasis in part through modulating Lipin1 stability.
SCF E3泛素连接酶复合物在正常细胞生理学和病理生理条件中发挥着关键作用。因此，鉴定含β-转导蛋白重复序列的蛋白（β-TRCP）底物对于理解SCF的生物学特性和功能至关重要。我们在β-TRCP底物筛选中使用了一种β-TRCP磷酸降解基序特异性抗体，并结合串联质谱法，鉴定出多个β-TRCP底物。其中一个底物是Lipin1，它是一种酶，也是固醇调节元件结合蛋白（SREBP）转录因子家族的抑制因子，这些转录因子可激活编码脂肪生成因子的基因。我们发现，SCF以一种需要雷帕霉素1（mTORC1）和酪蛋白激酶I（CKI）对Lipin1进行磷酸化的方式，特异性地与Lipin1相互作用并促进其多聚泛素化。在HepG2肝癌细胞中敲低β-TRCP会导致Lipin1蛋白丰度增加、SREBP依赖性基因表达受到抑制以及甘油三酯合成减弱。此外，β-TRCP1基因敲除小鼠的Lipin1蛋白丰度增加，并且免受高脂饮食诱导的肝脂肪变性。总之，这些数据揭示了β-TRCP在部分通过调节Lipin1稳定性来调控肝脏脂质代谢稳态方面的关键生理功能。
