Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
J Biol Chem. 2010 Feb 26;285(9):6595-603. doi: 10.1074/jbc.M109.068502. Epub 2009 Dec 28.
The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.
过氧化物酶体增殖物激活受体 γ（PPARγ）被认为是脂肪细胞分化的主调控因子，促进成熟脂肪细胞中的葡萄糖和脂质代谢。我们最近在基于 RNA 干扰的筛选中发现了酵母 Sterile 20（Ste20）蛋白激酶同源物 Map4k4，它是培养的脂肪细胞中 PPARγ 表达的抑制剂。在这里，我们表明，Map4k4 的 RNA 干扰介导的沉默在 3T3-L1 脂肪细胞中升高了 PPARγ1 和 PPARγ2 蛋白的水平，而不影响 PPARγ mRNA 水平，这表明 Map4k4 在转录后水平调节 PPARγ。PPARγ 的降解速率非常快，如在存在环己酰亚胺的情况下测量（t(1/2) = 2 h），但沉默 Map4k4 对 PPARγ 的降解没有影响。然而，Map4k4 的耗竭显着增强了 [(35)S] 蛋氨酸/半胱氨酸掺入蛋白质中，表明 Map4k4 信号降低了蛋白质翻译。我们展示了 Map4k4 的一个功能是抑制雷帕霉素敏感的哺乳动物雷帕霉素靶蛋白（mTOR）活性，降低 4E-BP1 磷酸化。此外，我们的结果表明，mTOR 和 4E-BP1 是 Map4k4 敲低后 PPARγ 蛋白表达增加所必需的。与这一概念一致，腺病毒介导的 Map4k4 表达降低了 PPARγ 蛋白水平和 mTOR 磷酸化。这些数据表明，Map4k4 通过减弱 mTOR 信号和 4E-BP1 依赖性机制，负向调节 PPARγ 的转录后表达。
