Drosophila protein phosphatase V regulates lipid homeostasis via the AMPK pathway.-猫眼课题宝

果蝇蛋白磷酸酶 V 通过 AMPK 途径调节脂质稳态。

DOI：

10.1093/jmcb/mjt050

发表时间：

2014

期刊：

J Mol Cell Biol

影响因子：

--

通讯作者：

中科院分区：

其他

文献类型：

--

作者：

Yin Dingzi;Huang Ping;Wu Jiarui;Song Haiyun

亲爱的编辑，蛋白质磷酸化对多种细胞过程至关重要。这种翻译后修饰由激酶介导的磷酸化和磷酸酶介导的去磷酸化调节。果蝇蛋白磷酸酶V （PpV）和酿酒酵母Sit4是哺乳动物PP6的同源物，属于丝氨酸/苏氨酸磷酸酶PP2A亚家族（Mann et al., 1993; Wang et al., 2012）。除了催化亚基，PP6全酶还含有一个调控亚基（人类中的PP6R1、PP6R2或PP6R3；酵母中的Sap4、Sap155、Sap185或Sap190；苍蝇中的CG10289）和一个支架亚基（Morales-Johansson等，2009）。对酿酒酵母的研究表明，Sit4敲除菌株在一个进化保守的激活位点降低了脂滴含量，增加了SNF1（哺乳动物和果蝇AMPK的同源物）的磷酸化（Bozaquel-Morais et al., 2010; Ruiz et al., 2011）。然而，其在后生动物中的作用尚未得到很好的探讨。为了研究PpV的生理作用，我们利用转基因RNAi蝇（PpVR-IR或PpVC-IR）普遍敲低PpV的调控或催化亚基。实时荧光定量PCR （qRT-PCR）分析显示，两种基因在幼虫中的敲除效率分别为60%和75%（数据未显示）。PpVR基因敲除后，幼虫体大小变化不明显，但在蛹期具有致死性。相反，敲低PpVc会导致体型和幼虫致死率严重降低（图1A）。甘油三酯（TAG）是果蝇幼虫营养储存的主要形式。敲除PpVc，而不敲除PpVR，导致这些幼虫中TAG水平显著降低（图1B）。AMPK通路抑制脂肪细胞分化和脂肪生成，同时促进脂质动员，在脂质分解代谢中发挥关键作用（Viollet and Andreelli, 2011; Hardie et al., 2012）。已有研究表明，Sit4参与酵母中SNF1/AMPK活性的调控（Bozaquel-Morais等，2010；Ruiz等，2011）。我们用磷酸化特异性抗体检测了在保守的Thr-172位点（代表AMPK的活性形式）磷酸化的AMPK水平。PpVR或PpVc的普遍敲低导致幼虫AMPK磷酸化水平明显升高（图1C）。PpVc RNAi表现出更强的作用，与更严重的表型一致。由于缺乏识别果蝇AMPK的抗体，我们无法监测AMPK的总水平。然而，PpVR或PpVc敲低均未导致AMPK mRNA水平升高（Supplementary Figure S1A），这表明AMPK磷酸化水平升高并非由于AMPK表达上调所致。果蝇脂肪体在功能上相当于哺乳动物的白色脂肪组织和肝脏，是TAG的主要储存部位。它还与其他器官沟通，调节整个身体的生长（Rajan和Perrimon， 2011）。当我们用cg-Gal4特异性敲除脂肪体中的PpVC或PpVR后，果蝇可以存活到成年，体型减小（图1D），体重减轻（补充图S1B）。同样在幼虫期，我们观察到通过敲低PpVC或PpVR，幼虫TAG水平降低（图1E），脂肪体AMPK磷酸化水平升高（图1F）。为了研究这些影响是否依赖于AMPK激活的增加，我们在脂肪体中同时表达了PpVC或PpVR的dsRNA，同时表达了靶向AMPK催化亚基（AMPK- ir）的转基因RNAi，这并没有减弱PpVC或PpVR的敲低效率（Supplementary Figure S1C）。敲低AMPK显著挽救了PpVC或PpVR敲低导致的体型（图1D）、体重（补充图S1B）和TAG水平（图1E）的下降，提示PpV通过AMPK途径调节果蝇脂质代谢和生长。我们进一步研究了PpV如何在细胞水平上影响脂肪体。我们在脂肪体中以克隆模式表达了抗PpVC或PpVR的dsRNA。与周围野生型细胞相比，敲低PpVC或PpVR的细胞细胞大小严重缩小（图1G和补充图S2A 2c）。我们也用PpVR的P元素插入等位基因（CG10289）诱导PpVR敲低克隆，得到了类似的结果（图1H），排除了RNAi观察到的表型是脱靶效应的可能性。与在全身观察到的结果相似，AMPK的同时敲低在很大程度上通过敲低PpV挽救了细胞大小的减少（图1G和补充图S2D 2f）。由于AMPK通路和mTOR通路相互拮抗以调节能量稳态和细胞生长（Inoki等，2012），我们测试了mTOR下游效应的组成活性S6K （Barcelo和Stewart， 2002）的表达是否可以挽救缩小的细胞大小。事实上，PpVC或PpVR对细胞大小的影响在很大程度上被激活的S6K逆转（图1G和补充图S2G 2i）。最后，我们测试了PpV是否是AMPK激活和脂质稳态的特异性磷酸酶。CG11597是丝氨酸/苏氨酸磷酸酶PP2A亚家族的另一个成员，与PpV的序列同源性最高。然而，用转基因RNAi系（CG11597- ir）普遍敲低CG11597对幼虫AMPK磷酸化水平没有影响（补充图S3A）。此外，以克隆模式表达CG11597 dsRNA对脂肪体中细胞大小没有影响（Supplementary Figure S3B2D）。综上所述，这些结果表明100 bbb [j] .分子细胞生物学杂志（2014），6,100 - 102 doi:10.1093/jmcb/mjt050, 2013年12月13日在线发表

Dear Editor, Protein phosphorylation is essential for multiple cellular processes. This posttranslational modification is regulated by kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation. Drosophila protein phosphatase V (PpV) and Saccharomyces cerevisiae Sit4 are homologs ofmammalian PP6, which belongs to the PP2A subfamily of serine/threonine phosphatases (Mann et al., 1993; Wang et al., 2012). In addition to the catalytic subunit, PP6 holoenzyme also contains a regulatory subunit (PP6R1, PP6R2, or PP6R3 in human; Sap4, Sap155, Sap185, or Sap190 in yeast; CG10289 in fly) and a scaffold subunit (Morales-Johansson et al., 2009). Studies in S. cerevisiae showed that Sit4 knockout strain had reduced lipid droplet content and increased phosphorylation of SNF1 (a homolog of mammalian and Drosophila AMPK) at an evolutionally conserved activation site (Bozaquel-Morais et al., 2010; Ruiz et al., 2011). However, its function in metazoan has not been well explored. To investigate the physiological role of PpV, we ubiquitously knocked down its regulatory or catalytic subunit with transgenic RNAi flies (PpVR-IR or PpVC-IR). Quantitative real-time PCR (qRT–PCR) analyses indicated 60% 2 75% knockdown efficiency for either gene in larvae (data not shown). Knockdown of PpVR did not show obvious changes in larval body size, but caused lethality at pupal stage. On the contrary, knockdown of PpVc resulted in severely reduced body size and larval lethality (Figure 1A). Triacylglyceride (TAG) is a main form for nutritional storage in Drosophila larvae. Knockdown of PpVc, but not PpVR, resulted in significantly reduced TAG levels in these larvae (Figure 1B). The AMPK pathway inhibits adipocyte differentiation and lipogenesis while promotes lipid mobilization, thus playing a key role in lipid catabolism (Viollet and Andreelli, 2011; Hardie et al., 2012). It has been shown that Sit4 contributes to the regulation of SNF1/AMPK activities in yeast (Bozaquel-Morais, et al., 2010; Ruiz, et al., 2011). We examined levels of AMPK phosphorylated at a conserved Thr-172 site (representing the active form of AMPK) with a phospho-specific antibody. Ubiquitous knockdown of either PpVR or PpVc caused a clear increase in AMPK phosphorylation levels in larvae (Figure 1C). The PpVc RNAi showed a stronger effect, consistent with the severer phenotype. We could not monitor the total AMPK levels due to lack of antibodies that recognize Drosophila AMPK. However, the mRNA levels of AMPK were not increased by PpVR or PpVc knockdown (Supplementary Figure S1A), suggesting that the elevated AMPK phosphorylation level did not result from upregulated AMPK expression. Drosophila fat body is functionally equivalent to mammalian white adipose tissue and liver, and is the main storage site for TAG. It also communicates with other organs and regulates the growth of whole body (Rajan and Perrimon, 2011). After we knocked down PpVC or PpVR specifically in the fat body with cg-Gal4, the flies were viable through adulthood, with reduced body size (Figure 1D) and weight (Supplementary Figure S1B). Also at the larval stage, we observed reductions in larval TAG levels (Figure 1E) and increases in AMPK phosphorylation levels in the fat body (Figure 1F) by PpVC or PpVR knockdown. To investigate whether these effects were dependent on increased AMPK activation, we expressed PpVC or PpVR dsRNA in the fat body simultaneously with a transgenic RNAi line targeting the AMPK catalytic subunit (AMPK-IR), which did not attenuate the knockdown efficiency of PpVC or PpVR (Supplementary Figure S1C). Knockdown of AMPK significantly rescued the reductions in body size (Figure 1D), body weight (Supplementary Figure S1B), and TAG levels (Figure 1E) caused by PpVC or PpVR knockdown, suggesting that PpV regulates Drosophila lipid metabolism and growth through the AMPK pathway. We further examined how PpV affected the fat body at cellular level. We expressed dsRNA against PpVC or PpVR in a clonal pattern in the fat body. Compared with surrounding wild-type cells, cells with knockdown of PpVC or PpVR had severely reduced cell size (Figure 1G and Supplementary Figure S2A 2 C). We also induced PpVR knockdown clones with a P element insertion allele of PpVR (CG10289 ) and obtained similar results (Figure 1H), excluding the possibility that the observed phenotype by RNAi was due to an off-target effect. Similar to that observed in whole body, simultaneous knockdown of AMPK largely rescued the decrease in cell size by knockdown of PpV (Figure 1G and Supplementary Figure S2D 2 F). As the AMPK pathway and the mTOR pathway antagonize to regulate energy homeostasis and cell growth (Inoki et al., 2012), we tested whether expression of a constitutively active S6K (Barcelo and Stewart, 2002), which is a downstream effector of mTOR, could rescue the reduced cell size. Indeed, the effect of PpVC or PpVR on cell size was largely reversed by activated S6K (Figure 1G and Supplementary Figure S2G 2 I). Lastly, we tested whether PpV is a specific phosphatase in AMPK activation and lipid homeostasis. CG11597 is another member of PP2A subfamily of serine/threonine phosphatases and has the highest sequence homology to PpV. However, ubiquitous knockdown of CG11597 with a transgenic RNAi line (CG11597-IR) had no effect on AMPK phosphorylation level in larvae (Supplementary Figure S3A). In addition, expression of CG11597 dsRNA in a clonal pattern had no effect on cell size in the fat body (Supplementary Figure S3B2D). Taken together, these results suggest that 100 | Journal of Molecular Cell Biology (2014), 6, 100–102 doi:10.1093/jmcb/mjt050 Published online December 13, 2013