Sqh is involved in the regulation of Ci stability in Hh signaling pathway.-猫眼课题宝

DOI：

10.1093/jmcb/mjv063

发表时间：

2015-12

期刊：

Journal of molecular cell biology

影响因子：

5.5

通讯作者：

中科院分区：

生物学1区

文献类型：

--

作者：

Chunying Liu;Y. Xiong;Jing Feng;Lei Zhang;Yun Zhao

在脊椎动物和无脊椎动物的发育过程中，Hedgehog （Hh）通路广泛参与细胞的生长和分化。Hh通路的故障可能导致各种人类疾病，包括出生缺陷和癌症。在果蝇中，Cubitus interruptus （Ci）是哺乳动物胶质母细胞瘤（glii）的同源物，是Hh通路的关键转录因子。Ci/Gli的活性和稳定性受hhh浓度的调控，进而精确诱导下游基因的表达。先前的研究表明，在Hh缺失的情况下，全长Ci （Ci155）通过cul1slimb为基础的E3连接酶介导的蛋白水解(Aza-Blanc et al., 1997; Jia et al., 2002; Price and Kalderon, 2002；Zhang et al., 2013)，而在高水平Hh存在的情况下，Ci155作为转录激活因子开启下游基因的表达，包括hib/rdx，随后由Cul3-HIB/ rdx为基础的E3连接酶完全降解Ci155 （Zhang et al., 2006）。然而，需要揭示更多的机制来指导Ci部分或完全降解。在本研究中，我们推测Sqh蛋白可能在Ci155裂解位点附近的区域（氨基酸697）与Ci155结合，从而在空间上阻碍了Ci n端与蛋白酶体的相互作用，从而保护Ci155不被完全降解。为了鉴定可能参与Ci部分降解的未知蛋白，我们生成了MBPCiZnF （Ci锌指结构域），并使用经过Hh处理和未经过Hh处理的Clone8细胞裂解液纯化了MBPCiZnF，然后进行了质谱分析（补充图S1）。通过RNAi Drosophila， Sqh进行额外的表型筛选后，选择了在Hh缺失时被CiZnF拉下的肌球蛋白调节轻链蛋白进行进一步研究。Sqh调节收缩环功能，是细胞分裂所必需的（Karess et al., 1991）。IP结果表明，在S2细胞中Hh缺失或存在的情况下，Sqh可以与Ci75和CiZnF相互作用，但不与Ci155相互作用（图1A和supplementary图S2）。这表明Sqh可以与Ci155结合，阻碍其进入蛋白酶体并在Hh存在下被降解。然而，这个过程是非常动态的，在IP实验中很难赶上。或者，在Hh存在的情况下，Sqh可以通过未知的机制与Ci155间接相互作用，从而实现Sqh对Ci155稳定性的调控。利用不同的SqhRNAi果蝇品系来评估Ci及其下游基因的表达水平。免疫荧光染色显示，sqh缺失导致Ci155和Ci75表达减少，dpp表达上调（图1B和C以及补充图S3）。qRT-PCR结果显示，sqhRNAi果蝇的sqh表达明显降低，dpp明显升高，ci和ptc均无明显变化（图1E）。这表明，由sqh缺失引起的Ci下降发生在蛋白水平。此外，在MS1096驱动的sqhRNAi和HA-Ci155组合果蝇中，通过western blotting检测翼盘中Ci155和Ci75蛋白水平的变化（图1D）。使用Image-Pro Plus 6.0软件计算Ci155/Ci75比值（图1D’）。通过qRT-RCR检测组合果蝇的RNAi效率，结果显示sqh表达下调50%（图1D”）。与对照组相比，Sqh降低导致Ci155和Ci75均降低，且Ci155/Ci75比值升高，表明Ci75的降低幅度大于Ci155。结果表明，Sqh对Ci155和Ci75的降解具有保护作用，并能促进Ci155部分降解为Ci75。在sqhRNAi果蝇中，由于sqh缺失导致的Ci75的更明显的减少可能解释了dpp随着Ci155蛋白的减少而上调。此外，对MS1096驱动的sqhRNAi果蝇的翅盘进行了TUNEL实验。未检测到明显的凋亡信号（图1F和补充图S4），说明Ci的降低和靶基因dpp的激活不是由凋亡引起的，而是影响了Ci155的稳定性和加工过程。在sqhRNAi果蝇中，Ci155的减少和dpp的上调可以通过Myc-Sqh的过表达来挽救（图1G），排除了sqh敲低造成脱靶效应的可能性，证实了这些表型确实是由蛋白sqh的降低引起的。先前的研究表明，Sqh的两个氨基酸T20和S21可以被磷酸化。在果蝇发育过程中，一旦被磷酸化，Sqh会招募更多的肌动蛋白参与组织和器官的形成（Corrigall et al., 2007; Zhang and Ward, 2011）。为了进一步阐明差异磷酸化的Sqh在Hh信号传导中的不同作用，我们分别在果蝇中过表达野生型Sqh、磷酸化的模拟TSDD和未磷酸化的模拟TSAA。免疫荧光染色检测Ci155、总Ci蛋白及靶基因dpp水平。Sqh过表达不影响Ci155（图1H）或dpp（补充图S5）的表达。然而，TSDD过表达导致Ci155、dpp减少，相应区域过度增殖584 bbb [j] .分子细胞生物学杂志，2015,7(6),584 - 587 doi:10.1093/jmcb/mjv063

Dear Editor, Hedgehog (Hh) pathway is widely involved in cell growth and differentiation during both vertebrate and invertebrate development. Malfunction of Hh pathway could cause various human diseases, including birth defects and cancers. In Drosophila, Cubitus interruptus (Ci), the homologue of mammal Glioblastomas (Gli), is a key transcription factor of Hh pathway. The activity andstabilityof Ci/Gli areregulatedbyHhconcentration, and further precisely induce downstream gene expressions. Previous studies have suggested that in the absence of Hh, full-length Ci (Ci155) is partially degraded into the repressor form Ci (Ci75), which blocks downstream gene expressions (Chen and Jiang, 2013), through Cul1Slimb-based E3 ligase-mediated proteolysis (Aza-Blanc et al., 1997; Jia et al., 2002; Price and Kalderon, 2002; Zhang et al., 2013), while in the presence of high-level Hh, Ci155 acts as a transcription activator to turn on the expression of downstream genes, including hib/rdx, followed with complete degradation of Ci155 by Cul3-HIB/ RDX-based E3 ligase (Zhang et al., 2006). However, more mechanisms directing Ci to partial or complete degradation need to be revealed. In this study, we speculate that Spaghetti squash (Sqh) protein may bind to Ci155 at the region near the cleavage site (amino acid 697), which spatially impedes the interactions between Ci N-terminus and proteasomes, thus protecting Ci155 from complete degradation. To identify unknown proteins that might be involved in Ci partial degradation, MBPCiZnF (Ci zinc finger domains) was generated and purified for pull-down assay using the lysate of Clone8 cells with/without Hh treatment and subsequent mass spectrometric analysis (Supplementary Figure S1). After additional phenotype screening by using RNAi Drosophila, Sqh, a myosin regulatory light chain protein pulled down by CiZnF in the absence of Hh was selected for further study. Sqh regulatesthe contractile ring function and is required for cytokinesis (Karess et al., 1991). The IP results indicated that Sqh could interact with both Ci75 and CiZnF in the absence/presence of Hh in S2 cells, but did not interact with Ci155 (Figure 1A and SupplementaryFigure S2). Thissuggests that Sqh could bind to Ci155, impeding its entering into proteasomes and being degraded in the presence of Hh. However, this process is very dynamic and hard to catch up in IP experiments. Alternatively, Sqh could indirectly interact with Ci155 through an unknown mechanism in the presence of Hh, which realizes the regulation of Ci155 stability by Sqh. Different lines of SqhRNAi Drosophila were used to evaluate the expression levels of Ci and downstream genes. Immunofluorescence staining indicated that loss-of-sqh resulted in the decrease of both Ci155 and Ci75, with an upregulation of dpp expression (Figure 1B and C and Supplementary Figure S3). qRT-PCR showed that sqh expression was clearly reduced, while dpp was obviously increased in sqhRNAi Drosophila, and both ci and ptc did not change distinctly (Figure 1E). This suggests that Ci decrease caused by loss-of-sqh happens at the protein level. Moreover, in the sqhRNAi and HA-Ci155 combination Drosophila driven by MS1096, the changes of Ci155 and Ci75 protein levels in the wing discs were detected by western blotting (Figure 1D). The ratio of Ci155/Ci75 was calculated by Image-Pro Plus 6.0 (Figure 1D′). The RNAi efficiency of the combination Drosophila was tested by qRT-RCR, showing that sqh expression was downregulated for 50% (Figure 1D′′). Comparedwiththecontrolgroup, Sqh reduction resulted in the decrease of both Ci155 and Ci75, and the ratio of Ci155/Ci75 was raised, indicating more reduction of Ci75 than Ci155. These results indicated that Sqh could protect Ci155 and Ci75 from degradation, and promote the partial degradation of Ci155 into Ci75. The more distinct reduction of Ci75 due to the loss-of-sqh may explain the upregulation of dpp along with the reduction of protein Ci155 in sqhRNAi Drosophila. Furthermore, TUNEL assay was carried out in the wing discs of sqhRNAi Drosophila driven by MS1096. No obvious apoptosis signals were detected (Figure 1F and Supplementary Figure S4), suggesting that the reduction of Ci and activation of target gene dpp were not caused by apoptosis, but rather the stability and processing of Ci155 were influenced. The decrease of Ci155 and upregulation of dpp in sqhRNAi Drosophila could be rescued by overexpression of Myc-Sqh (Figure 1G), which excludes the possibility of off-target effect by sqh knockdown and confirms that these phenotypes are indeed caused by the reduction of protein Sqh. Previous studies indicated that two amino acids T20 and S21 of Sqh can be phosphorylated. Once phosphorylated, Sqh recruits more actin to participate in the formation of tissues and organs during Drosophila development (Corrigall et al., 2007; Zhang and Ward, 2011). To further clarify the different roles of differentially phosphorylated Sqh in Hh signaling, wild-type Sqh, phosphorylated mimic TSDD, and unphosphorylated mimic TSAA, respectively, were overexpressed in Drosophila. Immunofluorescence staining was performed to evaluate the levels of Ci155 and total Ci protein, as well as the target gene dpp. Sqh overexpression did not affect the expression of either Ci155 (Figure 1H) or dpp (Supplementary Figure S5). However, TSDD overexpression resulted in the reduction of Ci155, dpp, and overproliferation of the corresponding region 584 | Journal of Molecular Cell Biology (2015), 7(6), 584–587 doi:10.1093/jmcb/mjv063 Published online October 30, 2015