Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing

Fengjie LI; Yongqin JIA; Zhen LIANG; Bin HU; Simin HE; Yanzhou WANG; Zeyu YANG; Zhiqing LIANG

doi:10.16016/j.2097-0927.202110107

您当前的位置：

首页 >

文章列表页 >

Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing

Basic Medicine | 更新时间：2026-01-09

- Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing
- Journal of Army Medical University Vol. 44, Issue 6, Pages: 513-521(2022)
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)第一附属医院妇产科
  2.410031 长沙，中南大学生命科学学院分子生物学研究中心
  3.410028 长沙，中南大学湘雅公共卫生学院
  4.400013 重庆，重庆市人民医院甲状腺乳腺外科
- 作者简介：
- 基金信息：
  
  the National Key Research and Development Program of China(2019YFC1005202)
- DOI：10.16016/j.2097-0927.202110107
  CLC： R345; R394.3; R737.31
- Received：19 October 2021，
  
  Revised：2021-12-08，
  
  Published：30 March 2022
- 稿件说明：
移动端阅览
Fengjie LI, Yongqin JIA, Zhen LIANG, et al. Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing[J]. Journal of Army Medical University, 2022, 44(6): 513-521.
DOI：

Fengjie LI, Yongqin JIA, Zhen LIANG, et al. Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing[J]. Journal of Army Medical University, 2022, 44(6): 513-521. DOI： 10.16016/j.2097-0927.202110107.

摘要

目的

通过蛋白质磷酸化修饰组学质谱测序，分析蛋白激酶WNK2在卵巢癌细胞中调控的生物过程。

方法

用WNK2干扰慢病毒感染SKOV3细胞系，药筛获得稳转细胞株，提取总蛋白进行蛋白质磷酸化修饰组学质谱测序，根据生物信息学分析干扰WNK2前后SKOV3细胞中蛋白质磷酸化水平的变化，筛选磷酸化差异蛋白，KEGG及GO富集分析获取差异蛋白在卵巢癌细胞中调控的生物学过程并进行实验验证。

结果

通过生物信息学分析磷酸化修饰组学质谱数据，结果显示干扰WNK2后，SKOV3细胞中共有745个蛋白磷酸化水平显著下降，其中包含1 078个磷酸化修饰位点发生变化，328个蛋白磷酸化水平显著上升，共415个磷酸化修饰位点发生变化。将磷酸化水平发生变化的差异蛋白进行KEGG、GO富集分析，发现WNK2主要参与了细胞骨架形成、信号传导、细胞组成、激酶活性调控等生物过程。干扰或过表达WNK2后，利用鬼笔环肽进行细胞骨架染色，结果显示，干扰WNK2后，细胞骨架断裂，排列紊乱；过表达WNK2后，细胞骨架重排增强。

结论

WNK2通过调控vimentin等蛋白的磷酸化水平参与卵巢癌细胞骨架形成过程。

Abstract

Objective

To analyze the biological processes regulated by protein kinase WNK2 in ovarian cancer cells based on protein phosphorylation modified mass spectrometry.

Methods

Ovarian cancer cell line SKOV3 was transfected with lentiviral vector to knockdown WNK2 and the obtained cells were further screened by drug screening. Then the total protein was extracted for protein phosphorylation modification mass spectrometry analysis. Bioinformatics analysis was adopted to detect the phosphorylation changes before and after WNK2 knockdown

and differential proteins were screened out. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to explore the functions of differentially phosphorylated proteins

and experiments were performed to verify the biological processes regulated by WNK2.

Results

Phosphorylation modified mass spectrometry indicated that WNK2 knockdown significantly decreased the phosphorylation of 745 proteins with 1 078 altered protein sites

and increased the phosphorylation of 328 proteins with 415 altered protein sites. KEGG and GO enrichment analyses showed WNK2 influences biological processes such as cytoskeleton organization

signal transduction

cell composition

and regulation of kinase activity. Phalloidin staining indicated after WNK2 knockdown resulted in broken and disordered cytoskeleton

while its overexpression promoted cytoskeleton remodeling.

Conclusion

WNK2 is involved in the cytoskeleton formation in ovarian cancer cells by regulating the phosphorylation of vimentin and other proteins.

关键词

Keywords

references

BRAY F, FERLAY J, SOERJOMATARAM I, et al . Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J ] . CA Cancer J Clin, 2018, 68(6): 394-424. DOI: 10.3322/caac.21492.

SIEGEL R L, MILLER K D, JEMAL A. Cancer statistics, 2020[J]. CA Cancer J Clin, 2020, 70(1): 7-30. DOI: 10.3322/caac.21590.

LAHIRY P, TORKAMANI A, SCHORK N J, et al . Kinase mutations in human disease: interpreting genotype-phenotype relationships[J ] . Nat Rev Genet, 2010, 11(1): 60-74. DOI: 10.1038/nrg2707.

MAOZ A, CICCONE M A, MATSUZAKI S, et al . Emerging serine-threonine kinase inhibitors for treating ovarian cancer[J ] . Expert Opin Emerg Drugs, 2019, 24(4): 239-253. DOI: 10.1080/ 14728214.2019.1696773.

CICCONE M A, MAOZ A, CASABAR J K, et al . Clinical outcome of treatment with serine-threonine kinase inhibitors in recurrent epithelial ovarian cancer: a systematic review of literature[J ] . Expert Opin Investig Drugs, 2016, 25(7): 781-796. DOI: 10.1080/13543784.2016.1181748.

XU B, ENGLISH J M, WILSBACHER J L, et al . WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain Ⅱ[J ] . J Biol Chem, 2000, 275(22): 16795-16801. DOI: 10.1074/jbc.275.22.16795.

GALLOLU KANKANAMALAGE S, KARRA A S, COBB M H. WNK pathways in cancer signaling networks[J]. Cell Commun Signal, 2018, 16(1): 72. DOI: 10.1186/s12964-018-0287-1.

MONIZ S, JORDAN P. Emerging roles for WNK kinases in cancer[J]. Cell Mol Life Sci, 2010, 67(8): 1265-1276. DOI: 10.1007/s00018-010-0261-6.

SATO A, SHIMIZU M, GOTO T, et al . WNK regulates Wnt signalling and β-Catenin levels by interfering with the interaction between β-Catenin and GID[J ] . Commun Biol, 2020, 3(1): 1-10. DOI: 10.1038/s42003-020-01386-2.

JUN P, HONG C, LAL A, et al . Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-gradespecific[J ] . Neuro Oncol, 2009, 11(4): 414-422. DOI: 10.1215/15228517-2008-096.

DUTRUEL C, BERGMANN F, ROOMAN I, et al . Early epigenetic downregulation of WNK2 kinase during pancreatic ductal adenocarcinoma development[J ] . Oncogene, 2014, 33(26): 3401-3410. DOI: 10.1038/onc.2013.312.

XU T, WU K, ZHANG L, et al . Long non-coding RNA LINC00858 exerts a tumor-promoting role in colon cancer via HNF4α and WNK2 regulation[J ] . Cell Oncol (Dordr), 2020, 43(2): 297-310. DOI: 10.1007/s13402-019-00490-8.

MONIZ S, MARTINHO O, PINTO F, et al . Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness[J ] . Hum Mol Genet, 2013, 22(1): 84-95. DOI: 10.1093/hmg/dds405.

ZAMARAEV A V, VOLIK P I, SUKHIKH G T, et al . Long non-coding RNAs: a view to kill ovarian cancer[J ] . Biochim Biophys Acta Rev Cancer, 2021, 1876(1): 188584. DOI: 10.1016/j.bbcan.2021.188584.

MONIZ S, VERÍSSIMO F, MATOS P, et al . Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2[J ] . Oncogene, 2007, 26(41): 6071-6081. DOI: 10.1038/sj.onc.1210706.

SERYSHEVA E, BERHANE H, GRUMOLATO L, et al . Wnk kinases are positive regulators of canonical Wnt/β-catenin signalling[J ] . EMBO Rep, 2013, 14(8): 718-725. DOI: 10.1038/embor.2013.88.

ALVARADO-KRISTENSSON M, ROSSELLÓ C A. The biology of the nuclear envelope and its implications in cancer biology[J]. Int J Mol Sci, 2019, 20(10): 2586. DOI: 10.3390/ijms20102586.

SHORT C A, SUAREZ-ZAYAS E A, GOMEZ T M. Cell adhesion and invasion mechanisms that guide developing axons[J]. Curr Opin Neurobiol, 2016, 39: 77-85. DOI: 10.1016/j.conb.2016.04.012.

MA X D, DANG Y M, SHAO X W, et al . Ubiquitination and long non-coding RNAs regulate actin cytoskeleton regulators in cancer progression[J ] . Int J Mol Sci, 2019, 20(12): 2997. DOI: 10.3390/ijms20122997.

JUNG H, YOON S R, LIM J, et al . Dysregulation of rho GTPases in human cancers[J ] . Cancers, 2020, 12(5): 1179. DOI: 10.3390/cancers12051179.

ALLEN A, GAU D, FRANCOEUR P, et al . Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells[J ] . J Biol Chem, 2020, 295(46): 15636-15649. DOI: 10.1074/jbc.ra120.013963.

JIU Y, PERÄNEN J, SCHAIBLE N, et al . Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA[J ] . J Cell Sci, 2017, 130(5): 892-902. DOI: 10.1242/jcs.196881.

YILMAZ M, CHRISTOFORI G. EMT, the cytoskeleton, and cancer cell invasion[J]. Cancer Metast Rev, 2009, 28(1/2): 15-33. DOI: 10.1007/s10555-008-9169-0.

HALL A. The cytoskeleton and cancer[J]. Cancer Metast Rev, 2009, 28(1/2): 5-14. DOI: 10.1007/s10555-008-9166-3.

LANIER M H, KIM T, COOPER J A. CARMIL2 is a novel molecular connection between vimentin and actin essential for cell migration and invadopodia formation[J]. Mol Biol Cell, 2015, 26(25): 4577-4588. DOI: 10.1091/mbc.e15-08-0552.

潘燕, 韩婧, 张晔, 等. Vimentin在肿瘤转移中的作用及药物研究进展[J]. 生理科学进展, 2010, 41(6): 413-416.

PAN Y, HAN J, ZHANG Y, et al . Role ofvimentin in tumor metastasis and drug research[J ] . Prog Physiol Sci, 2010, 41(6): 413-416.

LIAO G, WANG R, REZEY A C, et al . MicroRNA miR-509 regulates ERK1/2, the vimentin network, and focal adhesions by targeting Plk1[J ] . Sci Rep, 2018, 8(1): 12635. DOI: 10.1038/s41598-018-30895-8.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Alkaline phosphatase, liver/bone/kidney, inhibits proliferation of ovarian cancer cell line SKOV3

Role of hypoxia in promoting migration and cell fusion of urine-derived stem cells

Focused low-intensity pulsed ultrasound upregulates Vimentin protein expression to inhibit chondrocyte apoptosis

Store-operated calcium entry involves in regulating the hyperpermeability of endothelial cells induced by ox-LDL

Transcriptomic sequencing reveals that WNK2 promotes reactive oxygen species production in ovarian cancer cells

Related Author

Lijuan CHENG

Shijie ZHAN

Bin LIU

Hua LINGHU

张楠楠

胡超群

何昀

毕杨

Related Institution

Department of Obstetrics and Gynecology, the First Affiliated Hospital of Chongqing Medical University

Department of Pathology, College of Basic Medical Sciences, Chongqing Medical University

⁰