浏览全部资源
扫码关注微信
A549细胞中DBNLS232磷酸化调控丝状伪足形成及细胞迁移运动
DBNLS232 regulates formation of filopodia and promotes cell migration in A549 cells
- 陆军军医大学学报 2023年45卷第17期 页码：1797-1805
- 作者机构：
1.400016 重庆，重庆医科大学：分子医学与肿瘤研究中心
2.400016 重庆，重庆医科大学：生物化学与分子生物学教研室
- 作者简介：
[ 张春冬，E-mail: zhangcd@cqmu.edu.cn ZHANG Chundong, E-mail: zhangcd@cqmu.edu.cn
- 基金信息：国家自然科学基金面上项目(82173243)
DOI：10.16016/j.2097-0927.202301032
中图分类号： R394.2;R73-37;R734.2收稿：2023-01-31，
修回：2023-04-12，
纸质出版：2023-09-15
- 稿件说明：
移动端阅览
尹讯, 张涛, 李瑶, 等. A549细胞中DBNLS232磷酸化调控丝状伪足形成及细胞迁移运动[J]. 陆军军医大学学报, 2023,45(17):1797-1805.
Xun YIN, Tao ZHANG, Yao LI, et al. DBNLS232 regulates formation of filopodia and promotes cell migration in A549 cells[J]. Journal of Army Medical University, 2023, 45(17): 1797-1805.
尹讯, 张涛, 李瑶, 等. A549细胞中DBNLS232磷酸化调控丝状伪足形成及细胞迁移运动[J]. 陆军军医大学学报, 2023,45(17):1797-1805. DOI： 10.16016/j.2097-0927.202301032.
Xun YIN, Tao ZHANG, Yao LI, et al. DBNLS232 regulates formation of filopodia and promotes cell migration in A549 cells[J]. Journal of Army Medical University, 2023, 45(17): 1797-1805. DOI： 10.16016/j.2097-0927.202301032.
摘要
目的
2
分析脑发育调节蛋白样蛋白(Drebrin-like，DBNL)对肺腺癌细胞迁移运动的影响，及其特异位点磷酸化修饰在丝状伪足形成中的作用。
方法
2
基于shRNA表达载体建立稳定敲减DBNL表达的A549细胞，分析其对癌细胞迁移运动的影响；综合利用在线蛋白质组数据库、定点突变和间接免疫荧光分析A549细胞中过表达野生型DBNL及3个位点突变型(S146E、T165E、S232E和S232A)对微丝骨架形态、丝状伪足形成及细胞迁移运动的影响；通过免疫沉淀实验确定A549细胞中S232位点发生磷酸化修饰。
结果
2
肺癌细胞中稳定敲减DBNL表达显著抑制细胞迁移运动(
P
<
0.001)。通过CPTAC蛋白组数据库分析发现，肺腺癌样本中相较于癌旁组织DBNL蛋白水平表达差异不明显(
P
>
0.05)；但3个丝氨酸/苏氨酸磷酸化位点S146、T165和S232修饰化水平明显上调(
P
<
0.001)。野生型及模拟磷酸化突变体S146E、T165E、S232E过表达在A549细胞中，免疫荧光检测发现S232E富
集于膜周凸起，且显著促进癌细胞丝状伪足形成(
P
<
0.001)；而表达磷酸化失活型S232A则诱导其弥散状分布在细胞质中，并抑制丝状伪足形成(
P
<
0.01)。最后利用免疫共沉淀及通用型丝氨酸磷酸化抗体证明A549细胞中DBNL的S232位点被磷酸化修饰；并基于在线磷酸化序列激酶预测分析发现，p38和CDK5等激酶是S232位点的潜在修饰激酶。
结论
2
肺腺癌细胞A549中磷酸化DBNL
S232
调控丝状伪足形成并促进细胞迁移运动。
Abstract
Objective
2
To investigate the role of site-specific phosphorylation of adaptor protein Drebrin-like (DBNL) in regulating the formation of filopodia and the migration and movement of lung cancer A549 cells.
Methods
2
DBNL was stably knocked down by shRNA in A549 cells
and then its effects on migration and movement of lung cancer cells were analyzed. Online proteome database
site-directed mutagenesis and indirect immunofluorescence assay were used integratively to analyze the effect of wild type DBNL and 3 mutant types (S146E and T165E
S232E and S232A) on F-actin arrangement
formation of filopodia and migration and movement in A549 cells. Co-immunoprecipitation assay was applied to confirm phosphorylation of DBNL S232.
Results
2
DBNL knockdown significantly inhibited the migration and movement of A549 cells (
P
<
0.001). CPTAC database analysis found no significant difference in total protein of DBNL between the adjacent and primary tissues (
P
>
0.05)
however
the phosphorylation of DBNL S146
T165 and S232 were predominantly increased in lung cancer tissue when compared to adjacent tissue (
P
<
0.001). In addition
indirect-immunofluorescence assay indicated that DBNL S232E which was enriched around the membrane that mimic phosphorylation improved the formation of filopodia and the movement of cells (
P
<
0.001)
but not DBNL wild type and other mutations (T165E
S232E). Furthermore
unphosphorylated mutation DBNL S232A obviously suppressed the formation of filopodia (
P
<
0.01). Finally
prediction for phosphorylation on online found that p38 and CDK5 kinases could potentially phosphorylate DBNL S232.
Conclusion
2
Phosphorylation of DBNL S232 in lung cancer A549 cells promotes the formation of filopodia and enhances the movement of lung cancer cells.
关键词
Keywords
references
ADAMS S J, STONE E, BALDWIN D R, et al . Lung cancer screening[J ] . Lancet, 2023, 401(10374): 390-408. DOI: 10.1016/S0140-6736(22)01694-4.
ZHAO W, ZHOU W, RONG L, et al . Epidermal growth factor receptor mutations and brain metastases in non-small cell lung cancer[J ] . Front Oncol, 2022, 12: 912505. DOI: 10.3389/fonc.2022.912505.
LAPPALAINEN P, KOTILA T, JÉGOU A, et al . Biochemical and mechanical regulation of actin dynamics[ J ] . Nat Rev Mol Cell Biol, 2022, 23(12): 836-852. DOI: 10.1038/s41580-022-00508-4.
MYLVAGANAM S, FREEMAN S A, GRINSTEIN S. The cytoskeleton in phagocytosis and macropinocytosis[J]. Curr Biol, 2021, 31(10): R619-R632. DOI: 10.1016/j.cub.2021.01.036.
GALLOP J L. Filopodia and their links with membrane traffic and cell adhesion[J]. Semin Cell Dev Biol, 2020, 102: 81-89. DOI: 10.1016/j.semcdb.2019.11.017.
HOUDUSSE A, TITUS M A. The many roles of myosins in filopodia, microvilli and stereocilia[J]. Curr Biol, 2021, 31(10): R586-R602. DOI: 10.1016/j.cub.2021.04.005.
ASEERVATHAM J. Cytoskeletal remodeling in cancer[J]. Biology, 2020, 9(11): 385. DOI: 10.3390/biology9110385.
吴彬, 王俊杰, 任志祥, 等. PLXDC2调控丝状伪足形成促进胃癌侵袭转移的机制研究[J]. 陆军军医大学学报, 2022, 44(8): 774-781. DOI: 10.16016/j.2097-0927.202112121.
WU B, WANG J J, RENZ X, et al . Plexin-domain containing 2 promotes invasion and metastasis of gastric cancer by regulating filopodia formation[J ] . J Army Med Univ, 2022, 44(8): 774-781. DOI: 10.16016/j.2097-0927.202112121.
DAKUP P P, FENG S, SHI T J, et al . Targeted quantification of protein phosphorylation and its contributions towards mathematical modeling of signaling pathways[J ] . Molecules, 2023, 28(3): 1143. DOI: 10.3390/molecules28031143.
HUANG J N, ZHANG P Y, SOLARI F A, et al . Molecular proteomics and signalling of human platelets in health and disease[J ] . Int J Mol Sci, 2021, 22(18): 9860. DOI: 10.3390/ijms22189860.
SINGH V, RAM M, KUMAR R, et al . Phosphorylation: implications in cancer[J ] . Protein J, 2017, 36(1): 1-6. DOI: 10.1007/s10930-017-9696-z.
HERRERA A, MENENDEZ A, TORROBA B, et al . Dbnl and β-catenin promote pro-N-cadherin processing to maintain apico-basal polarity[J ] . J Cell Biol, 2021, 220(6): e202007055. DOI: 10.1083/jcb.202007055.
BOATENG L R, CORTESIO C L, HUTTENLOCHER A. Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts[J]. J Cell Sci, 2012, 125(Pt 5): 1329-1341. DOI: 10.1242/jcs.096529.
YANG C Z, LI Z G, SHI Z, et al . Regulation of cell survival by the HIP-55 signaling network[J ] . Mol BioSyst, 2014, 10(6): 1393-1399. DOI: 10.1039/C3MB70552H.
LI Z J, PARK H R, SHI Z, et al . Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291- phospho-sensor motifs[J ] . Oncotarget, 2014, 5(10): 3197-3209. DOI: 10.18632/oncotarget.1900.
RASHID M M, SHATABDA S, HASAN M M, et al . Recent development of machine learning methods in microbial phosphorylation sites[J ] . Curr Genomics, 2020, 21(3): 194-203. DOI: 10.2174/1389202921666200427210833.
IZDEBSKA M, ZIELINSKA W, HAŁAS-WISNIEWSKA M, et al . Involvement of actin and actin-binding proteins in carcinogenesis[J ] . Cells, 2020, 9(10): 2245. DOI: 10.3390/cells9102245.
LAMBERT A W, PATTABIRAMAN D R, WEINBERG R A. Emerging biological principles of metastasis[J]. Cell, 2017, 168(4): 670-691. DOI: 10.1016/j.cell.2016.11.037.
CHUNG W L, EIBAUER M, LI W H, et al . A network of mixed actin polarity in the leading edge of spr eading cells[J ] . Commun Biol, 2022, 5(1): 1338. DOI: 10.1038/s42003-022-04288-7.
GAUTREAU A M, FREGOSO F E, SIMANOV G, et al . Nucleation, stabilization, and disassembly of branched actin networks[J ] . Trends Cell Biol, 2022, 32(5): 421-432. DOI: 10.1016/j.tcb.2021.10.006.
HAECKEL A, AHUJA R, GUNDELFINGER E D, et al . The actin-binding protein Abp1 controls dendritic spine morphology and is important for spine head and synapse formation[J ] . J Neurosci, 2008, 28(40): 10031-10044. DOI: 10.1523/JNEUROSCI.0336-08.2008.
BOATENG L R, BENNIN D, DE OLIVEIRA S, et al . Mammalian actin-binding protein-1/hip-55 interacts with FHL2 and negatively regulates cell invasion[J ] . J Biol Chem, 2016, 291(27): 13987-13998. DOI: 10.1074/jbc.M116.725739.
0
浏览量
0
下载量
0
CSCD
- 网络PDF下载
- Meta-XML下载
- BibTeX下载
- Endnote下载
- RefMan 下载
- NoteExpress下载
- NoteFirst下载
关联资源
相关文章
相关作者
相关机构
- 技术支持由北京北大方正电子有限公司提供 京ICP备09064830号-19
京公网安备11010802024621 - 本系统建议在Chrome、 IE9+ 以上版本浏览器阅读本站内容，360浏览器请切换至极速模式
- Cookies帮助我们提供服务并提供个性化体验。使用本网站，即表示您同意我们使用Cookies
