ARAP3-VAV2新结合方式鉴定与功能分析
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摘要
目的探索腺苷二磷酸糖基化因子6的鸟苷三磷酸酶激活蛋白3(ARAP3)与VAV鸟苷交换因子2(VAV2)新的作用方式及可能的生理功能。方法通过构建VAV2和ARAP3的突变体,在人子宫颈癌细胞(HeLa细胞)中共转VAV2的野生型和APAP3的突变体或者在人胚胎肾细胞(HEK-293T细胞)中共转ARAP3的野生型和VAV2的突变体,免疫共沉淀揭示二者的相互作用方式;在HeLa细胞中分别过表达VAV2和ARAP3野生型或突变体,荧光标记实验观察它们对纤维状肌动蛋白(F-actin)的调控作用。结果除了肉瘤基因同源2(SH2)结构域以外,VAV2的N端(1-660氨基酸)区域亦能介导其与ARAP3的相互作用,HeLa细胞中过表达VAV2和ARAP3均能影响细胞内F-actin的形成。结论 VAV2多个结构域均能介导其与ARAP3相互作用,两者之间的相互作用可能是促进大鼠肉瘤同源癌基因A(Rho A)活性的动态变化或循环,从而调节细胞内F-actin动态性以及肿瘤细胞的迁移和侵染能力。
Objective To study the new interaction model between adenosine diphosphate ribosylation factor 6guanosine triphosphatase activating protein 3( ARAP3) and VAV guanine nucleotide exchange factor 2( VAV2),further to analyze their possible physiological functions. Methods Eukaryotic expression wild type and mutantion plasmids of ARAP3 and VAV2 were constructed respectively. The co-immunoprecipitation assays with transfected human cervical carcinoma cells( HeLa cells) and human embryonic kidney cells( HEK-293 T cells) were used to detect the interaction model between ARAP3 and VAV2. The fluorescence labeling experiments were used to test the effect of overexpression of ARAP3,VAV2 and their mutants on fibrous actin( F-actin) assembly. Results Nterminal region of VAV2( 1-660aa) could also mediate its interaction with ARAP3 besides sarcoma gene homology 2( SH2) domain of VAV2. Overexpression of VAV2 and its mutants in HeLa cell respectively could regulate the Factin organization as same as ARAP3. Conclusion Several domains of VAV2 mediate its interaction with ARAP3,which may promote the dynamic changes of rat sarcoma homologus oncogene A( RhoA) activity and F-actin assembly,even cell migration and cell invasion.
Objective To study the new interaction model between adenosine diphosphate ribosylation factor 6guanosine triphosphatase activating protein 3( ARAP3) and VAV guanine nucleotide exchange factor 2( VAV2),further to analyze their possible physiological functions. Methods Eukaryotic expression wild type and mutantion plasmids of ARAP3 and VAV2 were constructed respectively. The co-immunoprecipitation assays with transfected human cervical carcinoma cells( HeLa cells) and human embryonic kidney cells( HEK-293 T cells) were used to detect the interaction model between ARAP3 and VAV2. The fluorescence labeling experiments were used to test the effect of overexpression of ARAP3,VAV2 and their mutants on fibrous actin( F-actin) assembly. Results Nterminal region of VAV2( 1-660aa) could also mediate its interaction with ARAP3 besides sarcoma gene homology 2( SH2) domain of VAV2. Overexpression of VAV2 and its mutants in HeLa cell respectively could regulate the Factin organization as same as ARAP3. Conclusion Several domains of VAV2 mediate its interaction with ARAP3,which may promote the dynamic changes of rat sarcoma homologus oncogene A( RhoA) activity and F-actin assembly,even cell migration and cell invasion.
引文
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[8]Wu B,Wang F,Zhang J,et al.Identification and structural basis for a novel interaction between Vav2 and Arap3[J].J StructBiol,2012,180(1):84-95.
[2]Peng F,Zhang B,Ingram A J,et al.Mechanical stretch-induced Rho A activation is mediated by the RhoGEF Vav2 in mesangial cells[J].Cell Signal,2010,22(1):34-40.
[3]Bustelo X R.Vav family exchange factors:an integrated regulatory and functional view[J].Small GTPases,2014,5(2):9.
[4]Krugmann S,Anderson K E,Ridley S H,et al.Identification of ARAP3,a novel PI3K effector regulating both Arf and Rho GT-Pases,by selective capture on phosphoinositide affinity matrices[J].Mol Cell,2002,9(1):95-108.
[5]Havel L S,Kline E R,Salgueiro A M.Vimentin regulates lung cancer cell adhesion through a VAV2-Rac1 pathway to control focal adhesion kinase activity[J].Oncogene,2015,34(15):1979-90.
[6]Li J J,Sun Z J,Yuan Y M,et al.EphB3 stimulates cell migration and metastasis in a kinase-dependent manner through Vav2-Rho GTPase axis in papillary thyroid cancer[J].J Biol Chem,2017,292(3):1112-21.
[7]Rosenberg B J,Gil-Henn H,Mader C C,et al.Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells[J].Mol Biol Cell,2017,28(10):1347-60.
[8]Wu B,Wang F,Zhang J,et al.Identification and structural basis for a novel interaction between Vav2 and Arap3[J].J StructBiol,2012,180(1):84-95.