β-arrestin1激活JNK信号通路促进慢性髓细胞白血病细胞K562增殖
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摘要
目的以CML K562细胞为研究对象,探索β-arrestin1促进CML细胞增殖的相关信号通路。方法以β-arrestin1慢病毒载体感染CML K562细胞,形成稳定的K562-siβ1和K562-β1细胞,及非特异性si RNA对照K562-Ctrl细胞。以此为研究对象,利用细胞计数与CCK-8实验检测细胞增殖能力;Western blot检测蛋白表达;免疫共沉淀(Co-IP)实验检测蛋白间的相互作用。结果细胞计数与CCK-8实验结果显示K562-β1细胞增殖与细胞存活率能力显著高于K562-Ctrl,而K562-siβ1显著低于K562-Ctrl。Western blot结果表明β-arrestin1特异性增强磷酸化JNK表达,JNK抑制剂SP600125能抑制p-JNK表达和K562细胞增殖;免疫共沉淀实验表明β-arrestin1能与Src结合。结论 CML K562细胞中β-arrestin1与Src结合,促进JNK信号通路激活,从而促进细胞增殖。
Objective To investigate the signaling pathways involved in β-arrestin1-induced proliferation of K562 cells. Methods We established stable cell lines K562-siβ1 and K562-β1 by lentivirus-mediated β-arrestin1 knock-down or overexpression in K562 cells, with cells transfected with non-specific si RNA as the control(K562-Ctrl). The proliferation of these cells were evaluated by cell counting and CCK-8 assays. Western blotting was used to detect the expression of JNK and p-JNK in the cells, and co-immunoprecipitation(Co-IP) assay was employed to investigate the interaction between β-arrestin1 and Src.Results K562-β1 cells showed significantly greater but K562-siβ1 cells had significantly lower proliferation ability and cell survival rate than K562-Ctrl cells. Western blotting showed that β-arrestin1 specifically enhanced the expression of p-JNK, and the JNK inhibitor SP600125 obviously suppressed p-JNK and cell proliferation of K562 cells. Co-IP assay revealed the binding of β-arrestin1 to Src. Conclusions In K562 cells, β-arrestin1 activates JNK signaling pathway by binding to Src to promote the cell proliferation.
Objective To investigate the signaling pathways involved in β-arrestin1-induced proliferation of K562 cells. Methods We established stable cell lines K562-siβ1 and K562-β1 by lentivirus-mediated β-arrestin1 knock-down or overexpression in K562 cells, with cells transfected with non-specific si RNA as the control(K562-Ctrl). The proliferation of these cells were evaluated by cell counting and CCK-8 assays. Western blotting was used to detect the expression of JNK and p-JNK in the cells, and co-immunoprecipitation(Co-IP) assay was employed to investigate the interaction between β-arrestin1 and Src.Results K562-β1 cells showed significantly greater but K562-siβ1 cells had significantly lower proliferation ability and cell survival rate than K562-Ctrl cells. Western blotting showed that β-arrestin1 specifically enhanced the expression of p-JNK, and the JNK inhibitor SP600125 obviously suppressed p-JNK and cell proliferation of K562 cells. Co-IP assay revealed the binding of β-arrestin1 to Src. Conclusions In K562 cells, β-arrestin1 activates JNK signaling pathway by binding to Src to promote the cell proliferation.
引文
[1]Rowley JD.Letter:a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining[J].Nature,1973,243:290-3.
[2]Sinclair A,Latif AL,Holyoake TL.Targeting survival pathways in chronic myeloid leukaemia stem cells[J].Br J Pharmacol,2013,169(8):1693-707.
[3]Lefkowitz RJ,Whalen EJ.Beta-arrestins:traffic cops of cell signaling[J].Curr Opin Cell Biol,2004,16(2):162-8.
[4]Goldberg AD,Allis CD,Bernstein E.Epigenetics:a landscape takes shape[J].Cell,2007,128(4):635-8.
[5]Di W,Wu J.Involvement of b-arrestins in cancer progression[J].Mol Biol Rep,2013,40(2):1065-71.
[6]Mcdonald PH,Chow CW,Miller WE,et al.beta-Arrestin 2:A receptor-regulated MAPK scaffold for the activation of JNK3[J].Science,2000,290(5496):1574-7.
[7]Luttrell LM,Roudabush FL,Choy EW,et al.Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds[J].Proc Natl Acad Sci USA,2001,98(5):2449-54.
[8]Bruchas MR,Macey TA,Lowe JD,et al.Kappa opioid receptor activation of p38 MAPK is GRK3-and arrestin-dependent in neurons and astrocytes[J].J Biol Chem,2006,281(26):18081-9.
[9]刘慧,龙娟,谭俊杰,等.β-arrestin1和β-arrestin2在白血病中的表达及意义[J].解放军医学杂志,2010,35(2):177-81.
[10]Li K,Qi X.b-Arrestin1 promotes the progression ofchronic myeloid leukaemia by regulating BCR/ABL H4 acetylation[J].Br J Cancer,2014,111(3):568-76.
[11]Sasaki DT,Murray BW.SP600125,an anthrapyrazolone inhibitor of Jun N-terminal kinase[J].Proc Natl Acad Sci USA,2001,98(24):13681-6.
[12]Cuenda A,Cohen P.PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo[J].J Biol Chem,1995,270(46):27489-94.
[13]Benovic JL,Kühn H,Weyand I,et al.Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase:potential role of an analog of the retinal protein arrestin(48-k Da protein)[J].Proc Natl Acad Sci USA,1987,84(24):8879-82.
[14]Morrison DK,Davis RJ.Regulation of map kinase signaling modules by scaffold proteins in mammals[J].Annu Rev Cell Dev Biol,2003,19(3):91-118.
[15]Downward J.The ins and outs of signaling[J].Nature,2001,411(6839):759-62.
[16]Shenoy SK.Transduction of receptor signals by beta-arrestins[J].Science,2005,308(3):512-7.
[17]Lefkowitz RJ.GRKs and beta-arrestins:roles in receptor silencing,trafficking and signaling[J].Trends Endocrinol Metab,2006,17(2):159-65.
[18]davis RJ.Signal transduction by the JNK group of MAP kinases[J].Cell,2000,103(1):239-52.
[19]Karin M,Gallagher E.From JNK to pay dirt:jun kinases,their biochemistry,physiology and clinical importance[J].IUBMB Life,2005,57(4/5):283-95.
[20]Abe J,Haendeler J.Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive Oxygen species[J].J Biol Chem,2000,275(16):11706-12.
[21]Vita JA,Berk BC.c-Jun n-terminal kinase activation by Hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation[J].J Biol Chem,2001,276(19):16045-50.
[22]Luttrell LM,Ferguson SS,Daaka Y,et al.Beta-arrestin-dependent formation of beta(2)adrenergic receptor Src protein kinase complexes[J].Science,1999,283(542):655-61.
[23]Zalevsky J,Thoma MS.β-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[J].Cell Biol.2000b,148(6):1267-81.
[24]Andrews JD,Kelvin AA.Regulation of tyrosine kinase activation and granule release throughβ-arrestin by CXCRI[J].Nat Immunol,2000,1(3):227-33.
[2]Sinclair A,Latif AL,Holyoake TL.Targeting survival pathways in chronic myeloid leukaemia stem cells[J].Br J Pharmacol,2013,169(8):1693-707.
[3]Lefkowitz RJ,Whalen EJ.Beta-arrestins:traffic cops of cell signaling[J].Curr Opin Cell Biol,2004,16(2):162-8.
[4]Goldberg AD,Allis CD,Bernstein E.Epigenetics:a landscape takes shape[J].Cell,2007,128(4):635-8.
[5]Di W,Wu J.Involvement of b-arrestins in cancer progression[J].Mol Biol Rep,2013,40(2):1065-71.
[6]Mcdonald PH,Chow CW,Miller WE,et al.beta-Arrestin 2:A receptor-regulated MAPK scaffold for the activation of JNK3[J].Science,2000,290(5496):1574-7.
[7]Luttrell LM,Roudabush FL,Choy EW,et al.Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds[J].Proc Natl Acad Sci USA,2001,98(5):2449-54.
[8]Bruchas MR,Macey TA,Lowe JD,et al.Kappa opioid receptor activation of p38 MAPK is GRK3-and arrestin-dependent in neurons and astrocytes[J].J Biol Chem,2006,281(26):18081-9.
[9]刘慧,龙娟,谭俊杰,等.β-arrestin1和β-arrestin2在白血病中的表达及意义[J].解放军医学杂志,2010,35(2):177-81.
[10]Li K,Qi X.b-Arrestin1 promotes the progression ofchronic myeloid leukaemia by regulating BCR/ABL H4 acetylation[J].Br J Cancer,2014,111(3):568-76.
[11]Sasaki DT,Murray BW.SP600125,an anthrapyrazolone inhibitor of Jun N-terminal kinase[J].Proc Natl Acad Sci USA,2001,98(24):13681-6.
[12]Cuenda A,Cohen P.PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo[J].J Biol Chem,1995,270(46):27489-94.
[13]Benovic JL,Kühn H,Weyand I,et al.Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase:potential role of an analog of the retinal protein arrestin(48-k Da protein)[J].Proc Natl Acad Sci USA,1987,84(24):8879-82.
[14]Morrison DK,Davis RJ.Regulation of map kinase signaling modules by scaffold proteins in mammals[J].Annu Rev Cell Dev Biol,2003,19(3):91-118.
[15]Downward J.The ins and outs of signaling[J].Nature,2001,411(6839):759-62.
[16]Shenoy SK.Transduction of receptor signals by beta-arrestins[J].Science,2005,308(3):512-7.
[17]Lefkowitz RJ.GRKs and beta-arrestins:roles in receptor silencing,trafficking and signaling[J].Trends Endocrinol Metab,2006,17(2):159-65.
[18]davis RJ.Signal transduction by the JNK group of MAP kinases[J].Cell,2000,103(1):239-52.
[19]Karin M,Gallagher E.From JNK to pay dirt:jun kinases,their biochemistry,physiology and clinical importance[J].IUBMB Life,2005,57(4/5):283-95.
[20]Abe J,Haendeler J.Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive Oxygen species[J].J Biol Chem,2000,275(16):11706-12.
[21]Vita JA,Berk BC.c-Jun n-terminal kinase activation by Hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation[J].J Biol Chem,2001,276(19):16045-50.
[22]Luttrell LM,Ferguson SS,Daaka Y,et al.Beta-arrestin-dependent formation of beta(2)adrenergic receptor Src protein kinase complexes[J].Science,1999,283(542):655-61.
[23]Zalevsky J,Thoma MS.β-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2[J].Cell Biol.2000b,148(6):1267-81.
[24]Andrews JD,Kelvin AA.Regulation of tyrosine kinase activation and granule release throughβ-arrestin by CXCRI[J].Nat Immunol,2000,1(3):227-33.