丝氨酸/苏氨酸蛋白激酶突变体MEKK3~(KI)的构建及功能鉴定
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摘要
有丝分裂原活化蛋白激酶激酶激酶MEKK3(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase3)是一种丝氨酸/苏氨酸蛋白激酶,属于MAP3K(mitogen-activated protein kinase kinase kinase)家族,其在细胞增殖、凋亡、炎症反应、肿瘤的发生中发挥了重要的作用。利用分子克隆手段构建激酶功能失活(kinase inactive)型突变体MEKK3KI(391位赖氨酸突变为甲硫氨酸,K391→M391)的真核表达载体,并通过检测MEKK3KI蛋白对NudC的磷酸化作用及对细胞周期的影响以鉴定其生物活性。根据GenBank提供的人源MEKK3的cNA序列(NM_203351),扩增出野生型MEKK3(MEKK3~(WT))的目的基因,用重叠延伸PCR定点突变技术扩增激酶功能失活型MEKK3KI目的基因,并将其分别克隆至带有FLAG标签的真核表达载体p CMV-tag-2c和带有GFP标签的真核表达载体pEGFP-C1中。DNA序列分析结果显示,MEKK3KI基因序列成功将391位点的赖氨酸(K)突变为甲硫氨酸(M),表明突变体构建成功;免疫印迹分析显示,MEKK3~(WT)、MEKK3KI重组体均在He La细胞中高效表达;体外模拟磷酸化实验结果显示,野生型MEKK3~(WT)在体外可以磷酸化NudC,而激酶功能失活型突变体MEKK3KI无法将其磷酸化;激光共聚焦显微镜观察发现,野生型MEKK3在HeLa细胞中过表达,细胞核形态异常,与凋亡细胞核形态相似,而失活型MEKK3过表达不会导致细胞核明显变化。总之,本实验成功构建了野生型、失活型MEKK3的真核表达载体,同时发现其可以磷酸化NudC,促进细胞凋亡,为进一步揭示MEKK3生物学功能奠定了基础。
Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3( MEKK3) is a kind of serine/threonine protein kinase in the MAP3 K( mitogen-activated protein kinase kinase kinase) family,which plays an important role in cell proliferation,apoptosis,inflammatory response and tumor development.MEKK3 mutant of MEKK3 KI( K391→M391,conversion of lysine to methionine) was constructed by molecular cloning technology,and the activity of the mutant was examined by testing NudC's phosphorylation with MEKK3 KI,while it's role in cell apoptosis was detected. According to the cDNA sequence of human MEKK3( GenBank,NM_203351),we obtained the fragments of wild-type MEKK3( MEKK3~(WT)) by RT-PCR,and the kinase inactive MEKK3( MEKK3 KI) by overlap extension PCR and site-specific mutagenesis,Then we cloned these fragments into the eukaryotie expression vector pCMV-tag-2c and pEGFP-C1 respectively. The DNA sequence analysis showed that the lysine mutation at the MEKK3~(WT)site was methion. indicating that the mutant was successfully constructed.The immunoblotting analysis indicated that the recombinants of MEKK3~(WT)and MEKK3 KIwere successfully expressed in HeLa cells. Experiment of phosphorylation in vitro was used to assay the kinase activity of MEKK3~(WT)and MEKK3 KI. The data showed that MEKK3~(WT)phosphorylated NudC,where as the MEKK3 KIfailed to phosphorylate NudC. Then,the abnormal nuclear morphology was observed by confocal laser microscopy when GFP-MEKK3~(WT)/KI were over-expressed in HeLa cells,while this phenomenon suggested that MEKK3~(WT)could cause cell apoptosis and MEKK3 KIcouldn't. In conclusion,the eukaryotic expression vector of wild-type,inactive MEKK3 were successfully constructed,it was also found to phosphorylate NudC and promote apoptosis,the study providing useful tools for biological functional studies of MEKK3.
Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3( MEKK3) is a kind of serine/threonine protein kinase in the MAP3 K( mitogen-activated protein kinase kinase kinase) family,which plays an important role in cell proliferation,apoptosis,inflammatory response and tumor development.MEKK3 mutant of MEKK3 KI( K391→M391,conversion of lysine to methionine) was constructed by molecular cloning technology,and the activity of the mutant was examined by testing NudC's phosphorylation with MEKK3 KI,while it's role in cell apoptosis was detected. According to the cDNA sequence of human MEKK3( GenBank,NM_203351),we obtained the fragments of wild-type MEKK3( MEKK3~(WT)) by RT-PCR,and the kinase inactive MEKK3( MEKK3 KI) by overlap extension PCR and site-specific mutagenesis,Then we cloned these fragments into the eukaryotie expression vector pCMV-tag-2c and pEGFP-C1 respectively. The DNA sequence analysis showed that the lysine mutation at the MEKK3~(WT)site was methion. indicating that the mutant was successfully constructed.The immunoblotting analysis indicated that the recombinants of MEKK3~(WT)and MEKK3 KIwere successfully expressed in HeLa cells. Experiment of phosphorylation in vitro was used to assay the kinase activity of MEKK3~(WT)and MEKK3 KI. The data showed that MEKK3~(WT)phosphorylated NudC,where as the MEKK3 KIfailed to phosphorylate NudC. Then,the abnormal nuclear morphology was observed by confocal laser microscopy when GFP-MEKK3~(WT)/KI were over-expressed in HeLa cells,while this phenomenon suggested that MEKK3~(WT)could cause cell apoptosis and MEKK3 KIcouldn't. In conclusion,the eukaryotic expression vector of wild-type,inactive MEKK3 were successfully constructed,it was also found to phosphorylate NudC and promote apoptosis,the study providing useful tools for biological functional studies of MEKK3.
引文
1 Lange-Carter C,Pleiman C,Gardner A,et al. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf[J].Science,1993,260(5106):315-319
2 Blank J L,Pr G,Elliott E M,et al. Molecular cloning of mitogenactivated protein/ERK kinase kinases(MEKK)2 and 3[J]. The Journal of Biological Chemistry,1996,271(10):5361-5368
3 Nakamura K,Johnson G L. PB1 domains of MEKK2 and MEKK3interact with the MEK5 PB1 domain for activation of the ERK5 pathway[J]. The Journal of Biological Chemistry,2003,278(39):36989-36992
4 Cheng J,Yu L,Zhang D,et al. Dimeriza-tion through the catalytic domain is essential for MEKK2 activation.[J]. The Journal of Biological Chemistry,2005,280(14):13477-13482
5 Fritz A,Brayer K J,Mccormick N,et al. Phosphorylation of serine 526 is required for MEKK3 activity,and association with 14-3-3blocks dephosphorylation[J]. The Journal of Biological Chemistry,2006,281(10):6236-6245
6 Konno H,Yamamoto T,Yamazaki K,et al. TRAF6 establishes innate immune respo-nses by activating NF-kappaB and IRF7upon sensing cytosolic viral RNA and DNA[J]. Plos One,2009,4(5):e5674
7 Bing Su. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3[J]. Chinese Cell&Stem Cell Transplantation,2009,5(1):98-103
8 Cheng J,Yu L,Zhang D,et al. Dimerization through the catalytic domain is essential for MEKK2 activation[J]. The Journal of Biological Chemistry,2005,280(14):13477-13482
9 Xiao Y,Zheng Y,Tan P,et al. Overexpression of nuclear distribution protein(hN-UDC)causes proapoptosis and differenti-ation in Dami megakaryocytes[J]. Cell Proliferation,2013,46(5):576-585
10 Blonska M,You Y,Geleziunas R,et al. Restoration of NF-kappaB activation by tumornecrosis factor alpha receptor com-plex-targeted MEKK3 in receptor-inter-actingprotein-deficient cells[J]. Molecular and Cellular Biology,2004,24(24):10757-10765
11 Lu J,Liu L,Zheng M,et al. MEKK2 and MEKK3 suppress Hedgehog pathway dependent medulloblastoma by inhibiting GLI1 function[J]. Oncogene,2018,37(28):3864-3878
12 Matitau A E,Scheid M P. Phosphorylation of MEKK3 at threonine 294 promotes 14-3-3 association to inhibit nuclear factor kappaB activation[J]. The Journal of Biological Chemistry,2008,283(19):13261-13268
13 Zhang D,Facchinetti V,Wang X,et al. Identification of MEKK2/3 serine phosphorylation site targeted by the toll-like receptor and stress pathways[J]. European Molecular Biology Organization Journal,2006,25(1):97-107
14 Stevens M V,Broka D M,Parker P,et al. MEKK3 initiates TGFβ2-dependent EMT during endocardial cushion morphogenesis[J].Circulation Research,2008,103(12):1430-1440
15 Garner A P,Weston C R,Todd D E,et al. Delta MEKK3:ER*activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint[J]. Oncogene,2002,21(53):8089-8104
16 Xu L G,Li L Y,Shu H B. TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis[J]. The Journal of Biological Chemistry,2004,279(17):17278-17282
17 Bai J A,Jie H,Wei S,et al. GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis[J]. Apoptosis,2016,21(12):1386-1397
18 刘寿贵,刘磊,周晓健,等. siRNA沉默MEKK3基因促进TRAIL诱导乳腺癌MCF-7细胞的凋亡[J].中国肿瘤生物治疗杂志,2008,15(6):552-556Liu Shougui, Liu Lei, Zhou Xiaojian, et al. SiRNA silencing MEKK3 gene promotes apoptosis of breast cancer Mc F-7 cells induced by TRAIL[J]. Chinese Journal of Cancer Biotherapy,2008,15(6):552-556
19 Linares J F,Duran A,Reina-Campos M,et al. Amino acid activation of m TOR-C1 by a PB1-domain-driven kinase co-mplex Cascade[J]. Cell Reports,2015,12(8):1339-1352
20 Zhou T,Aumais J P,Liu X,et al. A role for Plk1 phosphorylation of NudC in cytokinesis[J]. Developmental Cell,2003,5(1):127-138
21 Chiu Y H,Xiang X,Dawe A L,et al. Deletion of nudC,a nuclear migration gene of aspergillus nidulans,causes morphological and cell wall abnorma-lities and is lethal[J]. Molecular Biology of the Cell,1997,8(9):1735-1749
22 Cappello S,Monzo P,Vallee R B. NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development[J]. Developmental Biology,2011,357(2):326-335
23 Tang Y S,Zhang Y P,Xu P. hNUD prom-otes the cell proliferation and differentiation in a leukemic cell line via activation of the thrombopoietin receptor(Mpl)[J]. Leukemia,2008,22(5):1018-1025
24 Xiao Y,Zheng Y,Tan P,et al. Overex-pression of nuclear distribution protein(hNUDC)causes pro-apoptosis and differentiation in Dami megakaryocytes[J]. Cell Proliferation, 2013, 46(5):576-585
25 Lin S H,Nishino M,Luo W,et al. Inhib-ition of prostate tumor growth by overexpression of NudC,a microtubule motor-associated protein[J]. Oncogene,2004,23(14):2499-2506
2 Blank J L,Pr G,Elliott E M,et al. Molecular cloning of mitogenactivated protein/ERK kinase kinases(MEKK)2 and 3[J]. The Journal of Biological Chemistry,1996,271(10):5361-5368
3 Nakamura K,Johnson G L. PB1 domains of MEKK2 and MEKK3interact with the MEK5 PB1 domain for activation of the ERK5 pathway[J]. The Journal of Biological Chemistry,2003,278(39):36989-36992
4 Cheng J,Yu L,Zhang D,et al. Dimeriza-tion through the catalytic domain is essential for MEKK2 activation.[J]. The Journal of Biological Chemistry,2005,280(14):13477-13482
5 Fritz A,Brayer K J,Mccormick N,et al. Phosphorylation of serine 526 is required for MEKK3 activity,and association with 14-3-3blocks dephosphorylation[J]. The Journal of Biological Chemistry,2006,281(10):6236-6245
6 Konno H,Yamamoto T,Yamazaki K,et al. TRAF6 establishes innate immune respo-nses by activating NF-kappaB and IRF7upon sensing cytosolic viral RNA and DNA[J]. Plos One,2009,4(5):e5674
7 Bing Su. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3[J]. Chinese Cell&Stem Cell Transplantation,2009,5(1):98-103
8 Cheng J,Yu L,Zhang D,et al. Dimerization through the catalytic domain is essential for MEKK2 activation[J]. The Journal of Biological Chemistry,2005,280(14):13477-13482
9 Xiao Y,Zheng Y,Tan P,et al. Overexpression of nuclear distribution protein(hN-UDC)causes proapoptosis and differenti-ation in Dami megakaryocytes[J]. Cell Proliferation,2013,46(5):576-585
10 Blonska M,You Y,Geleziunas R,et al. Restoration of NF-kappaB activation by tumornecrosis factor alpha receptor com-plex-targeted MEKK3 in receptor-inter-actingprotein-deficient cells[J]. Molecular and Cellular Biology,2004,24(24):10757-10765
11 Lu J,Liu L,Zheng M,et al. MEKK2 and MEKK3 suppress Hedgehog pathway dependent medulloblastoma by inhibiting GLI1 function[J]. Oncogene,2018,37(28):3864-3878
12 Matitau A E,Scheid M P. Phosphorylation of MEKK3 at threonine 294 promotes 14-3-3 association to inhibit nuclear factor kappaB activation[J]. The Journal of Biological Chemistry,2008,283(19):13261-13268
13 Zhang D,Facchinetti V,Wang X,et al. Identification of MEKK2/3 serine phosphorylation site targeted by the toll-like receptor and stress pathways[J]. European Molecular Biology Organization Journal,2006,25(1):97-107
14 Stevens M V,Broka D M,Parker P,et al. MEKK3 initiates TGFβ2-dependent EMT during endocardial cushion morphogenesis[J].Circulation Research,2008,103(12):1430-1440
15 Garner A P,Weston C R,Todd D E,et al. Delta MEKK3:ER*activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint[J]. Oncogene,2002,21(53):8089-8104
16 Xu L G,Li L Y,Shu H B. TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis[J]. The Journal of Biological Chemistry,2004,279(17):17278-17282
17 Bai J A,Jie H,Wei S,et al. GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis[J]. Apoptosis,2016,21(12):1386-1397
18 刘寿贵,刘磊,周晓健,等. siRNA沉默MEKK3基因促进TRAIL诱导乳腺癌MCF-7细胞的凋亡[J].中国肿瘤生物治疗杂志,2008,15(6):552-556Liu Shougui, Liu Lei, Zhou Xiaojian, et al. SiRNA silencing MEKK3 gene promotes apoptosis of breast cancer Mc F-7 cells induced by TRAIL[J]. Chinese Journal of Cancer Biotherapy,2008,15(6):552-556
19 Linares J F,Duran A,Reina-Campos M,et al. Amino acid activation of m TOR-C1 by a PB1-domain-driven kinase co-mplex Cascade[J]. Cell Reports,2015,12(8):1339-1352
20 Zhou T,Aumais J P,Liu X,et al. A role for Plk1 phosphorylation of NudC in cytokinesis[J]. Developmental Cell,2003,5(1):127-138
21 Chiu Y H,Xiang X,Dawe A L,et al. Deletion of nudC,a nuclear migration gene of aspergillus nidulans,causes morphological and cell wall abnorma-lities and is lethal[J]. Molecular Biology of the Cell,1997,8(9):1735-1749
22 Cappello S,Monzo P,Vallee R B. NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development[J]. Developmental Biology,2011,357(2):326-335
23 Tang Y S,Zhang Y P,Xu P. hNUD prom-otes the cell proliferation and differentiation in a leukemic cell line via activation of the thrombopoietin receptor(Mpl)[J]. Leukemia,2008,22(5):1018-1025
24 Xiao Y,Zheng Y,Tan P,et al. Overex-pression of nuclear distribution protein(hNUDC)causes pro-apoptosis and differentiation in Dami megakaryocytes[J]. Cell Proliferation, 2013, 46(5):576-585
25 Lin S H,Nishino M,Luo W,et al. Inhib-ition of prostate tumor growth by overexpression of NudC,a microtubule motor-associated protein[J]. Oncogene,2004,23(14):2499-2506