Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can direct...Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.展开更多
Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided int...Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.展开更多
Brassinosteroids(BRs)are essential phytohormones that broadly regulate plant growth,development,and adaptation to biotic and abiotic stresses.In Arabidopsis,apoplastic BR molecules are perceived by a plasma membrane-l...Brassinosteroids(BRs)are essential phytohormones that broadly regulate plant growth,development,and adaptation to biotic and abiotic stresses.In Arabidopsis,apoplastic BR molecules are perceived by a plasma membrane-localized receptor complex comprising the ligand-binding receptor BRI1 and the co-receptor BAK1.While negative regulators of the BR receptor complex,such as BKI1,BIR3,and PUB12/13,have been well characterized,how BRI1 and BAK1 are positively modulated in the BR pathway remains largely unknown.In this study,a genetic screen involving overexpression of RLP genes in the bak1-3 bkk1-1 double mutant reveals that enhanced RLP51 expression partially suppresses the BR-deficient phenotypes of bak1-3 bkk1-1.RLP51 overexpression also partially rescues the weak bri1 mutant allele,bri1-301.Although the rlp51 single mutant exhibits wild-type-like phenotypes,it enhances BR-defective phenotypes in bri1-301 and bak1 serk1 mutants.RLP51 is next found to interact with both BRI1 and BAK1 without affecting BRI1–BAK1 interaction.Critically,co-expression of RLP51 with BRI1 or BAK1 significantly increases BRI1 and BAK1 protein abundances.RLP51 appears to promote protein synthesis rather than stabilize BRI1 and BAK1 proteins.Thus,our study identifies RLP51 as a positive regulator of BR signaling that enhances the protein levels of BRI1 and BAK1.展开更多
After cellular damage caused by wounding or pathogens,Arabidopsis thaliana endogenous elicitor peptides(Peps)are released into the apoplast,enhancing innate immunity by directly binding to the membrane-localized leuci...After cellular damage caused by wounding or pathogens,Arabidopsis thaliana endogenous elicitor peptides(Peps)are released into the apoplast,enhancing innate immunity by directly binding to the membrane-localized leucine-rich repeat receptor kinase PEP RECEPTOR1(PEPR1).Ligand binding induces PEPR1 heterodimerization with the co-receptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1(BAK1),followed by PEPR1internalization,both essential for a subset of Pep1-induced responses.However,the role of BAK1 in Pep1-triggered PEPR1 endocytosis remains unclear.Here,we show that the ligandinduced PEPR1 endocytosis depends on its kinase activity and requires BAK1 C-terminal tail phosphorylation,which is equally indispensable for immune signaling and BAK1 internalization.Using a GFP insertional mutagenesis approach,we generated a partially functional GFP-tagged BAK1 to demonstrate that,following Pep1 elicitation,BAK1 and PEPR1 are endocytosed together with similar dynamics.Our findings identify the BAK1 function as a prerequisite for PEPR1 internalization.展开更多
To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plan...To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plants carry an early immune responsive gene FRK1(flg22-induced receptor-like kinase 1)promoter fused with a luciferase(LUC)reporter.A series of mutants with altered pFRK1::LUC activity were identified and named as Arabidopsis genes governing immune gene expression(aggie).The aggie5 mutant showed reduced pFRK1::LUC induction in response to multiple MAMPs treatments.Moreover,the activation of the endogenous FRK1 gene by MAMPs was also compromised in aggie5.Significantly,the bacterial flagellin epitope flg22-induced MAPK activation,ROS production,and immunerelated gene expression were reduced in aggie5.Map-based cloning coupled with next-generation sequencing revealed that aggie5 encodes a kinase-inactive BAK1,which is involved in brassinosteroid responses,innate immunity,and cell death control.Consistently,the aggie5/bak1-16 mutant displayed compromised responses to BR treatment.Furthermore,the aggie5/bak1-16 mutant exhibited seedling lethality when combined with the mutation of its closest homolog BKK1/SERK4.The data suggest that BAK1 kinase activity is required for its multiple functions in plant immunity,development,and cell death control.The isolation and characterization of these Aggie genes will contribute to a better understanding of host immune signaling and provide genetic resources to improve crop resistance.展开更多
文摘目的 探讨miR-125b通过靶向抑制Bak1表达影响人髓系白血病(AML)细胞增殖的机制及意义。方法选择miR-125b mimics、miR-125b inhibitor、miR-125b NC分别转染人AML细胞株THP-1(miR-125b mimics组、miR-125b inhibitor组、NC组)。采用荧光定量PCR检测miR-125b mRNA相对表达量, MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western-blot法检测蛋白表达水平,利用生物信息学分析和荧光素酶双报告系统明确miR-125b的下游靶基因。结果 miR-125b mimics转染后THP-1细胞中miR-125b表达水平显著上调( P <0.05),miR-125b inhibitor转染后细胞中miR-125b水平则显著降低( P <0.05)。转染24 h、48 h后,MTT法检测显示miR-125b mimics组的细胞增殖活性显著低于miR-125b inhibitor组与NC组( P <0.05),细胞凋亡率显著高于miR-125b inhibitor组与NC组( P <0.05),后两组对比差异无统计学意义( P >0.05)。转染48 h后,Western-blot法检测显示miR-125b mimics组的 Bak1蛋白表达水平显著高于miR-125b inhibitor组与NC组( P <0.05),三组PI3K、AKT蛋白表达水平对比差异无统计学意义( P >0.05)。在HEK293细胞株中,miR-125b mimic转染48 h后可以显著降低Bak1萤火虫荧光素酶活性( P <0.05),而对于含有突变型载体的Bak1萤火虫荧光素酶活性无显著影响( P >0.05)。结论 过表达miR-125b可通过靶向抑制Bak1的活性,抑制人AML细胞株的增殖活性,促进细胞凋亡。
基金Acknowledgments This study was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China （Grants 30425029, 30421001, 90717001）. We greatly thank Prof Hong Ma （Penn. State University, USA） for critical reading and writing improvement and Prof Nam-Hai Chua （The Rockefeller University, USA） for helpful comments. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Prof Sheng Luan （University of California, Berkeley, USA） for providing the construct pATC940. We thank Prof Hong-Quan Yang （SIPPE, CAS） for providing LexA yeast two-hybrid system and Prof Zhi-Yong Wang （The Stanford University, USA） for providing the BRI1 antibody. We thank Mr Xiao-Shu Gao for the help on Confocal Laser Scanning Microscopy.
文摘Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.
基金Science and technology introduction and innovation project of Xinjiang uygur autonomous region people's hospital(No.20170305).
文摘Objective:To investigate the effects of silencing BAK1 and BCL2 gene expression on proliferation,invasion and metastasis of hepatocellular carcinoma(HCC)HepG2 cells.Methods:30 HCC HepG2 cells were randomly divided into groups and received the corresponding treatments,namely,control group,NC-siRNA group,BAK1-siRNA group,BCL2-siRNA group and BAK1+BCL2 group,with 6 strains in each group.ThenqRT-PCR,CCK8,Transwell chamber invasion and scratch assay were used to detect the expression,proliferation,invasion and metastasis of BAK1 and BCL2 genes in HepG2 cells.Results:The mRNA expression,cell proliferation rate,cell migration rate and cell invasion ability of BAK1 and BCL2 in HepG2 cells were lowest in the BAK1+BCL2 siRNA group,followed by BCL2-siRNA group,BAK1-siRNA group,NC-shRNA group and control group(P<0.05).The proliferation rate of HepG2 cells in the BAK1+BCL2 siRNA group decreased significantly with time(P<0.05).Conclusion:Silencing the expression of BAK1 and BCL2 genes can inhibit the proliferation and invasion of HCC HepG2 cells and promote their apoptosis.
基金supported by the National Natural Science Foundation of China(32370295 and 32170280 to K.H.)Foundation of Science and Technology of Gansu Province(22ZD6NA049)+3 种基金the Fundamental Research Funds for the Central Universities(lzujbky-2021-kb05 to Y.F.and lzujbky-2023-kb05 to P.L.)the Science and Technology Department of Gansu Province(24JRRA392 to K.H.and 23JRRA1132 to C.X.)China Postdoctoral Science Foundation(2024M751259 and GZB20240285 to B.L.)Science and Technology Program of Gansu Province(25JRRA718 to B.L.).
文摘Brassinosteroids(BRs)are essential phytohormones that broadly regulate plant growth,development,and adaptation to biotic and abiotic stresses.In Arabidopsis,apoplastic BR molecules are perceived by a plasma membrane-localized receptor complex comprising the ligand-binding receptor BRI1 and the co-receptor BAK1.While negative regulators of the BR receptor complex,such as BKI1,BIR3,and PUB12/13,have been well characterized,how BRI1 and BAK1 are positively modulated in the BR pathway remains largely unknown.In this study,a genetic screen involving overexpression of RLP genes in the bak1-3 bkk1-1 double mutant reveals that enhanced RLP51 expression partially suppresses the BR-deficient phenotypes of bak1-3 bkk1-1.RLP51 overexpression also partially rescues the weak bri1 mutant allele,bri1-301.Although the rlp51 single mutant exhibits wild-type-like phenotypes,it enhances BR-defective phenotypes in bri1-301 and bak1 serk1 mutants.RLP51 is next found to interact with both BRI1 and BAK1 without affecting BRI1–BAK1 interaction.Critically,co-expression of RLP51 with BRI1 or BAK1 significantly increases BRI1 and BAK1 protein abundances.RLP51 appears to promote protein synthesis rather than stabilize BRI1 and BAK1 proteins.Thus,our study identifies RLP51 as a positive regulator of BR signaling that enhances the protein levels of BRI1 and BAK1.
基金supported by special research funding from the Flemish Governmentfunding from the Student Program-Graduate Studies Plan Program from the Coordination for the Improvement of Higher Education Personnel(Brazil)for a joint doctorate fellowship at Ghent University and University of Sao Paulo to F.A.O.-.M.+8 种基金the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement(No.101023079‘ENDOLOGISTIC’)to S.Y.P.E.W.Latin American Fellows Program to F.A.O.-.M.the National Science Foundation(NSF)(MCB-1906060)to P.H.NSF(IOS-2049642)to L.S.the China Scholarship Council for a predoctoral fellowship(201706350153)a UGent BOF doctoral mandate(01CD7122)to X.X.the Research Foundation-Flanders(G003720N)to E.R.funded by Ghent University,FWOthe Flemish Government-department EWI。
文摘After cellular damage caused by wounding or pathogens,Arabidopsis thaliana endogenous elicitor peptides(Peps)are released into the apoplast,enhancing innate immunity by directly binding to the membrane-localized leucine-rich repeat receptor kinase PEP RECEPTOR1(PEPR1).Ligand binding induces PEPR1 heterodimerization with the co-receptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1(BAK1),followed by PEPR1internalization,both essential for a subset of Pep1-induced responses.However,the role of BAK1 in Pep1-triggered PEPR1 endocytosis remains unclear.Here,we show that the ligandinduced PEPR1 endocytosis depends on its kinase activity and requires BAK1 C-terminal tail phosphorylation,which is equally indispensable for immune signaling and BAK1 internalization.Using a GFP insertional mutagenesis approach,we generated a partially functional GFP-tagged BAK1 to demonstrate that,following Pep1 elicitation,BAK1 and PEPR1 are endocytosed together with similar dynamics.Our findings identify the BAK1 function as a prerequisite for PEPR1 internalization.
基金supported by the National Natural Science Foundation of China(32272504)Chinese Universities Scientific Fund(2024TC187)to F.C.
文摘To dissect the signaling networks that orchestrate immune gene expression,we developed a sensitive genetic screen using an ethyl methanesulfonate(EMS)-mutagenized population of Arabidopsis transgenic plants.These plants carry an early immune responsive gene FRK1(flg22-induced receptor-like kinase 1)promoter fused with a luciferase(LUC)reporter.A series of mutants with altered pFRK1::LUC activity were identified and named as Arabidopsis genes governing immune gene expression(aggie).The aggie5 mutant showed reduced pFRK1::LUC induction in response to multiple MAMPs treatments.Moreover,the activation of the endogenous FRK1 gene by MAMPs was also compromised in aggie5.Significantly,the bacterial flagellin epitope flg22-induced MAPK activation,ROS production,and immunerelated gene expression were reduced in aggie5.Map-based cloning coupled with next-generation sequencing revealed that aggie5 encodes a kinase-inactive BAK1,which is involved in brassinosteroid responses,innate immunity,and cell death control.Consistently,the aggie5/bak1-16 mutant displayed compromised responses to BR treatment.Furthermore,the aggie5/bak1-16 mutant exhibited seedling lethality when combined with the mutation of its closest homolog BKK1/SERK4.The data suggest that BAK1 kinase activity is required for its multiple functions in plant immunity,development,and cell death control.The isolation and characterization of these Aggie genes will contribute to a better understanding of host immune signaling and provide genetic resources to improve crop resistance.
