5 BAD JNK UV 515 c-jun c-jun N-terminal 1 2 kinase JNK / JNK JNK - / - stress-activated MAP BAD BAD - / - kinase SAPK Professor Lin MEF JNK - / - mito

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 520 DOI /j cnki cjbmb BAD JNK UV * JNK BAD bcl-2 BAD JNK JNK UV PARP ADP UV MEF JNK sirna BAD MEF UV UV MEF GST-BAD UV UV BAD JNK GST-BAD JNK BAD Thr-201 BAD JNK T201A BAD BAD - / - BAD T201 JNK c-jun BAD JNK BAD UV BAD BAD UV JNK BAD bcl-2 Q291 BAD and JNK Regulates UV-induced Apoptosis CHEN Min 1 SHI Wei 1 ZHAO Jun 1 HAN Zhi-Yuan 2 * 1 Guangzhou First People s Hospital Guangzhou China 2 Guangzhou Medical University Guangzhou China Abstract JNK and BAD bcl-2 associated death promoter are known to regulate apoptosis We demonstrated that BAD could be a substrate of JNK and involved in the regulation of ultraviolet UV induced apoptosis Western blotting for PARP poly ADP-ribose polymerase cleavage and flow cytometry revealed that JNK was activated in UV-induced apoptosis in MEF cells RNAi knock-down of BAD increased the sensitivity to apoptosis The JNK in the lysates from apoptotic cells phosphorylated GST-BAD By in vitro incubation of synthetic JNK with GST-BAD the results showed that BAD phosphorylation occurred at Thr-201 Transfection of wild type or T201A mutant of BAD showed that the BAD phosphorylation inhibited the JNK kinase activity and the phophosrylation of c-jun suggesting a negative regulation to JNK activity The data suggested that BAD might be a substrate of JNK to be phosphorylated upon UV exposure Phosphrylated BAD suppresses the activity of JNK kinase to attenuate apoptotic cell death Key words ultraviolet UV cellular apoptosis JNK signal pathway BAD bcl-2 associated death promoter No * hanzy armstrong@ gmail com Received October Accepted March Supported by National Natural Science Foundation of China No * Corresponding author hanzy armstrong@ gmail com

2 5 BAD JNK UV 515 c-jun c-jun N-terminal 1 2 kinase JNK / JNK JNK - / - stress-activated MAP BAD BAD - / - kinase SAPK Professor Lin MEF JNK - / - mitogen-activated protein kinase MAPK BAD - / - 10% DMEM 1 2 UV FAS TNFα BAD bcl-2 associated death 3 promoter BAD bcl-2 Lipofectmine 2000 Bcl BAD bcl-2 20 min r /min 20 min JNK BAD SDS-PAGE BAD UV UV 20 J MEF JNK - / - UV 1 JNK 1 1 GST-BAD ìci 32P-ATP 30 min 2 PARP actin JNK BAD Sigma c-jun Cell Signaling GST-BAD PAGE - 70 X- 2 h Annexin V /PI JNK GST-BAD 32 p ATP PerkinElmer 30 GSH-beads Caspase 3 Abcam Lipofectmine 2000 Invitrogen JNK GenScript sirna IDT sibad 5'-AGCUCCUGUUUGGAGUUUCA-3' sijnk 5'-UGAUUCAGAUGGAGUUAGA-3' sictrl 5'-GGAGCGCACCAUCUUCUUC-3' BAD BAD JNK cdna 1 6 pcdna3 1-Hygromycin + JNK 3 Thr-201 T201A SPSS11 0 ± JNK Stratgene t P < % CO % sirna SDS-PAGE GST-BAD yale 1 5 Annexin V /PI UV Annexin V /PI Annexin V PI Annexin V

516 31 2 UV JNK - / - 2 1 UV sirna JNK MEF Fig 1A PARP poly ADP-ribose polymerase DNA Fig 1B sirna JNK PARP PARP Caspase 8 Annexin V /PI Caspase PARP MEF JNK - / - 8 PRAR Fig 1C UV MEF 60% JNK - / -

3 UV JNK - / UV sirna JNK MEF Fig 1A PARP poly ADP-ribose polymerase DNA Fig 1B sirna JNK PARP PARP Caspase 8 Annexin V /PI Caspase PARP MEF JNK - / - 8 PRAR Fig 1C UV MEF 60% JNK - / - UV UV 20 J Caspase 3 Fig 1D UV PARP JNK - / - Caspase 3 Fig 1A MEF UV 15 h PARP JNK UV JNK MFE UV JNK - / - PARP Fig 1 Function of JNK pathway in UV induced cellular apoptosis A Wild type and JNK - / - MEFs were irradiated with UV 20J for the indicated hours Western blot analysis of PARP cleavage was performed with anti-parp antibody B Wild type MEFs were transfected with JNK1 sirna Twenty four hours later cells were treated with UV Protein levels of JNK and PARP were detected by Western blot C Wild type MEFs were transfected with JNK1 sirna and treated with UV 20J for 20 hours cells were stained with Annexin V /PI to analyze cellular apoptosis by flow cytometry D Wild type and JNK - / - cells were treated with UV 20J for 20 hours Caspase 3 activity was analyzed ** P < 0 01 *** P < 0 001

4 5 BAD JNK UV BAD UV 2B BAD PARP BAD UV sibad BAD MEF BAD UV sibad Annexin V / UV PI MEF BAD - / - Fig 2A BAD sirna Fig 2C BAD sirna MEF UV BAD - / - UV 10 ~ 20 h PARP 60% Caspase 3 Fig 2D sirna UV UV BAD - / - Caspase 3 BAD BAD MEF BAD - / - UV Fig BAD UV Fig 2 BAD involves in the regulation of UV induced apoptosis A Wild type MEFs were transfected with BAD sirna Twenty four hours later cells were treated with UV 20J for the indicated time Western blot analysis of PARP cleavage and BAD protein were performed with anti-parp and anti-bad antibody respectively B BAD - / - cells were transfected with BAD expressing plasmid Twenty four hours later cells were treated with UV and analyzed PARP cleavage C Wild type and BAD - / - cells were treated with UV 20J for 20 hours cells were stained with Annexin V /PI to analyze cell apoptosis by flow cytometry D Wild type and BAD - / - cells were treated with UV 20J for 20 hours Caspase 3 activity was analyzed ** P < 0 01 *** P < BAD BAD UV JNK JNK JNK Fig 3A BAD 204 UV MEF JNK GST-BAD UV BAD JNK - / - UV JNK

518 31 BAD Fig 3 The phosphorylation of BAD by JNK A Wild type and JNK - / - MEFs were treated with UV 20 J and incubated for 1 hour Immuno-precipitated IP was performed with anti-jnk antibody to

autoradiography Input GST-BAD was analyzed by Coomassie brilliant blue CBB staining B GST-BAD proteins phosphorylated by active JNK in vitro were tryptic digested and then analyzed by mass

5 BAD Fig 3 The phosphorylation of BAD by JNK A Wild type and JNK - / - MEFs were treated with UV 20 J and incubated for 1 hour Immuno-precipitated IP was performed with anti-jnk antibody to separate the JNK kinase from cell lysate JNK kinase was precipitated and incubated with GST- BAD for kinase assay described as the Methods section Phosphorylated substrate was detected by autoradiography Input GST-BAD was analyzed by Coomassie brilliant blue CBB staining B GST-BAD proteins phosphorylated by active JNK in vitro were tryptic digested and then analyzed by mass spectrometry Insert shows the recovered phosphorylated peptide fragment corresponding to Thr-201 JNK BAD JNK MEF JNK GST- BAD BAD Fig BAD - / - Fig 4A 3B JNK BAD Thr-201 Fig 4B BAD UV BAD JNK JNK JNK JNK BAD Thr-201 c-jun JNK 2 4 BAD JNK JNK JNK JNK UV 20J MEF BAD - / - Fig 4A BAD - / - JNK MEF JNK UV BAD - / - BAD JNK BAD JNK Fig 4 BAD affects JNK phosphorylation A Wild type and BAD - / - MEFs were irradiated with UV 20 J for the indicated time Western blot analysis of phosphorylated JNK was performed with anti-phospho-jnk antibody SE short exposure LE long exposure B BAD - / - cells were transfected with BAD expressing plasmid for 24 hours cells were treated with UV Western blot analyses of phospho-jnk phospho-c-jun and BAD were performed with specific antibodies 2 5 BAD JNK JNK BAD BAD Thr-201 T201A T201A BAD - / - UV Fig 5 BAD BAD T201A PARP Fig 5A Caspase 3 Fig 5B JNK c-jun Fig 5A BAD JNK BAD UV

5 BAD JNK UV 519 Fig 5 BAD phosphorylation affects JNK activity and cellular apoptosis A BAD - / - MEFs were transfected with wild type WT or Thr-201 mutant T201A BAD expressing plasmid After

6 5 BAD JNK UV 519 Fig 5 BAD phosphorylation affects JNK activity and cellular apoptosis A BAD - / - MEFs were transfected with wild type WT or Thr-201 mutant T201A BAD expressing plasmid After transfection for 24 hours cells were treated with UV 20 J for the indicated hours Western blot analyses of PARP phospho-jnk phospho-c- Jun and BAD were performed with specific antibodies respectively B BAD - / - MEFs were treated as described in A and 20 hours after irradiation Caspase 3 activity was analyzed * P < 0 05 UV 15 / 16 UV JNK JNK JNK c- jun c-jun JNK BAD JNK BAD Thr-201 BAD UV BAD BAD - / - sirna BAD Fig 2A 2B UV BAD UV BAD 2 3 BAD BAD BAD 3 BAD UV UV UV JNK Fig 1 cross talk 7 JNK BAD UV UV JNK BAD JNK 9-11 BAD Fig UV 4 JNK BAD UV Fig 5 JNK BAD BAD JNK JNK 12 JNK Fig 6 UV mitogen-activated protein kinase UV MAPK MAP kinase JNK JNK kinase MKK BAD BAD JNK JNK BAD JNK

7 JNK UV BAD BAD UV UV UV Bcl-2 J Fig 6 A schematic presentation of the mechanism of BAD cross talk with JNK in UV induced cellular apoptosis References 1 Vlahopoulos S Zoumpourlis V C JNK a key modulator of intracellular signaling J Biochemistry Ablp Y T Davis R J Signal transduction by the c-jun N- terminal kinase JNK -from inflammation to development J Curr Opin Cell Biol Chen L Willis S N Wei A et al Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function J Mol Cell Yang E Zha J Jockel J et al Bad a heterodimeric partner for Bcl-XL and Bcl-3 displaces Bax and promotes cell death J Cell Cleary M L Smith S D Sklar J Cloning and structural analysis of cdnas for bcl-2 and a hybrid bcl-2 / immunoglobulin transcript resulting from the t translocation J Cell Adachi M Imai K The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Cell Death Differ Harada H Grant S Apoptosis regulators J Rev Clin Exp Hematol Yu S W Andrabi S A Wang H et al Apoptosis-inducing factor mediates poly ADP-ribose PAR polymer-induced cell death J Pro Natl Acad Sci U S A Ji C Yang Y L Yang Z et al Perifosine sensitizes UVBinduced apoptosis in skin cells new implication of skin cancer prevention J Cell Signal Rodust P M Stockfleth E Ulrich C et al UV-induced squamous cell carcinoma- a role for antiapoptotic signaling pathways J Br J Dermatol suppl Katiyar S K Meeran S M Obesity increases the risk of UV radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling J Radic Biol Med Tournier C The 2 faces of JNK Signaling in Cancer J Genes Cancer Yamasaki T Kawasaki H Nishina H Diverse Roles of JNK and MKK Pathways in the Brain J J Signal Transduct doi /2012 / Haeusgen W Herdegen T Waetzig V The bottleneck of JNK signaling molecular and functional characteristics of MKK4 and MKK7 J Eur J Cell Biol Sehgal V Ram P T Genes Cancer Network Motifs in JNK Signaling J 16 Son Y Kim S Chung H T et al Reactive oxygen species in the activation of MAP kinase J Methods Enzymol Ebelt N D Cantrell M A Van Den Berg C L C-Jun N-terminal kinases mediate a wide range of targets in the metastatic cascade J Genes Cancer Rana A Rana B Mishra R et al Mixed lineage kinase-c-jun N-terminal kinase axis A potential therapeutic target in cancer J Genes Cancer

标题

环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨