【病毒外文文献】2004 Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells

Tyrosine dephosphorylation of STAT3 E6 Murakami Shigeru 1 National Tokyo Available online 14 Signal transducer and activator of transcription 3 p38 Extracellular signal regulated kinase 1 2 c Jun N terminal responses Recently we reported that p38 mitogen activated by dominant negative and antisense STAT3 inhibitors resulted in a decrease in cell viability and subsequent apoptosis 13 15 phosphorylated at tyrosine Tyr 705 and slightly phosphory FEBS 28924 FEBS Letters 577 2004 187 192 Corresponding author Fax 81 42 564 4881 E mail address tmizutan nih go jp T Mizutani lated at serine Ser 727 in Vero E6 cells was dephosphoryl ated at Tyr 705 by activation of p38 MAPK on SARS CoV infection Lack of transcriptional activity of STAT3 by viral infection may decrease anti apoptotic activity in the cells kinase 1 Introduction Severe acute respiratory syndrome SARS is a newly found infectious disease caused by a novel coronavirus SARS co ronavirus SARS CoV 1 2 In late 2002 SARS CoV spread from Guangdong Province in China to more than 30 countries The pathogenesis of SARS in vivo may be mediated by both the e ect of viral replication in the target cells and immune Thus STAT3 is thought to act as an anti apoptotic tran scription factor Recent studies indicated that interactions between STATs and some viral proteins cause degradation of STATs in virus infected cells For example V protein of measles virus forms complexes with STAT1 STAT2 and STAT3 and inhibits extracellular IL 6 and intracellular v Src STAT3 dependent signaling 16 Thus measles virus in duced degradation of STATs may provide a mechanism for virus induced cytokine inhibition that links innate immune evasion to adaptive immune suppression In the present study we found that STAT3 constitutively Edited by Hans Diete Abstract Severe acute respiratory syndrome SARS has become a global public health emergency p38 mitogen activated protein kinase MAPK and its downstream targets are activated in SARS coronavirus SARS CoV infected Vero E6 cells and activation of p38 MAPK enhances the cytopathic e ects of SARS CoV infection In addition weak activation of Akt cannot prevent SARS CoV infection induced apoptosis in Vero E6 cells In the present study we demonstrated that signal transducer and activator of transcription STAT 3 which is constitutively phosphorylated at tyrosine Tyr 705 and slightly phosphory lated at serine Ser 727 in Vero E6 cells was dephosphorylated at Tyr 705 on SARS CoV infection In addition to phosphory lation of p38 MAPK in virus infected cells other MAPKs i e extracellular signal regulated kinase ERK 1 2 and c Jun N terminal kinase JNK were phosphorylated Although inhibi tors of ERK1 2 and JNK PD98059 and SP600125 had no e ect on phosphorylation status of STAT3 inhibitors of p38 MAPK SB203580 and SB202190 partially inhibited dephos phorylation of STAT3 at Tyr 705 Tyr 705 phosphorylated STAT3 was localized mainly in the nucleus in mock infected cells whereas STAT3 disappeared from the nucleus in virus infected cells As STAT3 acts as an activator of transcription in the nucleus these results suggest that STAT3 lacks its activity on transcription in SARS CoV infected Vero E6 cells C211 2004 Federation of European Biochemical Societies Published by Elsevier B V All rights reserved Keywords Severe acute respiratory syndrome coronavirus Vero Tetsuya Mizutani a Shuetsu Fukushi a Masaaki Ichiro Kurane a a Special Pathogens Laboratory Department of Virology Musashimurayama b Department of Molecular Oncology Graduate School of Medicine Suita Osaka Received 23 July 2004 revised 27 September 0014 5793 22 00 C211 2004 Federation of European Biochemical Societies Published doi 10 1016 j febslet 2004 10 005 in SARS coronavirus infected cells b Toshio Hirano b Masayuki Saijo a Morikawa a Institute of Infectious Diseases Gakuen 4 7 1 208 0011 Japan Graduate School of Frontier Biosciences Osaka University Japan 2004 accepted 4 October 2004 October 2004 r Klenk protein kinase MAPK plays important roles in the cytopathic e ects and apoptosis in SARS CoV infected cells 3 Fur thermore weak activation of Akt cannot prevent apoptosis by SARS CoV infection 4 Thus it is necessary to examine the signaling pathways in SARS CoV infected cells in culture to understand the molecular mechanisms of its pathology in vivo MAPKs are signal transducers that respond to extracellular stimulation by cytokines growth factors viral infection and stressors and in turn regulate cell di erentiation proliferation survival and apoptosis 5 8 p38 MAPK is strongly activated by stressors and inflammatory cytokines Our previous study indicated that p38 MAPK phosphorylation in SARS CoV infected Vero E6 cells reached the maximal level at 18 h post infection h p i and cytopathic e ects CPEs were observed from 24 h p i 3 The CPEs were partially prevented by treatment with the p38 MAPK inhibitor SB203580 strongly suggesting that the p38 MAPK signaling pathway is involved in the control of cell death in SARS CoV infected Vero E6 cells On the other hand signal transducer and activator of transcription STAT proteins are transcription factors that mediate cytokines and growth factors Activation of all STAT proteins is induced by phosphorylation of a single tyrosine residue leading to dimerization via an intermolecular SH2 phosphotyrosine interaction 9 12 STAT3 is a major tran scription factor activated in response to cytokines such as interleukin 6 IL 6 and IL 10 Inhibition of STAT3 signaling by Elsevier B V All rights reserved virus infection SARS CoV which was isolated as Frankfurt 1 17 and lated to several signaling pathways that responded specifically to SARS CoV infection including p38 MAPK and Akt were found In the present study we examined whether signal transducer and activator of transcription 3 STAT3 was dysregulated on infection with SARS CoV as reported for measles virus infection 16 Vero E6 cells were infected with SARS CoV at m o i of 10 and cellular proteins were har vested at 6 12 18 and 24 h p i Western blotting analysis was performed using a series of anti STAT3 antibodies that rec ognized total STAT3 or Tyr 705 or Ser 727 phosphorylated forms of STAT3 In Vero E6 cells STAT3 was phosphorylated constitutively at Tyr 705 whereas Ser 727 was only slightly phosphorylated Fig 1 mock infection Constitutive activa tion of STAT3 was observed in breast carcinoma cell lines 19 Interestingly Tyr 705 phosphorylated STAT3 was not de tected after 18 h p i in SARS CoV infected Vero E6 cells even 188 T Mizutani et al FEBS Letters 577 2004 187 192 kindly provided by Dr J Ziebuhr was used in the present study In fection was usually performed with a multiplicity of infection m o i of 10 2 2 Treatment with inhibitors SB203580 and SB202190 as p38 MAPK inhibitors PD098059 as a MEK inhibitor and SP600125 as a JNK inhibitor were dissolved in dimethyl sulfoxide DMSO at a concentration of 10 or 20 mM All reagents were purchased from Calbiochem San Diego CA USA All test wells including mock treated controls were treated with 0 25 DMSO v v Vero E6 cells were inoculated with SARS CoV at m o i of 10 for 1 h and then cells were treated with inhibitors for 17 h 2 3 Subcellular fractionation SARS CoV infected or mock infected Vero E6 cells at 18 h p i were subjected to subcellular fractionation using a Subcellular Proteome Extraction Kit Calbiochem according to the manufacturer s in structions Each subcellular fraction was then analyzed by Western blotting 2 4 Western blotting After virus infection whole cell extracts were electrophoresed on either 12 5 or 10 20 gradient polyacrylamide gels and transferred onto PVDF membranes Immobilon P Millipore Bedford MA USA In the present study we applied two sets of samples to poly acrylamide gels and the membranes were divided into two halves after blotting The following antibodies obtained from Cell Signaling Technology Inc Beverly MA USA were used in the present study at a dilution of 1 1000 rabbit anti phospho STAT3 Tyr 705 anti body rabbit anti phospho STAT3 Ser 727 antibody rabbit anti p38 MAPK Thr180 Tyr182 antibody rabbit anti p38 MAPK antibody rabbit anti phospho p44 42 MAPK Thr202 Tyr204 ERK1 2 antibody rabbit anti p44 42 MAPK ERK1 2 antibody rabbit anti phospho SAPK JNK Thr183 Tyr185 antibody anti SAPK JNK antibody anti phospho MEK1 2 Ser217 221 antibody anti MEK1 2 antibody anti phospho SEK1 MKK4 Thr261 antibody anti SEK1 MKK4 antibody anti phospho MKK7 Ser271 Thr275 antibody anti MKK7 antibody anti phospho MKK3 MKK6 Ser189 207 antibody anti MKK3 antibody anti phospho Jak1 Tyr1022 1023 an tibody anti phospho JAK2 Tyr1007 1008 anti body and anti phospho Tyk2 Tyr1054 1055 antibody Mouse anti STAT3 antibody diluted 1 2500 mouse anti JAK1 antibody diluted 1 250 and mouse anti Tyk2 antibody diluted 1 1000 obtained from BD Biosciences Franklin Lakes NJ USA were also used Rabbit anti JAK2 antibody C 20 diluted 1 200 was obtained from Santa Cruz Biotechnology Santa Cruz CA USA After 15 h incubation with the above antibodies the membranes were washed with Tris borate saline containing 0 1 Tween 20 0 1 TBS Tween and the reactions were detected with a ProtoBlot II AP system Promega Co Madison WI USA as described previously 18 3 Results 3 1 Tyrosine dephosphorylation of STAT3 in SARS CoV infected cells As described in our previous report we compared the cel lular protein profiles of SARS CoV infected 18 h p i when apoptosis was not evident and mock infected Vero E6 cells by Western blotting using 125 antibodies to investigate cellular responses to SARS CoV infection 3 4 Cellular proteins re 2 Materials and methods 2 1 Cells and virus Vero E6 cells were routinely subcultured in 75 cm 3 flasks in Dul becco s modified Eagle s medium DMEM Sigma St Louis MO USA supplemented with 0 2 mM L glutamine 100 units ml penicillin 100 lg ml streptomycin and 5 v v fetal bovine serum FBS and maintained at 37 C176C in an atmosphere of 5 CO 2 For use in the ex periments the cells were split once onto 6 or 24 well tissue culture plate inserts and cultured until they reached 100 confluence The culture medium was changed to 2 FBS containing DMEM prior to though the total amount of STAT3 did not change until 24 h p i Fig 1 This result suggested that STAT3 Tyr 705 was dephosphorylated from 18 to 24 h p i On the other hand Ser 727 phosphorylated STAT3 was slightly increased at the same time points Based on the hypothesis that tyrosine phosphor ylation of STAT is necessary for its activation 9 12 these results suggest that the level of activation of STAT3 was de creased in SARS CoV infected Vero E6 cells Thus the mechanism of dysregulation of STAT3 by SARS CoV is dif ferent from that by measles virus in which Measles virus V protein forms complexes with STATs resulting in degradation of STAT3 3 2 Phosphorylation level of upstream kinases of STAT3 As a general explanation of signal transduction of STAT3 binding of IL 6 to the receptor induces dimerization of the common gp130 signal transduction subunit of the IL 6 family of cytokine receptors and then Janus kinases JAK1 and 2 and Tyk2 are phosphorylated at tyrosine residues through a conserved membrane proximal binding domain 20 The phosphorylated JAKs and Tyk2 create docking sites for STAT3 Dimeric STAT3 Tyr phosphorylated by JAKs and Tyk2 migrates to the nucleus where STAT3 activates tran scription of specific genes Thus JAK1 JAK2 Tyk2 and STAT3 are phosphorylated in response to IL 6 To test the hypothesis that Tyr 705 dephosphorylation of STAT3 by SARS CoV infection occurred due to a lack of signals from JAK1 2 and Tyk2 the phosphorylation status of these kinases was examined in virus infected or mock infected Vero E6 cells As shown in Fig 2 JAK1 JAK2 and Tyk2 were phosphor Fig 1 Infection of Vero E6 cells with SARS CoV a ected the signaling pathway of STAT3 Western blotting analysis of proteins from SARS CoV infected Vero E6 cells was performed using antibodies that rec ognized forms of STAT3 phosphorylated at Tyr 705 or Ser 727 ylated at low levels in mock infected cells whereas no signifi cant changes in phosphorylation level of these kinases were observed after virus infection These observations suggest that Tyr 705 dephosphorylation of STAT3 in virus infected cells occurred independent of its upstream kinases Therefore total activity of STAT3 may be low in Vero E6 cells However it can not be ruled out that the anti phospho JAK1 JAK2 and Tyk2 antibodies used in the present study are di cult to be recognized in the phosphorylated JAK1 JAK2 and Tyk2 in Vero E6 cells as the datasheet included no description of cross reactivity with monkey 3 3 Stimulation of Tyr 705 phosphorylation of STAT3 by cytokines Previous studies have indicated that at least six cytokines IL 2 IL 6 IL 10 IL 15 IL 17 and IL 22 can stimulate ac tivation of STAT3 21 24 Tyr 705 dephosphorylation of STAT3 to be phosphorylated in Vero E6 cells stimulated by SARS CoV infection and IL 22 Although the phosphoryla tion level of p38 MAPK was not changed in SARS CoV in fected Vero E6 cells by treatment with IL 22 for 20 min at 18 h p i the Tyr 705 phosphorylation of STAT3 increased Fig 4B These results suggest that signaling pathway via IL 22 is not regulated by p38 MAPK in Vero E6 cells 3 4 Tyr 705 phosphorylated STAT3 in the nucleus To determine the localization of Tyr 705 phosphorylated STAT3 in Vero E6 cells subcellular extraction was performed using a Subcellular Proteome Extraction Kit Calbiochem and then Western blotting analyses were performed As described above the amounts of total and Tyr 705 phosphorylated STAT3 were similar in cells grown in media containing 0 2 or 5 FCS The subcellular localization of STAT3 was not formed using anti phospho STAT3 Tyr antibody B C M N S and T indicate cytosolic organelle membrane nuclear cytoskeletal and total cellular fraction respectively Fig 4 IL 22 induces Tyr phosphorylation of STAT3 A Vero E6 cells were treated with IL 22 from 0 to 90 min Cellular proteins were sampled every 15 min Western blotting was performed using anti phospho STAT3 Tyr and Ser antibodies B SARS CoV infected Vero E6 cells were treated with IL 22 for 20 min at 18 h p i and then Western blot analysis was performed using anti phospho STAT3 Tyr and anti phospho p38 MAPK T Mizutani et al FEBS Letters 577 2004 187 192 189 STAT3 in virus infected cells may be due to a lack of stimu lation by these cytokines after 18 h p i Therefore it is im portant to identify the cytokines responsible for stimulating Tyr phosphorylation of STAT3 to understand the mechanism of Tyr 705 dephosphorylation of STAT3 in virus infected cells To investigate whether fetal calf serum FCS in the medium contains stimulators for STAT3 phosphorylation Vero E6 cells were cultured in DMEM containing 0 2 or 5 FCS for 18 h subjected to subcellular fractionation and then the proteins were analyzed by Western blotting using anti phospho specific antibodies As shown in Fig 3A there were no significant di erences in total amount of STAT3 in cultures with various concentrations of FCS in the medium This result suggests that FCS does not contain components that stimulate Tyr phosphorylation of STAT3 We next de termined cytokines and their receptors produced in Vero E6 cells using a GEArray Q Series Human Interleukin and Re ceptor Gene Array SuperArray Bioscience Corporation Frederick MD USA Expression level of IL 22 which has also been reported as a stimulator of STAT3 23 was very low and receptors for IL 22 IL 22RA1 and RA2 were ob tained as strong and weak signals respectively data not shown Therefore we examined whether IL 22 stimulated Tyr 705 and Ser 727 phosphorylation of STAT3 As shown in Fig 4A the level of Tyr 705 phosphorylated STAT3 increased 15 min after treatment with murine IL 22 200 ng ml Pepro Tech EC London UK whereas phosphorylation of Ser 727 was not enhanced suggesting that it is di cult for Ser 727 of Fig 2 Phosphorylation levels of upstream kinases of STAT3 in virus infected cells Western blotting analyses were performed using anti phospho JAK1 JAK2 and Tyk2 antibodies Fig 3 Subcellular localization of Tyr phosphorylated STAT3 A Vero E6 cells were incubated in DMEM containing 0 2 or 5 FCS for 18 h After subcellular fractionation Western blotting was per a ected by the concentration of FCS Fig 3A Total STAT3 was located mainly in the cytosol and also in membranes or ganelles and the nuclear fractions On the other hand Tyr 705 phosphorylated STAT3 appeared mainly in the nuclear fraction We next examined whether Tyr 705 phosphorylated STAT3 was not present in the nucleus in SARS CoV infected Vero E6 cells at 18 h p i As shown in Fig 3B Tyr 705 phosphorylated STAT3 had clearly disappeared from the nuclear fraction in virus infected cells This result strongly suggested that STAT3 did not act as a transcriptional enhancer in SARS CoV infected Vero E6 cells after 18 h p i 3 5 Phosphorylation of MAPKs in SARS CoV infected cells Our previous report indicated that SARS CoV infection to Vero E6 induced phosphorylation of p38 MAPK and its downstream targets HSP 27 eIF4E and CREB 3 Phos phorylation of these proteins was prevented by treatment of the cells with the p38 MAPK inhibitor SB20854 MAPKs were reported to induce Ser 727 phosphorylation of STAT3 25 To investigate whether other MAPKs i e ERK1 2 extracellular signal regulated kinase and JNK are also phosphorylated in SARS CoV infected Vero E6 cells the kinetics of phosphor ylation of ERK1 2 and JNK were analyzed by Western blot ting Vero E6 cells were infected with SARS CoV at m o i of 10 and the cell extracts were prepared at various time points after infection Western blotting analysis demonstrated that MKK4 7 respectively As shown in Fig 5 MKK3 6 MEK1 2 and MKK4 7 were phosphorylated in SARS CoV infected Vero E6 cells 3 6 Tyr dephosphorylation of STAT3 by p38 MAPK To investigate whether Tyr 705 phosphorylation is regulated by activated MAPKs in SARS CoV infected Vero E6 cells the infected cells were treated for 18 h with three MAPK inhibi tors SB203580 p38 MAPK inhibitor PD98059 MEK inhibitor and SP600125 JNK inhibitor Tyr 705 and Ser 727 phosphorylated STAT3 were then analyzed by Western blotting As shown in Fig 6A SB203580 did not a ect Ser 727 phosphorylation of STAT3 while SARS CoV induced de phosphorylation of STAT3 Tyr 705 was partially inhibited by SB203580 On the other hand neither PD98059 nor SP600125 a ected STAT3 phosphorylation To confirm whether inhibi tion of p38 prevents dephosphorylation of STAT3 Tyr 705 virus infected cells were treated with another p38 inhibitor SB202190 As shown in Fig 6B Tyr 705 dephosphorylation of STAT3 in the infected cells was also partially inhibited by SB202190 These results indicated that activated p38 MAPK in SARS CoV infected Vero E6 cells regulates Tyr 705 phos phorylation of STAT3 but not that of Ser 727 4 Discussion In the present and previous studies we reported that the 190 T Mizutani et al FEBS Letters 577 2004 187 192 levels of phosphorylated ERK1 2 and JNK were increased in SARS CoV infected cells Fig 5A Two phosphorylated forms of ERK ERK1 and ERK2 were detected at 12 h p i and accumulated continuously up to 24 h p i The kinetics of accumulation of phosphorylated ERK1 2 and JNK in the in fected cells were similar to that of accumulation of phos phorylated p38 MAPK We next examined whether the upstream MAPK kinases MAPKKs were also phosphory lated in SARS CoV infected Vero E6 cells The kinases of p38 ERK1 2 and JNK are known as MKK3 6 MEK1 2 and Fig 5 MAPKs phosphorylation in virus infected cells Western blot ting analysis of proteins from SARS CoV infected Vero E6 cells was performed using anti phospho ERK1 2 JNK MEK1 2 MKK4 MKK7 and MKK3 6 antibodies Fig 6 E ects of treatment of SARS CoV infected Vero E6 cells with p38 MAPK inhibitor A Vero E6 cells were infected with SARS CoV at m o i of 10 and then incubated with SB203580 PD98059 and SP600125 at concentrations from 10 to 50 lM for 17 h Western blotting