Tyrosine Kinase C-Abl Potentiates Interferon-mediated Antiviral Immunity Los ntawm STAT1 Phosphorylation
Nov 06, 2023
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Interferon (IFN)-induced activation ntawm lub teeb liab transducer thiab activator ntawm transcription (STAT) tsev neeg yog ib qho kev tshwm sim tseem ceeb hauv kev tiv thaiv kab mob. Ntawm no, peb qhia tau hais tias cov nonreceptor kinases c-Abl thiab Arg ncaj qha cuam tshuam nrog STAT1 thiab potentiate phosphorylation ntawm STAT1 ntawm Y701. c-Abl/Arg tuaj yeem kho STAT1 phosphorylation ywj siab ntawm Janus kinases thaum tsis muaj IFNg thiab potentiate IFNg-mediated STAT1 phosphorylation. Ntxiv mus, STAT1 dimerization, nuclear translocation, thiab downstream gene transcription yog tswj los ntawm c-Abl / Arg. c-Abl/Arg (abl1/abl2) deficiency ua rau muaj kev cuam tshuam cov tshuaj tiv thaiv kab mob hauv cov kab mob vesicular stomatitis. Piv nrog rau lub tsheb, kev tswj hwm ntawm c-Abl / Arg selective inhibitor AMN107 ua rau muaj kev tuag ntau ntxiv hauv cov nas uas kis tus kab mob khaub thuas tib neeg. Peb txoj kev tshawb fawb pom tau tias c-Abl ua lub luag haujlwm tseem ceeb hauv STAT1 kev ua kom pom tseeb txoj hauv kev thiab muab txoj hauv kev tseem ceeb rau kev tiv thaiv kab mob tiv thaiv kab mob.
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Taw qhia
Interferons (IFNs) ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob hauv lub cev tiv thaiv kab mob thiab kev tiv thaiv kab mob los ntawm kev tswj hwm kev ua kom Janus kinases (JAKs) thiab teeb liab transducer thiab activator ntawm transcription (STAT) thiab los ntawm reprogramming cellular gene qhia (Stark thiab Darnell, 2012). Kev sib txuas ntawm IFNs rau lawv cov receptors ua rau muaj qhov sib txawv ntawm phosphorylation ntawm peb JAKs (JAK1, JAK2, thiab TYK2) thiab kev nrhiav neeg ua haujlwm ntawm STATs, tso cai rau ib qho tyrosine residues nyob ze ntawm C-terminal kawg ntawm cov STATs phosphorylated los ntawm JAKs. (Platanias, 2005). Cov STATs ua haujlwm ces dimerize, tsiv mus rau hauv lub nucleus, thiab khi rau cov neeg txhawb nqa ntawm IFN-tej noob caj noob ces txhawm rau txhawb lawv cov ntawv sau. Lub tyrosine phosphorylation ntawm STATs yog ib qho tseem ceeb hauv JAK-STAT txoj hauv kev ntawm IFN signaling thiab koom nrog STAT dimerization, nuclear translocation, thiab DNA binding (Stark thiab Darnell, 2012). Yog li, tyrosine phosphorylation ua haujlwm raws li STAT ua kom hloov. Cov ntaub ntawv pov thawj tau los ntawm kev cuam tshuam ntawm JAK cov noob hauv nas qhia tias JAKs yog qhov tseem ceeb tshaj plaws ntawm STAT tyrosine kinases (Villarino li al., 2017). Txawm li cas los xij, tyrosine residues hauv STAT1, STAT3, thiab STAT5 kuj tau pom tias muaj phosphorylated hauv JAK-tsis muaj cov hlwb los ntawm cov kab mob epidermal loj hlob thiab platelet-derived kev loj hlob receptors nrog intrinsic tyrosine kinase kev ua (Leaman li al., 1996; Vignaiset. Ib., 1996). Tsis tas li ntawd, kev ua kom muaj zog ntawm STATs tau pom nyob rau hauv cov hlwb hloov pauv uas qhia txog oncogenic tyrosine kinases xws li v-Src, v-Abl, thiab BCR-Abl, tab sis seb STATs yog qhov ncaj qha substrates ntawm cov kinases tseem yuav txiav txim siab (Danial thiab Rothman. , 2000; Reddy et al., 2000). Cov tsiaj txhu Abelson kinase c-Abl encoded los ntawm c-abl (abl1) gene thiab Abl-related gene protein Arg encoded los ntawm arg (abl2) gene yog cov tswv cuab ntawm tsev neeg ntawm intracellular nonreceptor tyrosine kinases uas yuav tsum tau rau ntau yam txheej txheem ntawm tes, suav nrog kev loj hlob, apoptosis, adhesion, cell migration, thiab kev ntxhov siab (Bradley thiab Koleske, 2009; Colicelli, 2010; Greuber li al., 2013; Pendergast, 2002). Cov kev ua kinase ntawm c-Abl yog nruj tswj nyob rau hauv ib txwm physiological mob. Retroviral transduction thiab chromosomal translocation xwm txheej ua rau muaj kev qhia ntawm v-Abl lossis BCR-Abl, ib daim ntawv ntawm oncogenic ntawm Abl, raws li (Advani thiab Pendergast, 2002). Tsis tas li ntawd, lub hom phiaj tshem tawm ntawm c-abl noob hauv cov nas ua rau pleiotropic phenotypes cuam tshuam nrog kev tiv thaiv kab mob, nrog rau kev ua kom lub cev tsis zoo, splenic thiab thymic atrophy, lymphopenia, thiab nce kev kis kab mob (Schwartzberg li al., 1991). Ntxiv mus, kev kho mob ntawm BCR-Abl-positive chronic myeloid leukemia (CML) nrog Abl inhibitors STI571 (imatinib) thiab AMN107 (nilotinib) yog txuam nrog kev tiv thaiv kab mob hauv cov neeg mob (Dietz li al., 2004; Hochhaus li al., 2016; Mattiuzzi et al., 2003). Cov kev tshawb fawb yav dhau los tau pom tias cAbl ua lub luag haujlwm hauv kev tswj hwm ntawm TCR (T cell receptor)- thiab BCR (B cell receptor)-mediated signal transduction, development, proliferation, and cytokine production (Gu et al., 2009; Pendergast, 2002 ; Silberman et al., 2008). Abl-deficient nas kuj pom tau tias txo qis ntawm T/B hlwb (Gu li al., 2009; Liberatore thiab Goff, 2009; Schwartzberg li al., 1991). Txawm li cas los xij, cov txheej txheem hauv qab ntawm kev tiv thaiv tsis zoo uas tshwm sim thaum tsis muaj lossis inhibition ntawm c-Abl tseem tsis pom. Nws tau raug tshaj tawm tias JAK-STAT txoj kev taw qhia yog qhov tseem ceeb rau BCR-Abl-induced transformation (Carlesso li al., 1996; Danial et al., 1998; de Groot et al., 1999). Yog li, nws tsim nyog tshawb xyuas seb JAK-STAT tseem tuaj yeem pab txhawb rau c-Abl-innate tiv thaiv kab mob. Ntawm no, peb tshaj tawm tias STAT1 cuam tshuam nrog thiab yog phosphorylated los ntawm c-Abl nyob rau hauv ib tug JAK-ywj siab yam, los ntawm cov dimerization, nuclear localization, thiab downstream gene transcription ntawm STAT1 yog tswj. Cov kev tshawb pom no tau qhia tias, ntxiv rau JAK kinase, c-Abl yog qhov tseem ceeb rau kev ua kom tag nrho ntawm STAT1 thiab cov lus teb tom qab IFN-mediated antiviral.
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Kev hloov pauv ntawm interferon gamma-activated sequence downstream noob yog tswj los ntawm Abl kinase
Our previous study suggested that c-Abl widely participates in the regulation of gene transcription (Dong et al., 2017). To illustrate the role of c-Abl in gene expression, the transcription of approximately 22,000 genes in wild-type (WT) and c-abl / arg / MEFs (mouse embryonic fibroblasts) was detected using Affymetrix GeneChips (Mouse Genome 430 2.0) (GEO accession: GSE154568). A total of 1,744 differentially expressed genes (DEGs) with fold changes >4 tau pom nyob rau hauv c-abl/arg ob-knockout (DKO) thiab WT MEFs, ntawm uas 960 tau upregulated thiab 784 tau downregulated (Daim duab 1A, sab laug). Txhawm rau tshawb xyuas ntxiv txog lub peev xwm ntawm tes thiab txoj hauv kev cuam tshuam ntawm cov DEGs, peb tau ua Gene Ontology (GO) tsom xam (rau cov ntsiab lus hauv cov txheej txheem lom neeg, kev ua haujlwm molecular, thiab cellular tivthaiv) (Daim duab S1A). Cov genes downregulated los ntawm c-Abl thiab Arg feem ntau cuam tshuam nrog cov lus teb tiv thaiv kab mob (GO: 0009615, GO: 0051607, thiab GO: 0045071) thiab kev tiv thaiv kab mob (GO: 0002376, GO: 0045087). Kev soj ntsuam ntxiv tau pom tias IFN-cov noob tshwj xeeb (Liu et al., 2012) tau ua kom muaj txiaj ntsig ntawm cov noob qis (Figures 1A, txoj cai, thiab S1B). Txhawm rau txheeb xyuas qhov sib txawv ntawm qhov profile, cov ntawv sau tseg ntawm cov tshuaj tiv thaiv kab mob, suav nrog cxcl10, psmb9, tap1, thiab isg15, tau txiav txim siab los ntawm ntau qhov kev hloov pauv-polymerase saw cov tshuaj tiv thaiv (RT-PCR) thiab normalized rau qib psma5. , uas tsis yog tswj hwm los ntawm c-Abl. Piv nrog rau WT MEFs, c-abl / arg / MEFs tau nthuav tawm cov ntawv sau tseg qis ntawm cov noob no, tab sis qhov kev txo qis ntawm DKO tau raug thim rov qab los ntawm c-Abl kev cawm dim nyob rau hauv ib koob tshuaj (Daim duab 1B thiab S1C). Cov ntaub ntawv no qhia tias lub vaj huam sib luag ntawm IFN-induced noob tau tswj los ntawm c-Abl thiab Arg. Txhawm rau tshawb xyuas cov ntsiab lus tseem ceeb uas tswj hwm los ntawm Abl kinases hauv cov lus teb rau IFN, lub luciferase reporter system tau tsim los ntawm tap1 / psmb9 sib koom ob txoj kev txhawb nqa tswj hwm los ntawm IFNg (Daim duab 1C). Raws li qhov xav tau, c-Abl / Arg-selective inhibitor AMN107 cuam tshuam qhov kev ua haujlwm ntawm kev hloov pauv ntawm tap1 txhawb nqa (liab) (daim duab 1D). Qhov tseem ceeb, tsis zoo li kev tshem tawm ntawm NFkB-kev ua yeeb yam (xiav) lossis IFN qhov kev pom zoo ib ntus (daj) cov ntsiab lus, AMN107 kev kho mob muaj me ntsis yog tias muaj kev cuam tshuam rau tap1 kev txhawb nqa thaum lub gamma-activated sequence (GAS) lub caij (ntsuab) raug tshem tawm (Daim duab 1D ). Cov kev tshawb pom no qhia tau hais tias STAT1-targeted GAS cis-element koom nrog hauv c-Abl-regulated tap1 transcription, uas qhia tau hais tias STAT1 yog lub luag haujlwm rau c-Abl-mediated kev cai ntawm IFN- teb cov noob qhia.
c-Abl ncaj qha cuam tshuam nrog STAT1
IFN-induced TAP1 kev qhia yog tswj hwm los ntawm c-Abl, tejzaum nws los ntawm STAT1, qhia tias STAT1 yuav cuam tshuam nrog c-Abl. Txhawm rau kom paub tseeb tias qhov kev xav no, lysates ntawm MCF-7 cov hlwb raug rau cov tshuaj tiv thaiv-c-Abl immunoprecipitation tom qab immunoblotting nrog anti-STAT1 antibody. STAT1 muaj nyob rau hauv anti-c-Abl tab sis tsis yog IgG immunoprecipitates (Daim duab 2A). Tom ntej no, 293T hlwb tau cotransfected nrog Myc-cAbl thiab Flag-STAT1 lossis Flag-Vector ua tus tswj. Lub xub ntiag ntawm Myc-c-Abl nyob rau hauv anti-Flag immunoprecipitates npaj los ntawm cov hlwb coexpressing Chij-STAT1 tab sis tsis Flag-Vector pom ib qho kev koom tes ntawm ectopically qhia Myc-c-Abl thiab Flag-STAT1 (Daim duab 2B, sab laug). Ib qho txiaj ntsig zoo sib xws kuj tau txais hauv kev sim sib txuas (Daim duab 2B, txoj cai). Ntxiv mus, lwm tus tswv cuab ntawm Abl tsev neeg, Arg (Abl2), uas yog homologous heev rau c-Abl hauv nws cov N-terminal domain (NTD), kuj cuam tshuam nrog STAT1 (Daim duab S2A).
Daim duab 1. c-Abl tswj cov kev txhawb nqa ntawm IFN-tej noob caj noob ces ntawm GAS cov ntsiab lus
Tom ntej no, lysates npaj los ntawm 293T hlwb qhia Flag-c-Abl tau incubated nrog agarose-conjugated GST-STAT1 lossis GST ib leeg, thiab Flag-c-Abl tau kuaj pom hauv GST-STAT1 tab sis tsis yog GST adsorbates (Daim duab 2C). Txhawm rau txiav txim siab tsis ncaj binding mediated los ntawm lwm yam khoom hauv cell lysates, anti-Flag immunoprecipitates npaj los ntawm 293T hlwb qhia Flag-c-Abl tau raug rau SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), thiab cov proteins raug xa mus rau PVDF (polyvinylidene fluoride) daim nyias nyias thiab ces blotted nrog soluble GST-STAT1 los yog GST (raws li kev tswj). Cov txiaj ntsig tau pom tias GST-STAT1, tab sis tsis yog GST, khi c-Abl ncaj qha hauv vitro (Daim duab 2D). Txhawm rau txhais cov c-Abl-binding domain, agarose-conjugated thiab GST-fused tag nrho-ntev lossis truncated STAT1 (Daim duab 2E, sab sauv vaj huam sib luag) tau incubated nrog lysates ntawm 293T hlwb qhia Flag-c-Abl. Kev soj ntsuam ntawm cov adsorbates los ntawm immunoblotting nrog anti-Flag antibody pom tau hais tias cov amino acids 577-750 ntawm STAT1, uas tsim ib cheeb tsam uas muaj SH2 domain thiab transactivation domain, yog lub luag hauj lwm rau lub c-Abl kev sib cuam tshuam (Daim duab 2E, sab vaj huam sib luag) . Ib yam li ntawd, Flag-STAT1 immunoprecipitated los ntawm anti-Flag antibody yog fractioned los ntawm SDS-PAGE thiab raug xa mus rau PVDF membrane. Tom qab ntawd, daim nyias nyias tau blotted nrog soluble GST-c-Abl SH3 lossis GST-c-Abl SH2 thiab GST nkaus xwb. Cov txiaj ntsig tau pom tias c-Abl SH3 sau yog lub luag haujlwm tseem ceeb rau STAT1 koom haum (Daim duab 2F). Tsis tas li ntawd, qhov kev sib cuam tshuam ntawm endogenous c-Abl nrog STAT1 tau soj ntsuam los ntawm Duolink sib thooj ligation assay (PLA). STAT1: c-Abl complexes tau pom feem ntau nyob rau hauv cytoplasm, thiab tsim ntawm cov complexes muaj ntau yam potentiated los ntawm IFNg stimulation (Daim duab 2G). Ua ke, cov kev tshawb pom no qhia tau hais tias muaj kev sib raug zoo ntawm c-Abl thiab STAT1 ob leeg hauv vitro thiab hauv vivo.
Daim duab 2. c-Abl cuam tshuam nrog STAT1
c-Abl mediates JAK-kev ywj pheej STAT1 phosphorylation
Txhawm rau tshawb xyuas seb STAT1 yog ib qho substrate ntawm c-Abl, purified Nws-tagged STAT1 yog incubated nrog recombinant c-Abl (muaj cov catalytic domain ntawm amino acids 237-643) nyob rau hauv lub xub ntiag ntawm ATP rau ib tug in vitro kinase assay. Immunoblotting ntawm cov tshuaj tiv thaiv cov khoom nrog anti-phospho-tyrosine thiab anti-phospho-STAT1 Y701 tau pom tias STAT1 tau ncaj qha phosphorylated los ntawm c-Abl ntawm tyrosine residue(s), suav nrog Y701, hauv vitro (Daim duab 3A). Tom ntej no, peb pom tias Flag-STAT1 qhia hauv 293T hlwb yog tyrosine phosphorylated, tshwj xeeb tshaj yog nyob rau residue Y701, los ntawm Myc-c-Abl tab sis tsis yog kinase-inactive mutant Myc-c-Abl (K290R). Tsis tas li ntawd, thaum kho nrog c-Abl / Arg-selective inhibitor AMN107, c-Abl-mediated STAT1 phosphorylation yuav luag raug tshem tawm, uas qhia tau hais tias tyrosine phosphorylation ntawm STAT1 yog nyob ntawm kev ua haujlwm c-Abl kinase (Daim duab 3B). c-Abl-mediated STAT1 phosphorylation tau cuam tshuam loj heev thaum STAT1 Y701 tau hloov nrog phenylalanine, qhia tias Y701 yog qhov loj phosphosite ntawm Abl (Daim duab 3C). Ntxiv mus, STAT1 kuj tau phosphorylated los ntawm Arg (Daim duab S2B). Txhawm rau qhia ntxiv txog qhov tshwj xeeb STAT1 residues phosphorylated los ntawm c-Abl, Chij-STAT1 coexpressed nrog Myc-c-Abl tau raug rau cov kua chromatography ua ke nrog tandem loj spectrometry tsom xam. Ntxiv rau qhov zoo-txhais thiab ua haujlwm tseem ceeb STAT1 phosphosite Y701 (Schindler li al., 1992; Shuai et al., 1993), ob lwm phosphosites, Y106 thiab Y665, kuj tau txheeb xyuas (Daim duab S4A). Piv nrog rau WT STAT1, ob qho tib si Y106F thiab Y701F mutants pom kev cuam tshuam tyrosine phosphorylation (Daim duab 3C). Txawm li cas los xij, phosphorylation ntawm STAT1 Y701 tsis cuam tshuam los ntawm phosphorylation ntawm Y106 thiab Y665, qhia tias Y106 thiab Y665 tuaj yeem ua lub luag haujlwm sib txawv (Daim duab S4B). Ua ke, cov kev tshawb pom no qhia tau hais tias tyrosine residues ntawm STAT1, suav nrog cov ntaub ntawv yav dhau los tau tshaj tawm Y701, tuaj yeem phosphorylated los ntawm Abl tsev neeg kinases.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
JAKs feem ntau yog lub luag haujlwm rau IFN-induced STAT1 Y701 phosphorylation (Villarino li al., 2017). Raws li kev cia siab, STAT1 Y701 phosphorylation raug ntxias los ntawm IFNg thiab tau cuam tshuam loj heev los ntawm ruxolitinib, bispecific inhibitor ntawm JAK1 thiab JAK2 (Daim duab 3D). Qhov tseem ceeb, kev cuam tshuam Y701 phosphorylation kuj tau pom raws li Abl inhibition (Daim duab 3D, txoj kab 4), qhia tias c-Abl ib feem ua rau IFNg-induced STAT1 activation. Tsis tas li ntawd, STAT1 Y701 phosphorylation kuj raug ntxias los ntawm DPH, cell-permeable c-Abl activator, kom tsawg dua los ntawm IFNg, uas tuaj yeem thim rov qab los ntawm AMN107 (Daim duab 3E). Txhawm rau kom paub meej tias DPH-induced Abl activation, c-Abl Y412 phosphorylation, qhov taw qhia ntawm kev ua kom Abl, kuj tau kuaj pom (Daim duab 3E). Nws tau raug pom tias JAK2 ua kom tshwm sim ua ntej thiab xav tau rau kev ua kom tom ntej ntawm JAK1 (Briscoe li al., 1996). Txhawm rau tshem tawm cov txiaj ntsig ntawm JAK2 ntawm kev ua kom STAT1, peb tsim ib qho jak2-KO MCF-7 xov tooj ntawm tes ntawm CRISPR. Txo qis tab sis pom tau tias STAT1 Y701 phosphorylation tau pom nyob rau hauv jak2-KO MCF-7 hlwb piv rau hauv cov niam txiv MCF-7 hlwb, uas tuaj yeem ua tau zoo sib xws hauv lub xub ntiag ntawm Myc-c-Abl. tab sis tsis yog Myc-c-Abl (K290R) (Daim duab 3F) thiab tau ploj zuj zus ntxiv tom qab kev kho AMN107. Hauv c-abl / arg / cells, IFNg-induced phosphorylation ntawm STAT1 ntawm Y701 tau cuam tshuam zoo heev hauv lub sijhawm- thiab koob tshuaj-raws li (Daim duab 3G). Tag nrho cov kev soj ntsuam no qhia tau hais tias c-Abl kinase tuaj yeem ncaj qha phosphorylate thiab qhib STAT1 ywj siab ntawm IFNg-JAK txoj kev taw qhia thiab qhov tseem ceeb, ob lub kinases yuav muaj kev sib koom ua ke ntawm STAT1 phosphorylation ntawm Y701.
Daim duab 3. STAT1 yog phosphorylated los ntawm c-Abl
Daim duab 4. c-Abl txhawb STAT1 dimer tsim thiab nuclear ntshuam
c-Abl-mediated phosphorylation tswj STAT1 kev ua haujlwm hloov pauv
Lub tyrosine phosphorylation ntawm STATs yog ib kauj ruam tseem ceeb hauv JAK-STAT txoj hauv kev ntawm IFN signaling thiab koom nrog STAT dimerization, nuclear translocation, thiab DNA binding. Txhawm rau tshawb xyuas yog tias c-Abl-mediated phosphorylation tswj STAT dimerization, GFP-STAT1 thiab Flag-STAT1 tau coexpressed hauv 293T hlwb hauv lub xub ntiag lossis tsis muaj Myc-c-Abl. Cov theem ntawm GFP-STAT1 nyob rau hauv anti-Flag immunoprecipitates tau tshuaj xyuas los qhia STAT1 dimerization. Raws li pom hauv daim duab 4A, kev sib koom ua ke ntawm c-Abl muaj peev xwm tsim cov STAT1-STAT1 homodimers. Immunofluorescence microscopy ntawm MCF-7 cov hlwb ntxiv tau pom tias ablation ntawm c-abl/arg lossis kev kho mob nrog AMN107 cuam tshuam IFNg-induced STAT1 nuclear translocation (Figures 4B thiab 4C).
Tsis tas li ntawd, electromobility hloov kev soj ntsuam tau ua haujlwm los txheeb xyuas qhov kev txhawb nqa kev sib koom tes ntawm STAT1. Ib thaj tsam biotin-tagged IRF1 cov neeg txhawb nqa uas muaj STAT1 khi kev pom zoo ib ntus tau siv los ua qhov kev tshawb nrhiav (Aaronson thiab Horvath, 2002). Qhov kev sojntsuam no tau tsim nrog cov khoom siv hluav taws xob los ntawm 293T hlwb exogenously qhia STAT1 nrog lossis tsis muaj c-Abl, uas yog sib npaug raws li daim duab S5A. Raws li pom nyob rau hauv daim duab 4D, ntau IRF1 sojntsuam-bound complexes uas muaj STAT1 tau pom nyob rau hauv nuclear rho tawm (Daim duab 4D, txoj kab 1). Complex tsim tau nce me ntsis nrog IFNg kev kho mob (Daim duab 4D, txoj kab 2) thiab nce ntxiv hauv cov hlwb ectopically nthuav qhia c-Abl (Daim duab 4D, kab 3). Txawm li cas los xij, piv nrog WT STAT1, c-Abl pom me ntsis yog tias muaj kev cuam tshuam rau kev txhawb nqa kev khi ntawm STAT1 harboring Y701F (Daim duab 4D, kab 7 thiab 8), thaum Y106F thiab Y665F kev hloov pauv pom ze li tsis txawv nrog WT STAT1. Qhov tshwj xeeb ntawm cov complexes uas muaj STAT1 thiab qhov kev soj ntsuam kuaj tau raug lees paub raws li qhov sib ntxiv ntawm ib qho kev sib tw tsis muaj npe (Daim duab 4D, kab 9). Tsis tas li ntawd, qhov sib ntxiv ntawm cov tshuaj tiv thaiv STAT1 ua rau muaj kev tsim cov pab pawg supershifted (Daim duab 4D, kab 11). Thaum lub tagged probes tau incubated nrog nuclear rho tawm ntawm 293T hlwb raws li kev tswj, tsis muaj complexes tau pom (Daim duab 4D, kab 10). Cov txiaj ntsig no qhia tau tias c-Abl-mediated STAT1 Y701 phosphorylation potentiates qhov khi ntawm STAT1 nrog nws cov phiaj xwm txhawb nqa.
Daim duab 5. c-Abl tswj IFN-txog gene qhia
Tom qab ntawd, IFNg-induced transactivation ntawm loj STAT1- tswj cov noob, suav nrog ccl5, cxcl10, cxcl11, gbp2, ido1, ifi35, irf1, isg15, oasl, psmb9, thiab tap1, raug soj ntsuam hauv WT / c- arg DKO MCF-7 cell ntawm RT-PCR. Raws li qhov xav tau, kev hloov pauv cov noob tau raug cuam tshuam los ntawm c-abl/arg depletion (Daim duab 5A). Ntxiv mus, cov noob feem ntau tshwm sim los ntawm IFNg thiab IFNa ua tsis tiav los ntawm IFNs nyob rau hauv lub xub ntiag ntawm AMN107 (Figures 5B thiab 5C). Cov kev tshawb pom no qhia tau hais tias kev ua haujlwm hloov pauv ntawm STAT1 tau tswj hwm zoo los ntawm c-Abl.
c-Abl txhawb IFN-nyob ntawm cov tshuaj tiv thaiv kab mob
TAP1 thiab PSMB9 tau koom nrog hauv kev tsim thiab nthuav tawm cov peptides hauv MHC chav kawm I antigen processing pathway (Ghannam li al., 2014; Vitale li al., 1998). Raws li kev tshawb pom yav dhau los, qhov kev nthuav qhia antigen ntawm ovalbumin los ntawm JAWS II hlwb rau B3Z hlwb tau cuam tshuam loj heev los ntawm AMN107 vim tias txo qis IL2 ntau lawm (Daim duab 6A). Txhawm rau soj ntsuam ntxiv cov tshuaj tiv thaiv kab mob lom ntawm STAT1 kev tswj hwm los ntawm c-Abl, WT, thiab c-abl / arg / MEFs pretreated nrog lossis tsis muaj serially diluted IFNg tau kis tus kab mob vesicular stomatitis (VSV). Tus kab mob VSV ua rau muaj kev tuag ntau ntawm tes hauv c-abl / arg / MEFs thiab WT hlwb kho nrog AMN107 dua li hauv WT MEFs kho nrog tsheb (Daim duab 6B). IFNg kev kho mob tshwj xeeb txo qis ntawm tes tuag vim yog kis kab mob, tab sis nws muaj qhov cuam tshuam tsawg dua thaum muaj AMN107. Raws li qhov kev tshawb pom no, c-abl/arg ablation hauv MEFs ua rau muaj kev xav tsis zoo rau IFN stimulation, tab sis qhov txiaj ntsig no tseem ceeb rov qab los ntawm c-Abl cawm (Daim duab 6B). Tom ntej no, A549 hlwb kho nrog lossis tsis muaj AMN107 tau kis tus kab mob Newcastle tus kab mob (NDV) qhia GFP, thiab cov txiaj ntsig tau qhia tias cov kab mob sib kis kuj tseem muaj zog los ntawm AMN107 kev kho mob tab sis suppressed los ntawm c-Abl qhia (Daim duab 6C). Raws li kev tshawb pom tias DNA transfection ua rau kev ua kom lub endogenous IFN teb (Park li al., 2003), transfection nrog pcDNA vector inhibited viral proliferation. Qhov tseem ceeb, kev hloov pauv nrog Flag-c-Abl ua rau muaj kev cuam tshuam ntau dua ntawm kev kis tus kab mob ntau dua li kev hloov pauv nrog ib qho vector khoob (Daim duab 6C). Tsis tas li ntawd, IFNg kev kho mob muaj me ntsis, yog tias muaj, cuam tshuam rau kev kis tus kab mob hauv lub xub ntiag ntawm c-Abl-selective inhibitor AMN107 (Daim duab 6D). Cov ntaub ntawv no qhia tias c-Abl ua lub luag haujlwm tseem ceeb hauv STAT1- cov tshuaj tiv thaiv kab mob sib kis. Peb txuas ntxiv mus tshawb xyuas lub luag haujlwm ntawm c-Abl hauv kev kis kab mob hauv kev hu xov tooj / fl, Lck-Cre (c-abl-conditional knockout, c-abl-cKO) nas, uas c-abl tau raug tshem tawm hauv thymocytes (Daim duab S7A. ) txij li thaum germline knockout ntawm c-abl noob ua rau khiav thiab tuag nyob rau hauv thawj ob lub lis piam tom qab yug me nyuam (Koleske li al., 1998; Schwartzberg li al., 1991). Tom qab ntawd, WT thiab c-abl-cKO nas tau kis tus kabmob influenza A (IAV). Ib qho kev txheeb cais tsis tseem ceeb tshaj qhov kev tuag tau pom ntawm cov nas c-abl-cKO ntau dua li cov nas WT. WT nas tsis tu ncua tswj hwm AMN107, uas tswj hwm c-Abl / Arg kinases hauv txhua cov ntaub so ntswg, ua kom pom kev nce siab rau IAV (Daim duab 6E). Sib sau ua ke, cov kev tshawb pom no qhia tias c-Abl yuav tsum muaj rau kev tiv thaiv kab mob hauv cov tsiaj.
Daim duab 6. c-Abl txhawb IFN-nyob ntawm cov tshuaj tiv thaiv kab mob
Kev sib tham
IFNg yog ib qho tseem ceeb tshaj plaws tiv thaiv kab mob cytokines thiab ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob hauv lub cev tiv thaiv kab mob. Nyob rau hauv canonical IFNg signaling, raws li IFNg stimulation, JAK1 thiab JAK2 yog recruited mus rau lub cytoplasmic tails ntawm aggregated IFNg receptors (IFNGRs) thiab qhib los ntawm sequential autophosphorylation thiab transphosphorylation txheej xwm (Stark, 2007; Villarino, 2007). Tom qab ntawd, STAT1 docks rau IFNGR los ntawm lub koom haum ntawm nws cov SH2 sau nrog kev lees paub nyob rau hauv IFNGR (Y440DKPH444), nyob rau hauv uas Y440 yog phosphorylated los ntawm JAKs (Greenlund li al., 1995). Ua raws li nws cov phosphorylation ntawm Y701 los ntawm JAKs, STAT1 dimerizes thiab translocates mus rau hauv lub nucleus, qhov uas nws txhawb cov transcription ntawm ib tug array ntawm IFNg teb cov noob (Aaronson thiab Horvath, 2002; Stark thiab Darnell, 2012). Stat1 ablation hauv nas ua rau tsis muaj cov lus teb rau IFN thiab tsis muaj IFN-induced antimicrobial thiab tshuaj tua kab mob hauv hlwb (Meraz li al., 1996). Stat1 / nas kuj qhia tau tias muaj kev cuam tshuam ntau ntxiv rau kev txhim kho ntawm tus kheej thiab tshuaj (3-methylcholanthrene)-induced qog (Kaplan li al., 1998) thiab hypersensitivity rau qee yam kab mob inflammatory (Bettelli li al., 2004; Villarino et al., 2004; ., 2010). Ntxiv mus, STAT1 signaling shields T hlwb los ntawm ntuj killer cell-mediated cytotoxicity (Kang li al., 2019).Cov kev tshawb pom no qhia tias STAT1 mediates innate immune activation thiab qog tiv thaiv kab mob.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Schlatterer et al. tau tshaj tawm tias c-Abl, tab sis tsis yog Arg, tuaj yeem ua rau cov neuronal poob los ntawm kev ua kom STAT1 ua kom muaj zog thiab interferon ntau lawm. Txawm li cas los xij, lub luag haujlwm tseem ceeb rau c-Abl-dependent STAT1 kev ua kom tsis tau raug qhia meej (Schlatterer li al., 2012). Ntawm no, peb qhia tias c-Abl khi thiab phosphorylates STAT1 thiab cuam tshuam nrog STAT2 ncaj qha hauv vitro (Daim duab 2 thiab 3, S8A, S9A, thiab S10A). STAT1 Y701 yog tshwj xeeb tshaj yog phosphorylated los ntawm JAK kinases, uas tswj STAT1 dimer tsim (Schindler li al., 1992; Shuai li al., 1993) thiab nucleocytoplasmic shuttling thaum lub sij hawm IFNg signaling (Mao et al., 6. Zhong Xu et al., 2005). Peb txoj kev tshawb fawb pom tau tias c-Abl, lwm qhov kinase uas tsis yog JAKs, kuj pab txhawb rau STAT1 Y701 phosphorylation ntawm nws tus kheej (Daim duab 3). Txawm hais tias c-Abl-mediated Y701 phosphorylation tsis muaj zog npaum li JAKs, nws zoo li yuav tsum tau rau Y701 kom ncav cuag qhov siab tshaj plaws phosphorylation txij li qhov tsis zoo Y701 phosphorylation tau pom nyob rau hauv c-abl / arg / cell raws li IFNg stimulus, thiab IFNg- induced STAT1 Y701 phosphorylation yog inhibited los ntawm AMN107, los ntawm 1.25 mM mus rau 5 mM, nyob rau hauv ib tug npaum li cas (Daim duab 3G thiab S11A). Txawm li cas los xij, Y701 phosphorylation tsis cuam tshuam ntau los ntawm lub xeev phosphorylation ntawm Y106 thiab Y665, lwm ob phosphosites ntawm c-Abl, qhia tias potentiated STAT1 Y701 phosphorylation tsis tshwm sim los ntawm c-Abl-mediated ntau qhov chaw phosphorylation yog pos STAT1. ntaus nqi los txhim kho JAK kev ua haujlwm. Cov kev tshawb fawb yav dhau los tau pom tias kev ua haujlwm ntawm JAK1 thiab qhov sib txawv ntawm JAK2 tau pom nyob rau hauv BCR-Abl-expressing cells (Chai et al., 1997; Henderson et al., 1997; Shuai et al., 1996). Tsis tas li ntawd, BCR-Abl tau pom tias yuav tsum ua tib zoo qhib STAT1 thiab STAT2 los ntawm tyrosine phosphorylation ntawm JAK1, JAK2, thiab JAK3 (Henderson li al., 1997). Cov ntaub ntawv pov thawj tam sim no txhawb nqa tias Abl thiab JAKs tuaj yeem hais tau cov txiaj ntsig zoo ntawm STAT1 phosphorylation ntawm Y701 (Daim duab 3E).
Ntxiv rau Y701, phosphorylation ntawm Y106 thiab Y665 tau txheeb xyuas ib txhij (Figures 3C thiab S4A). Cov phosphosites no tsis koom nrog STAT1 Y701 phosphorylation lossis STAT1 homodimerization (Figures S4B thiab S4C). Txawm li cas los xij, Murphy thiab cov npoj yaig tau pom tias kev sib cuam tshuam ntawm NTDs (N-terminal protein sib cuam tshuam domain, amino acids 1-136) ntawm monomers yog qhov tsim nyog rau lub dimerization ntawm nonphosphorylated full-length STAT molecules (Ota li al., 2004). STAT1 mutants (F77A thiab/los yog L78A) ua tsis tau tejyam uas tsis yog phosphorylated dimers, thiab cov mutants muaj phenotype-persistent phosphorylation nyob rau hauv IFN-kho hlwb thiab tsis kam mus rau TC45-mediated dephosphorylation hauv vitro (Mertens li al., 200). Y106 hauv NTD, uas nyob ze F77 thiab L78, tej zaum yuav muaj lub luag haujlwm tswj STAT1 hauv lub xeev phosphorylated dimer thiab tswj STAT1 hauv cov nucleus nyob rau hauv ionizing hluav taws xob (Figures S6A thiab S6B). Ntxiv mus, SH2 domain cuam tshuam nrog phosphorylated tyrosine-muaj npe thaum phosphorylated homodimer tsim los ntawm IFN (Shuai li al., 1994). Y665, uas nyob hauv SH2 sau, tuaj yeem cuam tshuam rau dimerization. Cov haujlwm ntawm c-Abl-mediated phosphorylation qhov chaw (Y106 thiab Y665) xav tau kev tshawb nrhiav ntxiv.
Zoo ib yam li JAKs, c-Abl-mediated phosphorylation tswj STAT1 dimer tsim (xws li STAT1- STAT1 homodimer thiab STAT1-STAT2 heterodimer tsim (Figures 4A, S12A, thiab S12B)), nuclear translocation, DNA , thiab transcription ntawm IFN-stimulated noob. Abl cuam tshuam tau ua rau muaj peev xwm kis tau tus kab mob kis tau zoo thiab ua rau muaj tus kab mob tuag ntawm tes (Figures 6B, 6C, thiab 6D). Ntxiv mus, peb pom tias ionizing hluav taws xob (IR) ntxias STAT1 nuclear translocation thaum tsis muaj c-Abl inhibitor (Figures S6A thiab S6B). IR activates c-Abl ncaj qha (Pendergast, 2002) thiab tom qab ntawd STAT1- nyob ntawm IFNg signaling, uas tuaj yeem muab cov txheej txheem hauv qab los ntawm kev kho hluav taws xob pib ua IFN-cascading innate thiab yoog lub cev tiv thaiv kab mob ntawm cov qog (Burnette li al. ., 2011) thiab muaj qhov muaj zog tiv thaiv kab mob hauv lub cev los teb rau IR stimuli. Piv nrog rau WT cov phooj ywg littermates, nas nyob rau hauv uas c-abl tau conditionally knocked tawm hauv thymocytes pom ntau dua tab sis tsis yog qhov tseem ceeb ntawm kev tuag. Qhov tseem ceeb, cov nas tau tswj hwm tus c-Abl / Arg inhibitor AMN107 muaj kev tuag ntau dua (Daim duab 6E), uas qhia tau hais tias c-Abl qhia nyob rau hauv ntau yam ntaub so ntswg pab txhawb kev tiv thaiv kab mob hauv cov tsiaj. Los ntawm cov ntaub ntawv no, peb xav hais tias endogenous Abl muaj latent basal tsis kam mus rau tus kab mob kis tau tus kab mob thiab uas activated Abl stimulated los ntawm ionizing radiation shields hlwb. Abl kinases yog constitutively activated nyob rau hauv feem ntau cov neeg mob nrog CML. Abl kinase inhibitors STI571 thiab AMN107 sawv cev rau kev kho mob pem hauv ntej rau CML txoj kev kho. Txawm li cas los xij, kab mob ua pa sab sauv thiab kev tiv thaiv kab mob yog feem ntau pom, uas tej zaum yuav tshwm sim los ntawm kev tawm tsam ntawm STAT txoj kev tswj hwm lub cev tiv thaiv kab mob (Hochhaus li al., 2016; Mattiuzzi li al., 2003). Peb qhov kev tshawb pom tau ntxiv lub hauv paus theoretical los txhim kho txoj kev kho kom zoo.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Hauv cov ntsiab lus, tyrosine kinase c-Abl tau pom los koom nrog STAT1 hauv vivo thiab hauv vitro, los txhawb STAT1 phosphorylation ntawm Y701 ywj siab ntawm JAKs, kom muaj zog transactivation, thiab kho kom haum xeeb hauv lub cev tiv thaiv kab mob, tshwj xeeb tshaj yog nyob rau hauv cov ntsiab lus ntawm c -Abl txog kev ntxhov siab. Peb qhov kev tshawb nrhiav muab ib txoj hauv kev ntxiv rau kev ua kom STAT1, uas ua rau lub cev loj hlob ntawm kev paub txog kev tswj hwm ntawm IFN downstream signaling pathway.
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