張路遙,王麗麗,楊金剛,邢思寧,趙松,于穎,謝曉冬,馬東初
1.遼寧中醫(yī)藥大學(xué),遼寧 沈陽(yáng) 113021;2.沈陽(yáng)軍區(qū)總醫(yī)院 全軍腫瘤診治中心醫(yī)學(xué)實(shí)驗(yàn)科,遼寧 沈陽(yáng) 110016;3.沈陽(yáng)軍區(qū)總醫(yī)院 全軍腫瘤診治中心腫瘤科,遼寧 沈陽(yáng) 110016
S6K1 C端自抑制假底物結(jié)構(gòu)域磷酸化與Akt去磷酸化協(xié)同調(diào)節(jié)SP600125誘導(dǎo)巨核細(xì)胞系多倍體化
張路遙1,3,王麗麗2,楊金剛2,邢思寧2,趙松2,于穎2,謝曉冬3,馬東初2
1.遼寧中醫(yī)藥大學(xué),遼寧 沈陽(yáng) 113021;2.沈陽(yáng)軍區(qū)總醫(yī)院 全軍腫瘤診治中心醫(yī)學(xué)實(shí)驗(yàn)科,遼寧 沈陽(yáng) 110016;3.沈陽(yáng)軍區(qū)總醫(yī)院 全軍腫瘤診治中心腫瘤科,遼寧 沈陽(yáng) 110016
目的:探討JNK抑制劑SP600125誘導(dǎo)巨核細(xì)胞白血病系多倍體化的調(diào)控機(jī)制。方法:用SP600125和3種抑制劑(PD184352、U0126和LY294002)處理Dami和CMK細(xì)胞,用點(diǎn)突變技術(shù)對(duì)核糖體蛋白S6激酶1(S6K1)C端自抑制假底物結(jié)構(gòu)域和疏水基序突變構(gòu)建的S6K1質(zhì)粒轉(zhuǎn)染細(xì)胞,流式細(xì)胞術(shù)分析細(xì)胞的DNA倍性,Western印跡檢測(cè)S6K1、MAPK和Akt蛋白的表達(dá)及磷酸化修飾位點(diǎn)的變化。結(jié)果:當(dāng)單獨(dú)使用3種抑制劑時(shí),對(duì)Dami和CMK細(xì)胞的倍性無(wú)影響。當(dāng)SP600125與3種抑制劑聯(lián)合時(shí),盡管PD184352下調(diào)了p44/42 MAPK在Thr202/Tyr204位點(diǎn)的磷酸化,但其并不抑制SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化;相反,U0126抑制SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化,但并不下調(diào)p44/42 MAPK的磷酸化;而LY294002增加Akt的磷酸化,并阻斷SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化。這3種抑制劑均在SP600125誘導(dǎo)的多倍體Dami和CMK細(xì)胞中部分抑制S6K1的Thr421/Ser424磷酸化,但并不增加S6K1的Thr389磷酸化。轉(zhuǎn)染S6K1突變質(zhì)粒的Dami細(xì)胞,并沒(méi)有對(duì)SP600125誘導(dǎo)的多倍體化產(chǎn)生影響,同時(shí),無(wú)論是模擬磷酸化或去磷酸化的Thr389突變均對(duì)SP600125誘導(dǎo)的多倍體化無(wú)影響,且未顯現(xiàn)出與LY294002的協(xié)同作用。C端自抑制假底物結(jié)構(gòu)域突變質(zhì)粒(S6K1-D3E)可以進(jìn)一步阻斷SP600125誘導(dǎo)的已經(jīng)被LY294002部分阻斷的Dami細(xì)胞多倍體化。結(jié)論:S6K1的C端自抑制假底物結(jié)構(gòu)域磷酸化與Akt去磷酸化協(xié)同調(diào)節(jié)SP600125誘導(dǎo)巨核細(xì)胞白血病系的多倍體化。
核糖體蛋白S6激酶1(S6K1);Akt;巨核細(xì)胞;多倍體化
巨核細(xì)胞是產(chǎn)生血小板的前體細(xì)胞,多倍體化在巨核細(xì)胞發(fā)育過(guò)程中發(fā)揮重要作用[1-3]。我們前期研究發(fā)現(xiàn),核糖體蛋白S6激酶1(ribosomal protein S6 kinase 1,S6K1)參與Nocodazole(微管抑制劑)和SP600125(JNK抑制劑)誘導(dǎo)的巨核細(xì)胞白血病細(xì)胞系Dami和CMK多倍體化,伴隨著S6K1在Thr421/Ser424位點(diǎn)的磷酸化及Thr389位點(diǎn)的去磷酸化[4-5],并且H-89阻斷SP600125誘導(dǎo)的Dami和CMK細(xì)胞多倍體化伴隨著S6K1在Thr389位點(diǎn)磷酸化的增加,以及在Thr421/Ser424位點(diǎn)磷酸化的減少[5]。然而,S6K1在藥物誘導(dǎo)巨核細(xì)胞白血病細(xì)胞系多倍體化過(guò)程中的作用機(jī)制尚不十分清楚。另有報(bào)道脯氨酸介導(dǎo)有絲分裂原調(diào)節(jié)絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)磷酸化Thr421/Ser424,其位于S6K1的C端自抑制假底物結(jié)構(gòu)域[6]。此外,Akt,又稱蛋白激酶B(protein kinase B,PKB),通過(guò)哺乳動(dòng)物雷帕霉素靶蛋白復(fù)合物1(mammalian target of rapamycin complex-1,mTORC1)激活S6K1[7]。
本此,我們用PD184352(高效選擇性MEK1/2抑制劑)、U0126(MEK抑制劑)和LY294002(PI3K抑制劑)處理Dami和CMK細(xì)胞,用點(diǎn)突變技術(shù)構(gòu)建S6K1 C端自抑制假底物結(jié)構(gòu)域和疏水基序內(nèi)不同位點(diǎn)的突變質(zhì)粒,探討了S6K1與MAPK和Akt通路在SP600125誘導(dǎo)細(xì)胞多倍體化方面所起的作用。我們的研究表明,S6K1的C端自抑制假底物結(jié)構(gòu)域磷酸化與Akt去磷酸化協(xié)同調(diào)節(jié)SP600125誘導(dǎo)巨核細(xì)胞白血病系的多倍體化。
1.1 材料
人巨核細(xì)胞白血病細(xì)胞系Dami購(gòu)自美國(guó)國(guó)立細(xì)胞庫(kù)(ATCC,其編號(hào)為CRL-9792),CMK細(xì)胞從德國(guó)微生物和細(xì)胞培養(yǎng)保存中心獲得。二甲基亞砜(DMSO)、碘化丙啶(PI)購(gòu)自Sigma公司;SP600125購(gòu)自美國(guó)LC實(shí)驗(yàn)室;PD184352、U0126、LY294002購(gòu)自 Selleckchem公司;RPMI 1640培養(yǎng)基購(gòu)自Life Technologies公司;新生牛血清購(gòu)自杭州四季青生物工程材料有限公司;兔抗人S6K1抗體、兔抗人磷酸化S6K1(Thr389)抗體、兔抗人磷酸化S6K1(Thr421/Ser424)抗體、兔抗人p44/42 MAPK抗體、兔抗人磷酸化p44/42 MAPK(Thr202/Tyr204)抗體、兔抗人Akt抗體,兔抗人磷酸化Akt(Ser473)抗體、辣根過(guò)氧化物酶(HRP)標(biāo)記的山羊抗兔IgG和HRP標(biāo)記的山羊抗鼠IgG均購(gòu)自 Cell Signaling Technology公司;β-actin購(gòu)自Santa Cruz Biotechnology公司;Amersham ECL Prime Western印跡試劑盒購(gòu)自GE Healthcare公司;即用PCR擴(kuò)增試劑盒和SanPrep柱式質(zhì)粒DNA小量抽提試劑盒購(gòu)自生工生物工程(上海)有限公司;限制性核酸內(nèi)切酶DpnⅠ和大腸桿菌DH5α感受態(tài)細(xì)胞購(gòu)自TaKaRa公司;Amaxa核轉(zhuǎn)染儀和轉(zhuǎn)染試劑盒購(gòu)自Amaxa公司。
1.2 細(xì)胞培養(yǎng)與處理
CMK和Dami細(xì)胞均以2×105/mL的密度接種于T75培養(yǎng)瓶中,在含10%新生牛血清的培養(yǎng)液中生長(zhǎng),將培養(yǎng)瓶置于37℃、5%CO2的潮濕孵箱中培養(yǎng)。在3種抑制劑誘導(dǎo)倍體化實(shí)驗(yàn)中,分別將PD184352(2μmol/L)、U0126(10μmol/L)或LY294002(30μmol/L)加入培養(yǎng)基,連續(xù)培養(yǎng)72 h。在SP600125聯(lián)合3種抑制劑誘導(dǎo)多倍體化實(shí)驗(yàn)中,在加入SP600125(32μmol/L)前1 h,分別將PD184352(2μmol/L)、U0126(10μmol/L)或LY294002(30μmol/L)加入培養(yǎng)基,連續(xù)培養(yǎng)72 h。以終濃度為1%的DMSO作為溶媒對(duì)照組。孵育后,收集細(xì)胞,并用PBS洗滌3次。
1.3 細(xì)胞倍性分析
分別收集實(shí)驗(yàn)組和對(duì)照組細(xì)胞5×105個(gè),用80%冰甲醇于-20℃固定過(guò)夜,PBS洗滌3次,加入50 mg/mL PI染液,室溫避光孵育30 min,用流式細(xì)胞儀檢測(cè)分析DNA倍性。
1.4 Western印跡
收集細(xì)胞,加入含蛋白酶抑制劑的細(xì)胞裂解液,冰浴中超聲波破碎細(xì)胞,100℃煮沸5 min,4℃、12 000 r/min離心5 min,回收上清即為提取的蛋白樣品,采用二辛可寧酸(BCA)法測(cè)定蛋白濃度。取蛋白樣品上樣進(jìn)行SDS-PAGE,并濕轉(zhuǎn)至硝酸纖維素膜上,將膜在含5%脫脂牛奶的TBS-T中室溫孵育2 h。兔抗人S6K1抗體(1∶5000)、兔抗人p-S6K1(Thr389)抗體(1∶300)、兔抗人p-S6K1(Thr421/Ser424)抗體(1∶500)、兔抗人p44/ 42 MAPK抗體(1∶5000)、兔抗人磷酸化p44/42 MAPK(Thr202/Tyr204)抗體(1∶500)、兔抗人Akt抗體(1∶2000)、兔抗人磷酸化 Akt(Ser473)抗體(1∶300)、小鼠抗人β-actin抗體(1∶80 000)孵育,4℃搖床振蕩過(guò)夜;TBS-T洗滌3次,加入HRP標(biāo)記的山羊抗兔IgG(1∶40 000)或HRP標(biāo)記的山羊抗小鼠IgG(1∶40 000),室溫孵育2 h;TBS-T洗滌3次,暗室內(nèi)用ECL化學(xué)發(fā)光檢測(cè),X線膠片曝光。
1.5 定點(diǎn)突變
設(shè)計(jì)含S6K1相應(yīng)位點(diǎn)堿基突變的一對(duì)互補(bǔ)引物,采用PAGE純化方式由Invitrion公司合成,按照要求加入一定量的無(wú)菌水溶解引物,配置成100μmol/L的母液,使用時(shí)稀釋至1/10。采用即用PCR擴(kuò)增試劑盒,反應(yīng)體系包括模板質(zhì)粒1 μL(50 ng)、2條引物各1μL、水9.5μL、2×HiFi緩沖液12.5μL。PCR條件為94℃預(yù)變性1 min,94℃變性40 s、60℃退火1 min、68℃延伸7 min,進(jìn)行18個(gè)循環(huán)。PCR后加入1μLDpnⅠ酶,37℃酶切1 h,酶切產(chǎn)物10μL直接轉(zhuǎn)化DH5α感受態(tài)細(xì)胞,在氨芐西林平皿中篩選,挑取單克隆進(jìn)行質(zhì)粒抽提和測(cè)序,以確認(rèn)突變位點(diǎn)堿基取代是否正確,并且沒(méi)有其他突變被導(dǎo)入。T389E突變用引物1進(jìn)行;T389A突變用引物2進(jìn)行;D3E突變用2對(duì)引物,先用引物3進(jìn)行S418D+T421E+S424D突變,確定突變成功后,以突變質(zhì)粒為模板,用引物4進(jìn)行S411D突變;T389E/D3E突變用引物1,以S6K1-D3E為模板進(jìn)行;T389A/D3E突變用引物2,以S6K1-D3E為模板進(jìn)行。所有突變體見表1。
1.6 核轉(zhuǎn)染
Dami細(xì)胞用編碼S6K1-WT或S6K1-T389E、S6K1-T389A、S6K1-D3E、S6K1-T389E/D3E和S6K1-T389A/D3E突變體的質(zhì)粒瞬時(shí)轉(zhuǎn)染,參見前文[4]。簡(jiǎn)言之,用預(yù)冷的PBS緩沖液洗滌Dami細(xì)胞,然后將細(xì)胞重懸于指定的電穿孔緩沖液中,細(xì)胞懸液的終濃度為1.2×108/mL。每種突變質(zhì)粒2μg用0.1 mL細(xì)胞懸液混勻,將混合物轉(zhuǎn)移至一個(gè)2.0 mm電穿孔池中,用Amaxa核轉(zhuǎn)染儀進(jìn)行核轉(zhuǎn)染。轉(zhuǎn)染后,將細(xì)胞重懸于含10%胎牛血清的RPMI1640培養(yǎng)基中,孵育過(guò)夜。孵育后,轉(zhuǎn)染的Dami細(xì)胞用SP600125(32μmol/L)在有或無(wú)LY294002(30μmol/L)預(yù)處理1 h后,于37℃、5%CO2的潮濕孵箱中誘導(dǎo)72 h。孵育72 h后收集細(xì)胞,檢測(cè)細(xì)胞倍性。以未轉(zhuǎn)染突變質(zhì)粒,但用SP600125誘導(dǎo)的Dami細(xì)胞作為對(duì)照組。
表1 通過(guò)點(diǎn)突變產(chǎn)生的S6K1突變質(zhì)粒
1.7 統(tǒng)計(jì)學(xué)分析
所有實(shí)驗(yàn)均重復(fù)3次。采用SPSS13.0統(tǒng)計(jì)軟件分析實(shí)驗(yàn)數(shù)據(jù),數(shù)據(jù)用x±s表示,進(jìn)行t檢驗(yàn),P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。
2.1 PD184352、U0126和LY294002對(duì)巨核細(xì)胞系多倍體化的影響
用PD184352(高效選擇性MEK1/2抑制劑)、U0126(MEK抑制劑)和LY294002(PI3K抑制劑)來(lái)研究這些通路是否參與SP600125誘導(dǎo)Dami和CMK細(xì)胞多倍體?;?PD184352、U0126和LY294002對(duì)底物的選擇抑制濃度(數(shù)據(jù)未示),對(duì)應(yīng)的使用終濃度分別為2、10和30μmol/L。
與我們以往報(bào)道的結(jié)果一致,SP600125處理顯著增加Dami和CMK細(xì)胞的DNA多倍性。與對(duì)照組(DMSO處理)Dami和CMK細(xì)胞的DNA平均倍性(2.8±0.2和2.9±0.4)相比,SP600125處理組的Dami和CMK細(xì)胞DNA平均倍性分別增至9.3±0.6和9.9±0.2(圖1A、B)。意外的是PD184352不能抑制SP600125誘導(dǎo)Dami和CMK細(xì)胞多倍體化。PD184352與SP600125聯(lián)合處理Dami和CMK細(xì)胞的DNA平均倍性與SP00125單獨(dú)處理沒(méi)有明顯差別,分別為8.9±0.7和9.5±0.3(圖1A、B)。然而,U0126和LY294002分別部分阻斷SP6000125誘導(dǎo)Dami和 CMK細(xì)胞 的多倍體化。U0126與SP600125聯(lián)合處理導(dǎo)致Dami和CMK細(xì)胞的DNA平均倍性分別降至6.8±0.2和7.3±0.3(與SP00125處理組比,P<0.05)。LY294002與SP600125聯(lián)合處理導(dǎo)致Dami和CMK細(xì)胞DNA平均倍性分別降至6.6±0.1和7.2±0.2(與SP00125處理組比,P<0.05)。同時(shí),這些抑制劑自身并不能對(duì)Dami和CMK細(xì)胞的DNA含量分布產(chǎn)生影響(圖1C,CMK細(xì)胞數(shù)據(jù)未示)。該結(jié)果提示,PI3K和MAPK信號(hào)途徑,而不是MEK1/2信號(hào)途徑,可能參與SP600125誘導(dǎo)Dami和CMK細(xì)胞的多倍體化。
2.2 LY294002通 過(guò) 上 調(diào) Akt磷 酸 化 阻 斷SP600125誘導(dǎo)的多倍體化
圖1 流式細(xì)胞儀分析SP600125單獨(dú)處理或SP600125分別聯(lián)合PD184352、U0126和LY294002處理Dami和CMK細(xì)胞72 h,以及3種抑制劑PD184352、U0126和LY294002分別處理Dami細(xì)胞72 h的倍性變化
為進(jìn)一步探討PD184352、U0126和LY294002對(duì)相關(guān)信號(hào)轉(zhuǎn)導(dǎo)通路的影響,用Western印跡分析了相關(guān)信號(hào)分子的表達(dá)和修飾變化。如圖2所示,PD184352可以顯著抑制SP600125誘導(dǎo)的p44/ 42 MAPK的Thr202/Tyr204的磷酸化,U0126則無(wú)此作用,表明SP600125誘導(dǎo)的Dami和CMK細(xì)胞多倍體化不依賴于MAPK信號(hào)轉(zhuǎn)導(dǎo)路徑。LY294002可上調(diào)SP600125誘導(dǎo)多倍體化Dami和CMK細(xì)胞Akt Ser473的磷酸化,表明 PI3K/Akt通路在用LY294002預(yù)處理的SP600126誘導(dǎo)的多倍體Dami和CMK細(xì)胞中被激活。鑒于PI3K/Akt通路活化促進(jìn)細(xì)胞增殖,且LY294002處理可部分阻斷SP600125誘導(dǎo)的Dami和CMK細(xì)胞多倍體化,以及SP600125輕度下調(diào)Akt Ser473磷酸化(盡管并不十分明顯),因此,確保PI3K/Akt通路不過(guò)度活化有利于SP600125誘導(dǎo)Dami和CMK細(xì)胞多倍體化。值得注意的是,PD184352、LY294002和U0126均在SP600125誘導(dǎo)的多倍體Dami和CMK細(xì)胞中,部分抑制S6K1的Thr421/Ser424磷酸化。然而,這些抑制劑并不增加S6K1的Thr389磷酸化。這些數(shù)據(jù)表明S6K1單獨(dú)的Thr421/Ser424磷酸化可能不足以介導(dǎo)由SP600125誘導(dǎo)Dami和CMK細(xì)胞的多倍體化,其他信號(hào)通路可能參與此過(guò)程。
2.3 S6K1 C端自抑制假底物結(jié)構(gòu)域磷酸化與Akt去磷酸化協(xié)同調(diào)節(jié)SP600125誘導(dǎo)的多倍體化
圖2 Western印跡檢測(cè)SP600125單獨(dú)處理和SP600125分別聯(lián)合PD184352、U0126、LY294002處理Dami和CMK細(xì)胞72 h的蛋白表達(dá)及磷酸化修飾位點(diǎn)的變化
我們以往曾發(fā)現(xiàn)myc-d/ED3E-pRK5(包含Ser411→Asp、Ser418→Asp、Thr421→Glu、Ser424→Asp和Thr389→Glu突變質(zhì)粒)可以阻斷Nocodazole誘導(dǎo)的Dami細(xì)胞多倍體化[4]。SP600125誘導(dǎo) Dami和CMK細(xì)胞多倍體化伴隨著S6K1的Thr421/Ser424磷酸化和Thr389去磷酸化(圖2),而且,H-89通過(guò)占據(jù)S6K1的ATP結(jié)合位點(diǎn),下調(diào)Thr421/Ser424磷酸化和上調(diào)Thr389磷酸化,阻斷SP600125誘導(dǎo)Dami和CMK細(xì)胞多倍體化[5]。然而,PD184352雖然可以下調(diào)SP600125誘導(dǎo)多倍體化Dami和CMK細(xì)胞上Thr421/Ser424磷酸化,但其并不阻斷SP600125誘導(dǎo)Dami和CMK細(xì)胞的多倍體化(圖1、2)。因此,為了明確S6K1 C端自抑制假底物結(jié)構(gòu)域磷酸化和Thr389磷酸化修飾在SP600125誘導(dǎo)多倍體化方面所起的作用,我們構(gòu)建了C端自抑制假底物結(jié)構(gòu)域內(nèi)相關(guān)磷酸化位點(diǎn)和Thr389位點(diǎn)的突變質(zhì)粒,即S6K1-WT、S6K1-T389E、S6K1-T389A和S6K1-D3E(圖3),并分別用這些質(zhì)粒轉(zhuǎn)染Dami細(xì)胞,觀察了對(duì)SP600125誘導(dǎo)Dami細(xì)胞倍體化的影響。令我們意外的是,與SP600125誘導(dǎo)的Dami細(xì)胞倍體化(7.04±0.37)相比,無(wú)論是轉(zhuǎn)染野生型S6K1(S6K1-WT:7.27±0.37)或突變型 S6K1(S6K1-T389E:6.93±0.32;S6K1-T389A:6.86±0.51;S6K1-D3E:6.78±0.4)均不能影響SP600125誘導(dǎo)的Dami細(xì)胞倍體化。然而,當(dāng)S6K1-D3E與LY294002共同處理SP600125誘導(dǎo)的Dami細(xì)胞后,LY294002對(duì)倍體化的抑制作用(從7.04±0.37降至5.30± 0.18,P<0.05)可被 S6K1-D3E進(jìn)一步增強(qiáng)(從5.30±0.18降至4.07±0.14,P<0.05)(圖4A、B)。然而,無(wú)論是S6K1-D3E突變聯(lián)合Thr389位點(diǎn)的模擬磷酸化突變(S6K1-T389E/D3E),還是去磷酸化突變(S6K1-T389A/D3E)均未明顯增強(qiáng)或降低S6K1-D3E對(duì)SP600125誘導(dǎo)倍體化的抑制作用(圖4C)。鑒于LY294002誘導(dǎo)Akt的Ser473磷酸化,該結(jié)果提示S6K1 C端自抑制假底物結(jié)構(gòu)域的磷酸化參與SP600125誘導(dǎo)Dami細(xì)胞的多倍體化調(diào)控,并需要Akt Ser473去磷酸化的協(xié)同作用。
哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信號(hào)通路是調(diào)控細(xì)胞合成代謝的重要開關(guān)[8],已有研究發(fā)現(xiàn),其參與哺乳動(dòng)物細(xì)胞的多倍體化調(diào)控[9-10]。S6K1是mTOR下游的重要靶分子之一,其通過(guò)調(diào)控核糖體合成、蛋白質(zhì)合成、細(xì)胞周期進(jìn)程和代謝,在細(xì)胞生長(zhǎng)、增殖和分化中起重要作用[11-15]。我們以往的研究發(fā)現(xiàn),S6K1可能在藥物誘導(dǎo)巨核細(xì)胞白血病細(xì)胞系多倍體化過(guò)程中發(fā)揮重要作用[4-5]。然而,詳細(xì)的作用機(jī)制尚不十分清楚,而且S6K1活性的調(diào)控比較復(fù)雜,至少包括mTOR、生長(zhǎng)因子、激素和應(yīng)激有關(guān)的信號(hào)轉(zhuǎn)導(dǎo)途徑調(diào)控的8個(gè)磷酸化位點(diǎn)(即 Thr229、Ser371、Thr389、Ser404、Ser411、Ser418、Thr421和Ser424)[16-18]。因此,在前期研究基礎(chǔ)上,我們進(jìn)一步解析了S6K1的磷酸化修飾變化在藥物誘導(dǎo)巨核細(xì)胞白血病細(xì)胞系多倍體化過(guò)程中的調(diào)控機(jī)制。有研究報(bào)道,血清素誘導(dǎo)肺動(dòng)脈平滑肌生長(zhǎng),且15(S)-羥基花生四烯酸誘導(dǎo)血管生成需要S6K1在Thr421/Ser424位點(diǎn)的磷酸化,但該過(guò)程可以被LY294002顯著阻斷[19-20];脯氨酸介導(dǎo)有絲分裂原調(diào)節(jié)MAPK磷酸化Thr421/Ser424[6];在心肌細(xì)胞中U0126阻斷TPA或胰島素誘導(dǎo)的S6K1 Thr421/Ser424位點(diǎn)的磷酸化[21]。
圖3 S6K1突變質(zhì)粒結(jié)構(gòu)圖
圖4 S6K1突變質(zhì)粒對(duì)SP600125單獨(dú)處理和SP600125聯(lián)合LY294002處理Dami細(xì)胞72 h的倍性影響
在本研究中,我們發(fā)現(xiàn)SP600125誘導(dǎo)p44/42 MAPK在Thr202/Tyr204位點(diǎn)的磷酸化及Dami和CMK細(xì)胞的多倍體化。雖然PD184352和U0126均能顯著抑制S6K1在Thr421/Ser424的磷酸化,然而盡管PD184352下調(diào)了p44/42 MAPK在Thr202/Tyr204位點(diǎn)的磷酸化,但并不抑制SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化。與此相反,U0126抑制SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化,但并不下調(diào)p44/42 MAPK的磷酸化。這些結(jié)果表明SP600125誘導(dǎo)的Dami和CMK細(xì)胞多倍體化不依賴于MAPK信號(hào)轉(zhuǎn)導(dǎo)路徑。有趣的是,LY294002不僅抑制Thr421/Ser424的磷酸化,同時(shí)增加Akt的磷酸化,并阻斷SP600125誘導(dǎo)的Dami和CMK細(xì)胞的多倍體化,該結(jié)果提示LY294002可能通過(guò)上調(diào)Akt磷酸化阻斷SP600125誘導(dǎo)的多倍體化。值得注意的是,這3種抑制劑均在SP600125誘導(dǎo)的多倍體Dami和CMK細(xì)胞中部分抑制S6K1的Thr421/Ser424磷酸化,然而,它們并不增加S6K1的Thr389磷酸化。總之,上述結(jié)果提示S6K1在Thr421/Ser424位點(diǎn)的磷酸化可能在SP600125誘導(dǎo)Dami和CMK細(xì)胞的多倍體化中發(fā)揮重要作用,然而,S6K1單獨(dú)的Thr421/Ser424磷酸化可能不足以介導(dǎo)由SP600125誘導(dǎo)Dami和CMK細(xì)胞的多倍體化。
有文獻(xiàn)報(bào)道,通過(guò)復(fù)合多位點(diǎn)磷酸化S6K1的逐步活化是通過(guò)C端假底物結(jié)構(gòu)域中的4個(gè)位點(diǎn)(Ser411、Ser418、Thr421和Ser424)的磷酸化來(lái)啟動(dòng),其誘導(dǎo)構(gòu)象變化、疏水基序(HM)和T-loop位點(diǎn)[7,22]。雖然這4個(gè)C端位點(diǎn)的磷酸化有助于S6K1激活,但其并非關(guān)鍵。這4個(gè)位點(diǎn)突變?yōu)楸彼釟埢蛘邚腃端刪除101個(gè)氨基酸殘基可以適當(dāng)?shù)卣T導(dǎo)S6K1激活[7]。一般認(rèn)為,Thr389位點(diǎn)的磷酸化是S6K1活化的標(biāo)志[23]。盡管前期研究表明S6K1參與巨核細(xì)胞多倍體化的調(diào)控[4-5],但調(diào)控S6K1活性的磷酸化位點(diǎn)的磷酸化修飾和倍體化的關(guān)系尚不清楚。在本研究中,我們發(fā)現(xiàn)用S6K1-WT、S6K1-T389E、S6K1-T389A和S6K1-D3E分別轉(zhuǎn)染Dami細(xì)胞,并沒(méi)有對(duì)SP600125誘導(dǎo)的多倍體化Dami細(xì)胞產(chǎn)生影響,同時(shí),具有Thr389→Glu突變或Thr389→Ala突變的任何一種S6K1對(duì)SP600125單獨(dú)處理或聯(lián)合LY294002處理的Dami細(xì)胞多倍體化沒(méi)有影響。然而,S6K1-D3E可以進(jìn)一步阻斷SP600125誘導(dǎo)的已被LY294002部分阻斷的Dami細(xì)胞多倍體化。鑒于LY294002誘導(dǎo)Akt的Ser473磷酸化,阻斷SP00125誘導(dǎo)的多倍體化,而且S6K1-D3E包含Ser411→Asp、Ser418→Asp、Thr421→Glu和Ser424→Asp的突變位點(diǎn)均位于C端自抑制假底物結(jié)構(gòu)域內(nèi),提示Akt的Ser473位點(diǎn)去磷酸化和C端自抑制假底物結(jié)構(gòu)域內(nèi)上述位點(diǎn)的磷酸化在SP600125誘導(dǎo)的多倍體化方面發(fā)揮重要作用。然而,S6K1蛋白可分成數(shù)個(gè)重要結(jié)構(gòu)域,其通過(guò)復(fù)雜的多位點(diǎn)磷酸化來(lái)調(diào)控其活性。因此,仍需要進(jìn)一步的研究來(lái)解析巨核細(xì)胞白血病細(xì)胞系多倍體化期間S6K1的相關(guān)分子機(jī)制。
[1]Wilcox D A.Megakaryocyte-and megakaryocyte precursor-related gene therapies[J].Blood,2016,127(10): 1260-1268.
[2]Geddis A E. Megakaryopoiesis[J]. Semin Hematol, 2010,47(3):212-219.
[3]Trakala M,Rodríguez-Acebes S,Maroto M,et al. Functional reprogramming of polyploidization in megakaryocytes[J].Dev Cell,2015,32(2):155-167.
[4]Ma D,Yu H,Lin D,et al.S6K1 is involved in polyploidization through its phosphorylation at Thr421/ Ser424[J].J Cell Physiol,2009,219(1):31-44.
[5]王麗麗,楊金剛,李長(zhǎng)嶺,等.核糖體蛋白S6激酶1(S6K1)介導(dǎo)SP600125誘導(dǎo)的巨核細(xì)胞白血病細(xì)胞系多倍體化依賴于細(xì)胞分化程度[J].細(xì)胞與分子生物學(xué)雜志,2016,32(10):1336-1341.
[6]Mukhopadhyay N K,Price D J,Kyriakis J M,et al. An array of insulin-activated,proline-directed serine/ threonine protein kinases phosphorylate the p70 S6 kinase[J].J Biol Chem,1992,267:3325-3335.
[7]Magnuson B,Ekim B,Fingar D C.Regulation and function of ribosomal protein S6 kinase(S6K)within mTOR signalling networks[J].Biochem J,2012,441:1-21.
[8]Li T,Wang G.Computer-aided targeting of the PI3K/ Akt/mTOR pathway:toxicity reduction and therapeutic opportunities[J].Int J Mol Sci,2014,15(10):18856-18891.
[9]Li Y,Chen X,Tang X,et al.DNA synthesis during endomitosis is stimulated by insulin via the PI3K/Akt and TOR signaling pathways in the silk gland cells of Bombyx mori[J].Int J Mol Sci,2015,16(3):6266-6280.
[10]Sharma S,Yao H P,Zhou Y Q,et al.Prevention of BMS-777607-induced polyploidy/senescence by mTOR inhibitor AZD8055 sensitizes breast cancer cells to cytotoxic chemotherapeutics[J].Mol Oncol,2014,8(3):469-482.
[11]Tavares M R,Pavan I C,Amaral C L,et al.The S6K protein family in health and disease[J].Life, 2015,131:1-10.
[12]Yi S A,Um S H,Lee J,et al.S6K1 Phosphorylation of H2B mediates EZH2 trimethylation of H3:A determinant of early adipogenesis[J].Mol Cell,2016,62(3): 443-452.
[13]Wu H,Xiao Z,Wang K,et al.MiR-145 is downregulated in human ovarian cancer and modulates cell growth and invasion by targeting p70S6K1 and MUC1 [J].Biochem Biophys Res Commun,2013,441(4):693-700.
[14]Pavan I C,Yokoo S,Granato D C,et al.Different interactomes for p70-S6K1 and p54-S6K2 revealed by proteomic analysis[J]. Proteomics, 2016,16(20):2650-2666.
[15]Miyake S,Wakita H,Bernstock J D,et al.Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning[J].J Neurochem, 2015,135(5):943-957.
[16]Shin S,Wolgamott L,Yu Y,et al.Glycogen synthase kinase(GSK)-3 promotes p70 ribosomal protein S6 kinase(p70S6K)activity and cell proliferation[J].Proc Natl Acad Sci USA,2011,108(47):E1204-1213.
[17]Wang J,Zhong C,Wang F,et al.Crystal structures of S6K1 provide insights into the regulation mechanism of S6K1 by the hydrophobic motif[J].Biochem J,2013,454(1):39-47.
[18]Zhang H,Hoff H,Marinucci T,et al.Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts[J].Exp Cell Res,2000,259(1):284-292.
[19]Liu Y,Fanburg B L.Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1[J].Am J Respir Cell Mol Biol,2006, 34:182-191.
[20]Ohanna M,Sobering A K,Lapointe T,et al.Atrophy of S6K1(2/2)skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control[J].Nat Cell Biol,2005,7:286-294.
[21]Iijima Y,Laser M,Shiraishi H,et al.c-Raf/MEK/ ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells[J].J Biol Chem,2002,277:23065-23075.
[22]Fenton T R,Gout I T.Functions and regulation of the 70kDa ribosomal S6 kinases[J].Int J Biochem Cell Biol,2011,43(1):47-59.
[23]Doscas M E,Williamson A J,Usha L,et al.Inhibition of p70 S6 kinase(S6K1)activity by A77 1726 and its effect on cell proliferation and cell cycle progress[J].Neoplasia,2014,16(10):824-834.
SP600125-Induced Polyploidization of Megakaryocytic Cell Lines by the Regulation of S6K 1 C-Terminal Autoinhibitory Pseudosubstrate Domain Phosphorylation and Akt Dephosphorylation
ZHANG Lu-Yao1,3,WANG Li-Li2,YANG Jin-Gang2,XING Si-Ning2, ZHAO Song2,YU Ying2,XIE Xiao-Dong3*,MA Dong-Chu2*
1.Liaoning University of Chinese Medicine,Shenyang 113021;
2.Department of Experimental Medicine,Tumor Center of PLA,Norhern Hospital,Shenyang 110016;
3.Department of Oncology,Tumor Center of PLA,Norhern Hospital,Shenyang 110016;China
*Co-corresponding authors,MA Dong-Chu,E-mail:mdc580819@sina.com;XIE Xiao-Dong,E-mail:doctor-xxd@163.com
[Abstract]Objective:To investigate the mechanism of SP600125-induced polyploidization of megakaryocytic leukemia cell lines.Methods:Dami and CMK cells were treated with SP600125 and three inhibitors PD184352, U0126 and LY294002.Meanwhile,we constructed plasmid encoding mutated ribosomal protein S6 kinase 1(S6K1)by point mutagenesis targeting the C-terminal autoinhibitory pseudosubstrate domain and the hydrophobic motif of S6K1 to transfecte cells.The DNA ploidy was analyzed by flow cytometry.The expression and phosphorylation of S6K1,MAPK and Akt were detected by Western blot.Results:When three inhibitors were used alone,there was no effect on the ploidy of Dami and CMK cells.When SP600125 was combined with three inhibitors,PD184352 did not inhibit SP600125-induced polyploidization of Dami and CMK cells,although it decreased the phosphorylation of p44/42 MAPK at Thr202/Tyr204.In contrast,U0126 inhibited SP600125-induced polyploidization of Dami and CMK cells,but did not decreased p44/42 MAPK phosphorylation.While LY294002 increased the phosphorylation of Akt and blocked SP600125-induced polyploidization of Dami and CMK cells.Notably,these three inhibitors,in SP600125-induced polyploidization of Dami and CMK cells,partially inhibited the phosphorylation of S6K1 at Thr421/Ser424,but did not increase the phosphorylation of S6K1 at Thr389.Dami cells that transfected with S6K1 mutant plasmid did not affect SP600125-induced polyploidization.At the same time,either the phosphorylation or dephosphorylation mutations with Thr389sites had no effect on SP600125-induced polyploidization,and it did not show synergistic effect with LY294002.However,C-terminal autoinhibitory pseudosubstrate domain mutant plasmid(S6K1-D3E)could further block polyploidization of SP600125-induced polyploidization of Dami cells that had been partially blocked by LY294002.Conclusion:SP600125-induced polyploidization of megakaryocytic leukemia cell lines by the regulation of S6K1 C-terminal autoinhibitory pseudosubstrate domain phosphorylation and Akt dephosphorylation.
S6 kinase 1(S6K1);Akt;megakaryocytes;polyploidization
Q25
A
1009-0002(2017)03-0233-09
10.3969/j.issn.1009-0002.2017.03.002
2016-12-29
國(guó)家自然科學(xué)基金(61302003,31571398)
張路遙(1990-),女,碩士研究生,(E-mail)329730208@qq.com
馬東初,(E-mail)mdc580819@sina.com;謝曉冬,(E-mail)doctor-xxd@163.com