本文關(guān)鍵詞： 細(xì)胞凋亡 p53 絲氨酸蘇氨酸蛋白激酶 信號(hào)轉(zhuǎn)導(dǎo) SIRT　出處：《第三軍醫(yī)大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的 蛋白激酶MST1(mammalian Sterile 20-like kinase 1)在其氨基端有一個(gè)Ste20相關(guān)的激酶催化域,羧基端有一個(gè)調(diào)節(jié)區(qū)。MST1在細(xì)胞增殖、分化、形態(tài)和細(xì)胞骨架重排方面發(fā)揮了重要作用。先前已有研究指出氨基端催化域的缺失會(huì)使MST1被半胱氨酸蛋白酶3(caspase-3)在為數(shù)眾多的細(xì)胞凋亡刺激素的作用下所切割,例如由CD95/FasL觸發(fā)的死亡受體,或被星孢菌素(STS),神經(jīng)酰胺,熱休克和亞砷酸鹽處理。被切割后的MST1的氨基末端會(huì)轉(zhuǎn)移至核內(nèi),然后對(duì)于染色質(zhì)的固縮和隨后發(fā)生的細(xì)胞凋亡產(chǎn)生一定的作用。而且,有實(shí)驗(yàn)表明在細(xì)胞內(nèi)過表達(dá)MST1的情況下,已經(jīng)發(fā)現(xiàn)了其被切割的情況和因此誘發(fā)的細(xì)胞凋亡。并且已經(jīng)有報(bào)道證實(shí)第326和第349位的天冬氨酸是兩個(gè)主要的切割位點(diǎn)。突變掉這些切割位點(diǎn)后,MST1的激活,核轉(zhuǎn)位和誘導(dǎo)細(xì)胞凋亡的能力都會(huì)明顯減弱。最近已經(jīng)明確了在MST1的第八亞結(jié)構(gòu)域的第183位的蘇氨酸是其主要的磷酸激活位點(diǎn)并且這個(gè)蘇氨酸位點(diǎn)的自磷酸化對(duì)于MST1激酶的激活是必須的。Hippo是哺乳動(dòng)物的MST1在果蠅中的同源蛋白,并且已經(jīng)被大量實(shí)驗(yàn)證實(shí)其通過抑制轉(zhuǎn)錄和(或者)降解細(xì)胞周期蛋白E(cyclin E)和DIAPs或者磷酸化并抑制Yorkie來限制細(xì)胞生長(zhǎng)和增殖。在哺乳動(dòng)物中,MST1已經(jīng)被證實(shí)可以分別通過促細(xì)胞分裂劑激活性蛋白激酶激酶4/促細(xì)胞分裂劑激活性蛋白激酶激酶7(MKK4/MKK7)和促細(xì)胞分裂劑激活性蛋白激酶激酶3/促細(xì)胞分裂劑激活性蛋白激酶激酶6(MKK3/MKK6)激活c-Jun氨基末端激酶(JNK)和p38MAPK激酶信號(hào)通路。最近有報(bào)道稱JNK對(duì)于MST1的激活和由MST1介導(dǎo)的通過磷酸化MST1上的第82位絲氨酸引起的細(xì)胞凋亡是必須的。此外,JNK的顯性失活突變體能抑制MST1誘導(dǎo)的caspase的激活和從而產(chǎn)生的細(xì)胞凋亡,而p38的顯性失活突變體和p38抑制劑則不能抑制MST1誘導(dǎo)的細(xì)胞凋亡。 MST1還能通過磷酸化組蛋白H2B上相對(duì)保守的位點(diǎn)(哺乳動(dòng)物細(xì)胞上第14位絲氨酸,酵母菌上第10位絲氨酸)來誘導(dǎo)的細(xì)胞凋亡。我們實(shí)驗(yàn)室的研究表明MST1能通過磷酸化O亞型叉頭框3a(FOXO3a)上的第207位絲氨酸和FOXO1上對(duì)應(yīng)的第212位絲氨酸從而涉及依賴于FOXO的神經(jīng)元細(xì)胞凋亡過程。近期我們還發(fā)現(xiàn)磷酸肌醇3(phosphoinositide 3)激酶/Akt能磷酸化第120位蘇氨酸從而抑制MST1介導(dǎo)的細(xì)胞凋亡前信號(hào)通路。 沉默信息調(diào)節(jié)因子2相關(guān)酶1(Sirt1)是NAD+依賴的去乙酰化酶,它有相當(dāng)數(shù)量的底物并且參與了多種細(xì)胞進(jìn)程。去乙酰化這些靶蛋白導(dǎo)致其蛋白活性既有可能被抑制也有可能被激活,從而影響機(jī)體生理的許多方面,例如轉(zhuǎn)錄沉默,基因水平控制的壽命長(zhǎng)短,細(xì)胞的新陳代謝,能量的動(dòng)態(tài)平衡,DNA修復(fù)和細(xì)胞存活。P53作為一個(gè)關(guān)鍵腫瘤抑制基因,在反饋細(xì)胞所接受的眾多壓力信號(hào)包括DNA損傷,組織缺氧和異常增殖過程中發(fā)揮了極其重要的作用。P53維持基因組穩(wěn)定性的方式主要是依賴于p53誘導(dǎo)的細(xì)胞凋亡,包括抑制腫瘤生長(zhǎng)或是清除腫瘤。總的來說,p53應(yīng)對(duì)DNA損傷的生物活性是和其轉(zhuǎn)錄后的修飾狀態(tài)密切相關(guān)的,特別是特殊位點(diǎn)的磷酸化、乙�；头核鼗irt1已經(jīng)被報(bào)道能強(qiáng)烈與其底物p53結(jié)合,并能去乙�；痯53的第382位賴氨酸。Sirt1介導(dǎo)的去乙�；軐�(duì)抗依賴于p53的轉(zhuǎn)錄激活并能特定地抑制在DNA損傷或氧化應(yīng)激下引起的依賴于p53的細(xì)胞凋亡。 先前的研究已經(jīng)指出MST1促進(jìn)的細(xì)胞凋亡有可能是依賴于p53的,但是MST1-p53在細(xì)胞凋亡過程中信號(hào)通路的分子機(jī)制還有很大一部分都是未知。所以本研究旨在明確MST1在遺傳毒性因子誘導(dǎo)的依賴于p53的細(xì)胞凋亡過程中扮演的角色,并期待著能發(fā)現(xiàn)MST1、Sirt1和p53在這個(gè)過程中的相互調(diào)節(jié)機(jī)制。 方法 1.1 MST1和Sirt1對(duì)p53和p21轉(zhuǎn)錄活性的影響 A. H1299細(xì)胞在轉(zhuǎn)染14*p53人工熒光素酶報(bào)告基因的同時(shí)分別共轉(zhuǎn)了p53、MST1、MST1 K59R和Sirt1質(zhì)粒。細(xì)胞裂解液用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)轉(zhuǎn)錄活性。B. H1299細(xì)胞在轉(zhuǎn)染p21啟動(dòng)子熒光素酶報(bào)告基因的同時(shí)分別共轉(zhuǎn)了p53、MST1和Sirt1質(zhì)粒。細(xì)胞裂解液用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)轉(zhuǎn)錄活性。 1.2 MST1和Sirt1對(duì)p53介導(dǎo)的細(xì)胞凋亡的影響 A. U2OS細(xì)胞轉(zhuǎn)染編碼MST1和p53 shRNA或者空載體的質(zhì)粒,然后用Etoposide處理36小時(shí)后用流式細(xì)胞儀檢測(cè)Annexin-V染色的細(xì)胞凋亡情況。B. HCT116 p53+/+和HCT116 p53-/-細(xì)胞被穩(wěn)定轉(zhuǎn)染MST1,對(duì)照組穩(wěn)定轉(zhuǎn)染空載體,然后用CDDP處理36小時(shí)后用流式細(xì)胞儀檢測(cè)Annexin-V染色的細(xì)胞凋亡情況。C. HCT116 p53+/+和HCT116 p53-/-細(xì)胞轉(zhuǎn)染MST1的小干擾RNA,對(duì)照組轉(zhuǎn)染隨機(jī)小干擾RNA,然后用CDDP處理48小時(shí)后用流式細(xì)胞儀檢測(cè)Annexin-V染色的細(xì)胞凋亡情況。D. U2OS細(xì)胞在轉(zhuǎn)染MST1質(zhì)粒的同時(shí)共轉(zhuǎn)Sirt1質(zhì)粒或空載體。用Etoposide處理36小時(shí)后用流式細(xì)胞儀檢測(cè)Annexin-V染色的細(xì)胞凋亡情況。 2.1 MST1對(duì)Sirt1介導(dǎo)的p53去乙�；饔玫挠绊� A.穩(wěn)定轉(zhuǎn)染MST1的HCT116 p53+/+細(xì)胞和其穩(wěn)定轉(zhuǎn)染空載體的對(duì)照組細(xì)胞的裂解液用免疫印跡方法檢測(cè),所用的抗體包括抗p53-k382乙酰化抗體和抗p53抗體。上樣量通過內(nèi)參14-3-3β蛋白調(diào)整,從而保持一致。B.轉(zhuǎn)染HA-p53或同時(shí)轉(zhuǎn)染GFP-MST1的細(xì)胞中FLAG-Sirt1的免疫沉淀物用免疫印跡方法檢測(cè),所用的抗體包括抗HA抗體和抗FLAG抗體。C.轉(zhuǎn)染p53、p300、Sirt1、MST1質(zhì)粒的293T細(xì)胞的裂解液用免疫印跡方法檢測(cè),所用抗體有抗p53-k382乙酰化抗體等。 2.2 MST1如何通過和Sirt1作用增強(qiáng)p53乙酰化 A.體外MST1激酶實(shí)驗(yàn),重組MST1蛋白和其底物GST-Sirt1在32P-ATP存在的情況下進(jìn)行孵育。反應(yīng)后用聚丙烯酰胺凝膠電泳分離蛋白質(zhì)進(jìn)行熒光放射自顯影術(shù)測(cè)定。B.體外MST1激酶實(shí)驗(yàn),有活性的重組MST1蛋白在32P-ATP存在的情況下和不同的Sirt1蛋白片段(P1、P2、P3)進(jìn)行孵育。反應(yīng)后用聚丙烯酰胺凝膠電泳分離蛋白質(zhì)進(jìn)行熒光放射自顯影術(shù)測(cè)定。C.體外磷酸化實(shí)驗(yàn),有活性的MST1蛋白和Sirt1蛋白在不帶放射性的ATP存在的情況下進(jìn)行孵育,然后將磷酸化實(shí)驗(yàn)產(chǎn)物和乙�；痯53孵育進(jìn)行去乙酰化實(shí)驗(yàn)。反應(yīng)后用聚丙烯酰胺凝膠電泳分離蛋白質(zhì)并用免疫印跡方法檢測(cè),所用抗體有抗p53-K382乙酰化抗體、抗GST抗體和抗Sirt1抗體。 結(jié)果與結(jié)論 1.1 MST1通過抑制Sirt1活性增強(qiáng)p53轉(zhuǎn)錄活性。 在14*p53報(bào)告基因中,Sirt1能顯著抑制p53的表達(dá),而野生型MST1能明顯挽救Sirt1誘導(dǎo)的p53降低,但是ATP結(jié)合位點(diǎn)突變的激酶活性失活的MST1(MST1 K59R)不能挽救Sirt1誘導(dǎo)的p53降低。與之相似的,Sirt1抑制了依賴于p53的p21熒光素酶活性,而MST1可以逆轉(zhuǎn)Sirt1誘導(dǎo)的p21表達(dá)的降低。綜上所述,MST1通過抑制Sirt1來促進(jìn)p53的轉(zhuǎn)錄活性。 1.2 MST1促進(jìn)細(xì)胞凋亡是依賴于p53并且Sirt1能抑制MST1誘導(dǎo)的細(xì)胞凋亡。 U2OS細(xì)胞在干擾掉p53基因的情況下過表達(dá)MST1,用Etoposide處理后,細(xì)胞凋亡減少。我們?cè)贖CT116 p53-/-細(xì)胞中也發(fā)現(xiàn)MST1的穩(wěn)定表達(dá)不能增加Cisplatin誘導(dǎo)的細(xì)胞凋亡數(shù)目,但是在HCT116 p53+/+細(xì)胞中MST1的穩(wěn)定表達(dá)卻能增加Cisplatin誘導(dǎo)的細(xì)胞凋亡數(shù)目,并且我們還發(fā)現(xiàn)在HCT116 p53+/+細(xì)胞中干擾掉MST1后Cisplatin誘導(dǎo)的細(xì)胞凋亡明顯減少而在HCT116 p53-/-細(xì)胞中干擾掉MST1后Cisplatin誘導(dǎo)的細(xì)胞凋亡不能被降低。這些實(shí)驗(yàn)證據(jù)證明MST1在DNA損傷情況下促進(jìn)細(xì)胞凋亡是依賴于p53。同時(shí)在Sirt1過表達(dá)細(xì)胞中,MST1介導(dǎo)的細(xì)胞凋亡會(huì)減少。 2.1 MST1抑制Sirt1介導(dǎo)的p53去乙酰化。 我們?cè)隗w外MST1激酶實(shí)驗(yàn)中發(fā)現(xiàn)MST1不能直接磷酸化p53。而Western blot使用p53特異的乙�；贵w檢測(cè)結(jié)果表明在MST1過表達(dá)細(xì)胞中內(nèi)源p53乙酰化水平有所增高。然后我們用IP實(shí)驗(yàn)證明MST1和Sirt1有直接的相互作用。接著,我們觀察到MST1的過表達(dá)會(huì)減弱p53和Sirt1的相互作用。與之一致的,在MST1過表達(dá)的細(xì)胞中Sirt1介導(dǎo)的p53去乙酰化是被抑制的。綜上所述,MST1抑制了Sirt1介導(dǎo)的p53去乙酰化。我們同時(shí)發(fā)現(xiàn)在體外MST1也能減少Sirt1介導(dǎo)的FOXO3去乙酰化,表明MST1也可能調(diào)控Sirt1其他底物的生物活性。 2.2 MST1通過磷酸化Sirt1增加p53乙酰化。 體外MST1激酶實(shí)驗(yàn)中用重組Sirt1蛋白作為底物表明Sirt1能被MST1磷酸化。我們接著用重組GST融合蛋白分別編碼了3個(gè)不重疊的Sirt1區(qū)域(肽段P1-P3)并以此指出在Sirt1上的磷酸化區(qū)域。在體外激酶實(shí)驗(yàn)中顯示包含了C末端的第489-747位氨基酸片段的P3是磷酸化的主要區(qū)域。在體外磷酸化和去乙�；瘜�(shí)驗(yàn)中,MST1磷酸化Sirt1確實(shí)減少了Sirt1誘導(dǎo)的p53去乙酰化。通過以上實(shí)驗(yàn)我們闡明了MST1通過負(fù)調(diào)控Sirt1的去乙�；钚詠碚{(diào)節(jié)p53功能的分子機(jī)制。
[Abstract]:Background and purpose
Protein kinase MST1 (mammalian Sterile 20-like kinase 1) is a Ste20 kinase catalytic domain related in its N-terminal, C-terminal region of.MST1 has a regulation in cell proliferation, differentiation, play an important role in the morphology and cytoskeleton rearrangement. Previous studies have indicated that deletion of amino terminal catalytic domain of MST1 is cysteine proteinase 3 (caspase-3) by cutting in the numbers of apoptotic stimuli under the action, such as the death receptor triggered by CD95/FasL, or staurosporine (STS), ceramide, heat shock and arsenite treatment. N-tail cleavage of MST1 will transfer to the nucleus, cell then the apoptotic chromatin condensation and subsequent acts. Moreover, experiments have shown that in cells overexpressing MST1, has discovered the cleavage and therefore the cell apoptosis induced by and. And it has been reported that 326th and 349th of the aspartic acid is two major cleavage sites. These mutations fall cleavage sites, MST1 activation, nuclear translocation and apoptosis induction ability will be significantly weakened. Recently it has been defined in the MST1 of the eighth sub domains of 183rd threonine is the main the phosphoric acid activation site and the threonine sites auto phosphorylation of MST1 kinase is required for.Hippo is the mammalian MST1 in Drosophila homologous protein, has been extensively shown through the inhibition of transcription and (or) degradation of cyclin E (cyclin E) and DIAPs or phosphorylation and inhibition of Yorkie the inhibition of cell growth and proliferation. In mammals, MST1 has been proved respectively by the mitogen activated protein kinase kinase 4/ mitogen activated protein kinase kinase 7 (MKK4/MKK7) and mitogen activated protein kinase kinase 3/ mitogen activated protein kinase kinase 6 (MKK3/MKK6) activation of c-Jun N-terminal kinase (JNK) and p38MAPK kinase signal pathway. Recently it has been reported that JNK for MST1 activation and MST1 mediated by phosphorylation on MST1 eighty-second serine induced cell apoptosis is necessary. In addition, the dominant negative mutant of JNK activation can inhibit MST1 induced caspase and resulting in apoptosis, and the dominant negative mutant of p38 and p38 inhibitors can inhibit the cell apoptosis induced by MST1.
Can MST1 through phosphorylation of histone H2B on a relatively conserved sites (mammalian cells on serine fourteenth, yeast serine tenth) induces apoptosis. Our experiments show that MST1 can phosphorylate O subtype of forkhead box 3A (FOXO3a) corresponding to the 207th serine and FOXO1 212nd serine processes involved in neuronal apoptosis and FOXO dependent. Recently we have shown that phosphatidylinositol 3 kinase (phosphoinositide 3) /Akt apoptosis phosphorylation of threonine 120th inhibits MST1 mediated signal pathway.
Silent information regulator 2 (Sirt1) is the 1 enzyme NAD+ - dependent deacetylase, it has a considerable number of substrates and involved in various cellular processes. The deacetylation of these target proteins leads to its protein activity can be suppressed may also be activated, thus affecting many aspects of the physical body, for example transcriptional silencing, genetic control of aging cells, the dynamic balance of energy, The new supersedes the old., DNA repair and cell survival of.P53 as a key tumor suppressor gene, numerous stress signals received in the cellular response including DNA damage, hypoxia and abnormal proliferation of.P53 played a role in the maintenance of genome stability is extremely important the main way is dependent on the cell apoptosis induced by p53, including the inhibition of tumor growth or tumor eradication. In general, the biological activity of p53 in response to DNA damage and its repair after. A closely related state, especially the specific phosphorylation, acetylation and ubiquitination of.Sirt1 has been reported to strongly with the substrate binding to p53, and can deacetylate p53 382nd lysine.Sirt1 mediateddeacetylation antagonizes p53 dependent transcriptional activation and specific inhibition of apoptosis in response to DNA damage or oxidative stress dependent on p53.
Previous studies have shown that MST1 promotes cell death may be dependent on p53, but the molecular mechanism of MST1-p53 signaling pathway in apoptosis is still largely unknown. So the purpose of this study was to play MST1 in the process of apoptosis induced by specific genetic toxicity factor depends on the role of p53, and look forward to to find MST1, Sirt1 and p53 in the process of mutual adjustment mechanism.
Method
The effect of 1.1 MST1 and Sirt1 on the transcriptional activity of p53 and p21
At the same time, A. in H1299 cells transfected with 14*p53 luciferase reporter gene were constructed to p53, MST1, MST1 K59R and Sirt1 plasmid. Cell lysate with dual luciferase reporter system detection of.B. transcriptional activity and H1299 cell activation in transfected p21 promoter luciferase reporter gene respectively with the turn of p53, MST1 and Sirt1 activity of plasmid. The dual luciferase in cell lysates.
The effect of 1.2 MST1 and Sirt1 on p53 mediated apoptosis
A. U2OS and p53 shRNA cells transfected with MST1 encoding plasmid or empty vector, and then use the Etoposide after 36 hours of treatment were detected by flow cytometry with Annexin-V staining the apoptosis of.B. HCT116 p53+/+ and HCT116 p53-/- cells were transfected with MST1, the control group transfected with empty vector, small interfering RNA then treated with CDDP after 36 hours of use flow cytometry was used to detect Annexin-V staining cell apoptosis.C. HCT116 p53+/+ and HCT116 p53-/- cells transfected with MST1, the control group were transfected with small interfering RNA, then treated with CDDP after 48 hours were detected by flow cytometry with Annexin-V staining of cell apoptosis in.D. U2OS cells transfected by MST1 plasmid and Sirt1 plasmid were transferred or empty the carrier by Etoposide. After 36 hours of treatment by flow cytometry to detect cell apoptosis by Annexin-V staining.
The effect of 2.1 MST1 on the deacetylation of p53 mediated by Sirt1
The cell lysate group were detected by Western blot method A. stable transfection of MST1 HCT116 p53+/+ cells and stably transfected with the empty vector, using antibodies including anti p53-k382 acetylation and anti p53 antibodies. The sample volume by reference 14-3-3 beta protein adjustment, so as to maintain consistent.B. HA-p53 transfected or FLAG-Sirt1 transfected and immune precipitate GFP-MST1 cells by Western blot, using antibodies including anti HA antibody and anti FLAG antibody.C. was transfected into p53, P300, Sirt1, Western blot was used to detect the cell lysate of MST1 plasmid 293T, the antibody of anti p53-k382 antibody acetylation.
How to enhance p53 acetylation by 2.2 MST1 and Sirt1
A. MST1 kinase in vitro experiment, the recombinant MST1 protein and its substrate GST-Sirt1 were incubated in the presence of 32P-ATP. The determination of.B. in vitro MST1 kinase assay fluorescent autoradiography by polyacrylamide gel electrophoresis for protein separation after reaction, active recombinant MST1 protein in the presence of 32P-ATP and Sirt1 protein fragment (P1, P2 P3), were incubated. The determination of.C. phosphorylation in vitro experiment of fluorescent autoradiography by polyacrylamide gel electrophoresis for protein separation after the reaction, the presence of MST1 protein and Sirt1 protein activity in non radioactive ATP cases were incubated and then phosphorylated experimental products and acetylated p53 incubation and deacetylation experiment. After reaction with proteins separated by polyacrylamide gel electrophoresis and Western blot was used to detect the anti p53-K382 antibody, acetylation antibody, anti GST antibody And anti Sirt1 antibody.
Results and conclusions
1.1 MST1 enhanced the transcriptional activity of p53 by inhibiting the activity of Sirt1.
In the 14*p53 gene, the expression of Sirt1 was inhibited by p53, while the wild type MST1 can significantly save the Sirt1 induced decrease of p53, but the ATP binding site mutation of the kinase activity of inactivation of MST1 (MST1 K59R) can save the Sirt1 induced decrease of p53. Similarly, Sirt1 inhibited p53 dependent p21 luciferase activity, while MST1 can reduce the expression of reversed Sirt1 induced p21. In summary, MST1 to promote p53 transcriptional activity through inhibition of Sirt1.
1.2 MST1 promotes apoptosis is dependent on p53 and Sirt1 can inhibit apoptosis induced by MST1.
U2OS cells overexpressing MST1 in p53 gene interfered under the condition, after treatment with Etoposide, reducing apoptosis. We also found that HCT116 in p53-/- cells with stable expression of MST1 can increase the number of cell apoptosis induced by Cisplatin, but MST1 HCT116 in p53+/+ cells of stable expression can increase the number of cell apoptosis induced by Cisplatin and, we also found that the disturbance of cell apoptosis induced by Cisplatin MST1 off in HCT116 p53+/+ cells was significantly reduced in HCT116 p53-/- cells apoptosis induced by Cisplatin interference cannot be dropped after MST1 was reduced. These experimental evidence of MST1 in DNA damage conditions promoting cell apoptosis is dependent on p53. and overexpressed in Sirt1 cells, MST1 mediated apoptosis will be reduced.
2.1 MST1 inhibits Sirt1 mediated p53 deacetylation.
We found that in vitro MST1 kinase assay MST1 cannot directly phosphorylate p53. and acetylated Western antibody test results using blot specific p53 in MST1 showed that the expression level of endogenous p53 cells increased acetyl. Then we use IP experiments show that MST1 and Sirt1 have direct interaction. Then, we observed the expression of the interaction of p53 and Sirt1 MST1 will be reduced. And the same, in cells overexpressing MST1 in Sirt1 mediated deacetylation of p53 is suppressed. In summary, MST1 inhibited Sirt1 mediated p53 deacetylation. We also found that can reduce Sirt1 mediated deacetylation of FOXO3 in vitro MST1, indicating that MST1 might regulate Sirt1 other substrates of biological activity.
2.2 MST1 increases p53 acetylation by phosphorylated Sirt1.
In vitro MST1 kinase assay using recombinant Sirt1 protein as substrate showed that Sirt1 can be phosphorylated by MST1. Then we use recombinant GST fusion protein respectively encoding 3 non overlapping regions of Sirt1 (peptide P1-P3) and points out that it is the phosphorylated region on Sirt1. The in vitro kinase assay shows that contains 489-747 article at the end of the amino acid fragment of C P3 is the main region of phosphorylation. In vitro phosphorylation and acetylation experiments, the phosphorylation of MST1 Sirt1 did reduce Sirt1 induced p53 deacetylation. Through the above experiments we elucidated the molecular mechanism of MST1 negative regulation by Sirt1 deacetylation activity to regulate the function of p53.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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