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  本文關(guān)鍵詞：PRAK和SEPTIN8相互作用研究　出處：《南方醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 絲氨酸/蘇氨酸蛋白激酶 酵母雙雜交篩選 蛋白質(zhì)—蛋白質(zhì)相互作用 離體結(jié)合實(shí)驗(yàn) 免疫共沉淀 信號通路

【摘要】：絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是真核細(xì)胞中介導(dǎo)細(xì)胞外信號至細(xì)胞內(nèi)反應(yīng)的重要信號系統(tǒng)。p38是MAPK家族成員之一,通過磷酸化不同的底物而發(fā)揮其生物學(xué)功能。p38 MAPK的激活涉及到細(xì)胞生長、凋亡等過程,同時也涉及到炎癥和應(yīng)激反應(yīng)的調(diào)控、轉(zhuǎn)錄因子的調(diào)控、細(xì)胞骨架的重構(gòu)等,在多種疾病如心肌肥大、缺血／再灌注損傷、神經(jīng)元病變、感染性疾病過程中都發(fā)揮著重要的作用。 p38 MAPK的激活是一種由特定的激酶通過高度保守的三級激酶級聯(lián)激活的(MKKK/MKK/MAPK)過程。其特異性的活化基序(motif)蘇氨酸(threonine,Thr)-甘氨酸(glycine, Gly)-酪氨酸(throsine, Tyr)活化基序中Thr和Tyr雙位點(diǎn)同時被磷酸化修飾而激活。能磷酸化激活p38的蛋白激酶有絲裂原激活蛋白激酶激酶3 (mitogen-activated protein kinase kinase 3,MKK3)和MKK6,它們都可以被上游的MKKK如轉(zhuǎn)化生長因子β激活激酶1(transforming growth factor-β-activated kinase 1, TAK1)、凋亡信號通路調(diào)控激酶1(apoptosis signal-regulating kinase 1, ASK1)、含SH3結(jié)構(gòu)域富含脯氨酸的蛋白激酶(Srchomology domain 3-containing proline-rich protein kinase, SPRK)、p21激活激酶(p21-activated kinase, PAK)等激活。 PRAK又稱MK5,是一個受p38 MAPK調(diào)控的絲氨酸／蘇氨酸激酶,由471個氨基酸殘基組成,分子量約為54 kD。PRAK廣泛表達(dá)于各種組織,尤其是在外周血單核-巨噬細(xì)胞、腦、血管內(nèi)皮、肺臟、腎臟以及卵巢前列腺中高表達(dá)。PRAK通過與不同的蛋白結(jié)合而激活不同的下游信號轉(zhuǎn)導(dǎo)通路,如PRAK與兩性蛋白相互作用可通過Ras和Cdc42通路介導(dǎo)神經(jīng)元軸突的生長；通過p38通路PRAK和熱休克蛋白25/27(heat shock protein 25/27, HSP25/27)相互作用可調(diào)控細(xì)胞微絲的組裝,介導(dǎo)細(xì)胞氧化應(yīng)激時應(yīng)力纖維的形成進(jìn)而介導(dǎo)細(xì)胞應(yīng)激反應(yīng)。進(jìn)一步的研究表明PRAK參與了多種疾病的發(fā)生與發(fā)展,如PRAK在內(nèi)毒素休克的過程中以及神經(jīng)退行性病變的病理過程中起到關(guān)鍵作用；PRAK與兩性蛋白的相互作用不僅可以促進(jìn)神經(jīng)元軸突的生長,還可以介導(dǎo)腫瘤細(xì)胞的生長和遷移。 由此看來,PRAK參與細(xì)胞內(nèi)多條細(xì)胞信號轉(zhuǎn)導(dǎo)通路,與炎癥、腫瘤等多種疾病密切相關(guān),是信號轉(zhuǎn)導(dǎo)通路研究領(lǐng)域的熱點(diǎn),但目前對其在細(xì)胞內(nèi)的功能調(diào)控機(jī)制尚不太清楚。 信號轉(zhuǎn)導(dǎo)方面的研究,因此我們十分關(guān)心PRAK在細(xì)胞內(nèi)的確切功能及其可能的上游激酶和下游底物。我們實(shí)驗(yàn)室在前期的研究中采用人PRAK為誘餌,采用酵母雙雜交系統(tǒng)篩選人心臟cDNA文庫獲得其相互作用蛋白SEPTIN8(Sept8)。 人Sept8蛋白基因全長編碼序列為1,452 bp,其編碼的蛋白質(zhì)分子量約為55.8 kD,屬于保守的細(xì)胞骨架GTP酶家族成員。位于Sept8蛋白中間的核心結(jié)構(gòu)域是保守的GTP結(jié)合位點(diǎn),具有GTP酶活性；緊鄰GTP結(jié)合位點(diǎn)N端為相對保守的多堿性氨基酸結(jié)構(gòu)域；其N端為由數(shù)百個氨基酸殘基組成富含脯氨酸和疏水氨基酸殘基的結(jié)構(gòu)域,可能是SH3結(jié)合區(qū)域；此外其C端擁有一個較長的由α螺旋構(gòu)成的卷曲螺旋(coiled-coil)結(jié)構(gòu)域,可能是介導(dǎo)與其它蛋白質(zhì)相互結(jié)合的結(jié)構(gòu)域。通過生物信息學(xué)分析還發(fā)現(xiàn)Sept8蛋白序列中存在磷酸化修飾位點(diǎn),乙�；揎椢稽c(diǎn)和棕櫚酰化位點(diǎn)。 我們推測Sept8是與PRAK相互作用的候選分子,原因有三：第一,在體內(nèi)有Sept8和PRAK共同的表達(dá)細(xì)胞,如腦膠質(zhì)瘤細(xì)胞、腎細(xì)胞、前列腺細(xì)胞；第二,Sept8參與介導(dǎo)受體下游的信號轉(zhuǎn)導(dǎo)有先例,如G蛋白介導(dǎo)的信號轉(zhuǎn)導(dǎo)通路中通過Rho蛋白調(diào)節(jié)Sept8的通路等；第三,PRAK參與的信號通路與Sept8參與的信號通路有重疊,如Ras環(huán)節(jié)的參與,絲裂原活化蛋白激酶(MAPK)如JNK、ERK的活化,以及轉(zhuǎn)錄因子如核轉(zhuǎn)錄因子κB (NF-κB)等的活化。因此我們可以提出如下假設(shè)：Ras與其配體相互作用后,通過直接或間接的方式激活Raf,Raf-1可磷酸化MEK1/MEK2(MAP kinase/ERK kinase)上的兩個調(diào)節(jié)性絲氨酸,從而激活MEKs,通過下游激酶將信號傳遞給p38 MAPK信號通路的PRAK蛋白,通過與Sept8蛋白相互作用后,激活下游蛋白如p53或HSP27,進(jìn)而觸動細(xì)胞內(nèi)的氧化應(yīng)激使得效應(yīng)細(xì)胞細(xì)胞因子表達(dá)譜發(fā)生變化,產(chǎn)生相應(yīng)的生理學(xué)和病理學(xué)功能。 如果上述假設(shè)能夠得到證實(shí),對闡明PRAK和Sept8相互作用在細(xì)胞信號轉(zhuǎn)導(dǎo)過程中的作用具有重要意義,并且能夠以PRAK和Sept8相互作用為切入點(diǎn)揭示PRAK相關(guān)疾病的發(fā)病機(jī)制。為此我們對PRAK和Sept8的相互作用進(jìn)行了探討。 通過構(gòu)建人腦膠質(zhì)瘤cDNA文庫,并在文庫中利用特異性引物將Sept8基因擴(kuò)增出來,隨后分別克隆到pET14b和pcDNA3載體上。在體外(in vitro)結(jié)合研究中,首先分別在大腸桿菌株BL21(DE3)中對GST-PRAK和His-Sept8進(jìn)行了表達(dá),并分別利用谷胱甘肽親和樹脂和Ni-NTA親和樹脂對這兩種蛋白進(jìn)行了純化。在獲得純化蛋白的基礎(chǔ)上,進(jìn)行離體結(jié)合實(shí)驗(yàn)。結(jié)果表明,結(jié)合在Ni-NTA親和樹脂上的His-Sept8能將GST-PRAK pull-down下來,而相同條件下對單獨(dú)的GST蛋白沒有作用,說明Sept8在離體條件下能與PRAK結(jié)合。 在在體結(jié)合研究中,將HA-PRAK和FLAG-Sept8質(zhì)粒共轉(zhuǎn)染HEK293細(xì)胞中,將細(xì)胞裂解并進(jìn)行免疫共沉淀。分別用抗HA抗體偶agorose和抗FLAG抗體偶合瓊脂微粒分別從兩方面進(jìn)行的免疫共沉淀實(shí)驗(yàn),結(jié)果表明：Sept8和PRAK在細(xì)胞內(nèi)存在相互作用,而NaAsO2刺激可以進(jìn)一步增強(qiáng)他們之間的相互作用。上述結(jié)果提示,Sept8和PRAK之間的結(jié)合具有刺激依賴性。由于NaAsO2刺激能強(qiáng)烈激活p38通路,因而,PRAK與Sept8蛋白之間相互作用可能與p38通路的激活有關(guān)。 由于Sept8和PRAK之間的餓相互作用在受到NaAsO2刺激后結(jié)合增強(qiáng),我們采用免疫共沉淀進(jìn)一步分析了Sept8和PRAK之間相互結(jié)合與NaAsO2刺激的時間依賴性結(jié)合的時間過程。設(shè)立了200μmol／LNaAsO2刺激0 min、10 min、15 min、30 min、60 min、90 min、120 min和240 min組,結(jié)果發(fā)現(xiàn),它們之間的相互作用在受到刺激10 min后結(jié)合強(qiáng)度明顯增加,至240 min則結(jié)合減弱。 為了進(jìn)一步分析調(diào)控PRAK與Sept8相互作用的上游激酶,我們分別采用了p38 MAPK激酶抑制劑SB203580、ERK1/2激酶抑制劑PD98059和JNK激酶抑制劑SP600125預(yù)處理細(xì)胞,再采用免疫共沉淀分析NaAsO2處理對PRAK與Sept8相互作用的影響,結(jié)果顯示PD98059與SP600125對PRAK與Sept8的相互結(jié)合都沒有影響。而SB203580預(yù)處理30 min后,NaAsO2再刺激60 min,Sept8和PRAK兩者的結(jié)合增強(qiáng)。微管解聚劑Nocodazole不能阻斷Sept8與PRAK的結(jié)合。利用PRAK的無活性突變體PRAK(182A)、活性突變體PRAK(182D)(?)口失去ATP結(jié)合活性的突變體PRAK(KM)分別與Sept8進(jìn)行免疫共沉淀的結(jié)果表明：PRAK(182A)和PRAK(KM)在未受到刺激的情況下,能與Sept8結(jié)合,而在受到刺激后,PRAK (KM)結(jié)合反而顯著減弱；PRAK(182D)與Sept8的結(jié)合模式則與野生型PRAK類似。這些結(jié)果都提示我們,Sept8與PRAK的結(jié)合似乎與p38通路的激活有著密不可分的聯(lián)系。 最后,我們采用免疫熒光試驗(yàn)分析了內(nèi)源性的PRAK與Sept8在NIH/3T3細(xì)胞中的共定位情況,結(jié)果顯示內(nèi)源性的PRAK和Sept8在細(xì)胞未受到刺激時同時分布于細(xì)胞漿與細(xì)胞核內(nèi),局部存在共定位,而刺激可顯著增加他們之間的共定位,且形成了許多顆粒樣的結(jié)構(gòu),其生物學(xué)意義還有待進(jìn)一步的研究來揭示。 通過上述研究,可以得出以下結(jié)論：1.Sept8與PRAK在離體和在體情況下均能結(jié)合。2.Sept8與PRAK的結(jié)合具有刺激反應(yīng)性,NaAsO2應(yīng)激刺激能促進(jìn)兩者結(jié)合,提示兩者的結(jié)合可能在細(xì)胞對應(yīng)激刺激的反應(yīng)中有著重要的作用。 3.ERK1／2激酶抑制劑PD98059和JNK激酶抑制劑SP600125及微管解聚劑Nocodazole都不能阻斷Sept8與PRAK的結(jié)合,而p38激酶抑制劑SB203580可以影響兩者的結(jié)合。 4.PRAK的無活性突變體與失去ATP結(jié)合活性的突變體未受刺激時能與Sept8結(jié)合,失去ATP結(jié)合活性的突變體刺激后并不能進(jìn)一步提高它們之間的相互作用。提示這些突變體干擾了正常情況下PRAK與Sept8的結(jié)合。 6.正常情況下內(nèi)源性的Sept8與PRAK在NIH/3T3細(xì)胞中存在共定位。
[Abstract]:Mitogen activated protein kinase (mitogen-activated protein kinase, MAPK.P38) is an important signal system of eukaryotic cells and mediate extracellular signals to intracellular reaction is a member of the MAPK family, through phosphorylation of different substrates and the biological function of.P38 MAPK activation involved in cell growth, apoptosis, but also related to the the regulation of inflammation and stress response, transcription factor regulation, cytoskeleton remodeling, in a variety of diseases such as myocardial hypertrophy, ischemia / reperfusion injury, neuron disease, infectious disease process plays an important role.
The activation of p38 MAPK is a kind of activated by specific kinase by three kinase cascade of highly conserved (MKKK/MKK/MAPK) process. The specific activation motif (motif) threonine (threonine, Thr) - glycine (Glycine, Gly) - tyrosine (throsine, Tyr) activated Thr and Tyr motif in double at the same time were modified by phosphorylation and activation. Phosphorylation of p38 activated protein kinase mitogen activated protein kinase kinase 3 (mitogen-activated protein kinase kinase 3, MKK3) and MKK6, both of them can be upstream of the MKKK such as transforming growth factor activated kinase 1 (transforming growth factor- beta -activated kinase 1, TAK1), apoptosis signal transduction pathways regulating kinase 1 (apoptosis signal-regulating 1 kinase, ASK1), SH3 domain containing proline rich protein kinase (Srchomology domain 3-containing proline-rich protein kinase SPRK p21 (P2), activated kinase 1-activated kinase, PAK) and so on.
PRAK also called MK5, is a p38 MAPK regulated serine / threonine kinase, composed of 471 amino acid residues with a molecular weight of about 54 kD.PRAK are widely expressed in various tissues, especially in peripheral blood monocyte macrophages, endothelial cells, brain, lung,.PRAK binding activates downstream signal transduction pathway by using different proteins with different expression of kidney and ovarian prostate, such as PRAK and amphoteric protein interaction through Ras and Cdc42 pathway mediated neurite growth; through the p38 pathway of PRAK and heat shock protein 25/27 (heat shock protein 25/27, HSP25/27) interaction can be assembled actin regulatory cells, mediated cell oxidative stress and stress fiber formation and cell mediated stress response. Further research showed that PRAK is involved in the occurrence and development of many diseases, such as process and neural PRAK of endotoxic shock Degenerative lesions play a key role in the pathological process. The interaction between PRAK and amphoteric proteins can not only promote the growth of neuronal axons, but also mediate the growth and migration of tumor cells.
In view of this, PRAK is involved in multiple cellular signal transduction pathways, and is closely related to many diseases such as inflammation and tumor. It is a hot topic in the research field of signal transduction. However, the regulation mechanism of its function in cells is not clear.
Study of signal transduction, so we are very concerned about the exact function of PRAK in cells and the possible upstream kinase and downstream substrates in our laboratory. The previous study using PRAK as bait to screen human heart cDNA library to obtain the interaction proteins of SEPTIN8 by yeast two hybrid system (Sept8).
The full-length human Sept8 protein encoding gene sequence was 1452 BP, encoding the protein molecular weight is about 55.8 kD, which belongs to the conserved cytoskeletal GTP enzyme family members. The core domain in Sept8 protein is conserved GTP binding sites with GTP enzyme activity; close to the GTP binding sites for N terminal amino acid domains alkaline relatively conservative domain; the N terminal on the grounds of hundreds of amino acids and proline rich hydrophobic amino acid residues, probably SH3 binding domain; in addition the C terminal has a longer composed of alpha helical coiled coil (coiled-coil) domain, may be mediated by combination with other domains protein. Bioinformatics analysis also found that phosphorylation sites in Sept8 protein sequence, acetylation and palmitoylation sites.
We speculate that Sept8 is a candidate molecule interacting with PRAK, there are three reasons: first, there is the expression of Sept8 and PRAK in the body, such as brain glioma cells, renal cells, prostate cells; second, Sept8 is involved in the signal transduction mediated by receptor downstream of a precedent, such as Sept8 regulated by Rho protein signal transduction pathway G protein mediated in the pathway; third, signaling pathway and Sept8 PRAK participation overlap, such as the Ras link in mitogen activated protein kinase (MAPK) such as JNK, ERK and the activation of transcription factors such as nuclear factor kappa B (NF- K B) activation etc. so we can put forward the following hypothesis: Ras ligand interactions, the activation of Raf through direct or indirect way, Raf-1 phosphorylation of MEK1/MEK2 (MAP kinase/ERK kinase) two regulatory serine, which activates MEKs by downstream kinase The signal is transmitted to the p38 MAPK signal pathway of PRAK protein through interactions with Sept8 proteins after activation of downstream proteins such as p53 or HSP27, and then touch the intracellular oxidative stress the effect of cytokines expression changes, produce the corresponding physiological and pathological functions.
If the hypothesis is confirmed, it is significant to clarify the PRAK and Sept8 interaction in signal transduction, and pathogenesis of PRAK and Sept8 interaction as the starting point of the PRAK related diseases. The PRAK and Sept8 interaction are discussed.
By constructing a cDNA Library of human glioma, and in the library by using specific primers of Sept8 gene was amplified, then cloned into pET14b vector and pcDNA3 (in vitro). The in vitro binding experiment, firstly in Escherichia coli strain BL21 (DE3) of GST-PRAK and His-Sept8 were expressed respectively, and the glutathione affinity resin and Ni-NTA resin affinity purified of these two proteins. Based on purified proteins, in vitro binding experiments. The results show that the combined Ni-NTA affinity resin His-Sept8 GST-PRAK can be pull-down down, under the same conditions the GST protein alone had no effect, Sept8 and PRAK combined with in vitro.
In the in vivo binding experiment, HA-PRAK and FLAG-Sept8 plasmids were transfected into HEK293 cells, the cell lysis and immunoprecipitation respectively. Immunization with anti HA antibody and anti FLAG antibody coupled pairs agorose agar particles respectively from the two aspects of the co precipitation experiments, the results showed that Sept8 and PRAK interact in cells but, NaAsO2 stimulation can further enhance the interaction between them. These results suggest that the binding between Sept8 and PRAK is stimulus dependent. Because NaAsO2 can strongly stimulate the activation of the p38 pathway, and therefore, the interaction between PRAK and Sept8 protein between may be related to the activation of the p38 pathway.
Between the Sept8 and the PRAK hungry interaction combined with enhanced by NaAsO2 stimulation, we used a further analysis of the interaction between Sept8 and PRAK time process combined with NaAsO2 stimulation time dependent co immunoprecipitation. Set up 200 mu mol / LNaAsO2 stimulation 0 min, 10 min, 15 min, 30 min, 60 min, 90 min, 120 min and 240 min group, found that the interaction between them with stimulation intensity significantly increased after 10 min to 240 min, with decreased.
In order to further analyze the regulation of PRAK interaction with Sept8 upstream kinase, we used p38 MAPK kinase inhibitor SB203580, ERK1/2 kinase inhibitor PD98059 and JNK kinase inhibitor SP600125 pretreated cells, analyze the effect of NaAsO2 treatment on PRAK interaction with Sept8 by CO immunoprecipitation, results showed that PD98059 and SP600125 on PRAK and Sept8 mutual with no effect. The pretreatment of SB203580 30 min after NaAsO2 stimulation for 60 min, both Sept8 and PRAK increased. The microtubule depolymerizing agent Nocodazole does not block the combination of Sept8 and PRAK. PRAK mutant by PRAK (182A), the activity of mutant PRAK (182D) (?) and lost ATP binding activity the mutant PRAK (KM) were co precipitation and Sept8 immunization results showed that PRAK (182A) and PRAK (KM) without provocation, combined with Sept8, and in the stimulation After that, the binding of PRAK (KM) decreased significantly, and the binding mode of PRAK (182D) to Sept8 was similar to that of wild type PRAK. All these results indicate that the binding of Sept8 to PRAK seems to be closely related to the activation of p38 pathway.
Finally, we used immunofluorescence test analysis of PRAK and endogenous Sept8 in NIH/3T3 cells showed co localization of endogenous PRAK and Sept8 in the cells were not stimulated and distributed in the cytoplasm and the nucleus, the local co localization and stimulation could significantly increase the co localization between them, and the formation of a a lot of granular structure, its biological significance remains to be further studied to reveal.
Through the above research, we can draw the following conclusions: 1.Sept8 and PRAK in vitro and can combine.2.Sept8 with PRAK stimulation in body condition, NaAsO2 stress stimulation can promote the combination of the two, suggesting that the combination of the two in the cell may play an important role in the response to stress stimuli.
3.ERK1 / 2 kinase inhibitor PD98059 and JNK kinase inhibitor SP600125 and microtubule depolymerization agent Nocodazole can not block the binding of Sept8 to PRAK, while p38 kinase inhibitor SB203580 can affect the combination of Sept8 and PRAK.
The 4.PRAK mutant and the loss of the ATP binding activity of mutant unstimulated can bind to Sept8, loss of ATP binding activity of mutant stimulation did not further improve the interaction between them. These mutants interfere with the node PRAK and Sept8 under normal conditions.
6. under normal conditions, endogenous Sept8 and PRAK are Co located in NIH/3T3 cells.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

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相關(guān)期刊論文 前5條

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