PRAK和SEPTIN8相互作用研究

发布时间：2018-01-01 04:44

  本文关键词：PRAK和SEPTIN8相互作用研究　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 丝氨酸/苏氨酸蛋白激酶 酵母双杂交筛选 蛋白质—蛋白质相互作用 离体结合实验 免疫共沉淀 信号通路

【摘要】：丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是真核细胞中介导细胞外信号至细胞内反应的重要信号系统。p38是MAPK家族成员之一,通过磷酸化不同的底物而发挥其生物学功能。p38 MAPK的激活涉及到细胞生长、凋亡等过程,同时也涉及到炎症和应激反应的调控、转录因子的调控、细胞骨架的重构等,在多种疾病如心肌肥大、缺血／再灌注损伤、神经元病变、感染性疾病过程中都发挥着重要的作用。 p38 MAPK的激活是一种由特定的激酶通过高度保守的三级激酶级联激活的(MKKK/MKK/MAPK)过程。其特异性的活化基序(motif)苏氨酸(threonine,Thr)-甘氨酸(glycine, Gly)-酪氨酸(throsine, Tyr)活化基序中Thr和Tyr双位点同时被磷酸化修饰而激活。能磷酸化激活p38的蛋白激酶有丝裂原激活蛋白激酶激酶3 (mitogen-activated protein kinase kinase 3,MKK3)和MKK6,它们都可以被上游的MKKK如转化生长因子β激活激酶1(transforming growth factor-β-activated kinase 1, TAK1)、凋亡信号通路调控激酶1(apoptosis signal-regulating kinase 1, ASK1)、含SH3结构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又称MK5,是一个受p38 MAPK调控的丝氨酸／苏氨酸激酶,由471个氨基酸残基组成,分子量约为54 kD。PRAK广泛表达于各种组织,尤其是在外周血单核-巨噬细胞、脑、血管内皮、肺脏、肾脏以及卵巢前列腺中高表达。PRAK通过与不同的蛋白结合而激活不同的下游信号转导通路,如PRAK与两性蛋白相互作用可通过Ras和Cdc42通路介导神经元轴突的生长；通过p38通路PRAK和热休克蛋白25/27(heat shock protein 25/27, HSP25/27)相互作用可调控细胞微丝的组装,介导细胞氧化应激时应力纤维的形成进而介导细胞应激反应。进一步的研究表明PRAK参与了多种疾病的发生与发展,如PRAK在内毒素休克的过程中以及神经退行性病变的病理过程中起到关键作用；PRAK与两性蛋白的相互作用不仅可以促进神经元轴突的生长,还可以介导肿瘤细胞的生长和迁移。 由此看来,PRAK参与细胞内多条细胞信号转导通路,与炎症、肿瘤等多种疾病密切相关,是信号转导通路研究领域的热点,但目前对其在细胞内的功能调控机制尚不太清楚。 信号转导方面的研究,因此我们十分关心PRAK在细胞内的确切功能及其可能的上游激酶和下游底物。我们实验室在前期的研究中采用人PRAK为诱饵,采用酵母双杂交系统筛选人心脏cDNA文库获得其相互作用蛋白SEPTIN8(Sept8)。 人Sept8蛋白基因全长编码序列为1,452 bp,其编码的蛋白质分子量约为55.8 kD,属于保守的细胞骨架GTP酶家族成员。位于Sept8蛋白中间的核心结构域是保守的GTP结合位点,具有GTP酶活性；紧邻GTP结合位点N端为相对保守的多碱性氨基酸结构域；其N端为由数百个氨基酸残基组成富含脯氨酸和疏水氨基酸残基的结构域,可能是SH3结合区域；此外其C端拥有一个较长的由α螺旋构成的卷曲螺旋(coiled-coil)结构域,可能是介导与其它蛋白质相互结合的结构域。通过生物信息学分析还发现Sept8蛋白序列中存在磷酸化修饰位点,乙酰化修饰位点和棕榈酰化位点。 我们推测Sept8是与PRAK相互作用的候选分子,原因有三：第一,在体内有Sept8和PRAK共同的表达细胞,如脑胶质瘤细胞、肾细胞、前列腺细胞；第二,Sept8参与介导受体下游的信号转导有先例,如G蛋白介导的信号转导通路中通过Rho蛋白调节Sept8的通路等；第三,PRAK参与的信号通路与Sept8参与的信号通路有重叠,如Ras环节的参与,丝裂原活化蛋白激酶(MAPK)如JNK、ERK的活化,以及转录因子如核转录因子κB (NF-κB)等的活化。因此我们可以提出如下假设：Ras与其配体相互作用后,通过直接或间接的方式激活Raf,Raf-1可磷酸化MEK1/MEK2(MAP kinase/ERK kinase)上的两个调节性丝氨酸,从而激活MEKs,通过下游激酶将信号传递给p38 MAPK信号通路的PRAK蛋白,通过与Sept8蛋白相互作用后,激活下游蛋白如p53或HSP27,进而触动细胞内的氧化应激使得效应细胞细胞因子表达谱发生变化,产生相应的生理学和病理学功能。 如果上述假设能够得到证实,对阐明PRAK和Sept8相互作用在细胞信号转导过程中的作用具有重要意义,并且能够以PRAK和Sept8相互作用为切入点揭示PRAK相关疾病的发病机制。为此我们对PRAK和Sept8的相互作用进行了探讨。 通过构建人脑胶质瘤cDNA文库,并在文库中利用特异性引物将Sept8基因扩增出来,随后分别克隆到pET14b和pcDNA3载体上。在体外(in vitro)结合研究中,首先分别在大肠杆菌株BL21(DE3)中对GST-PRAK和His-Sept8进行了表达,并分别利用谷胱甘肽亲和树脂和Ni-NTA亲和树脂对这两种蛋白进行了纯化。在获得纯化蛋白的基础上,进行离体结合实验。结果表明,结合在Ni-NTA亲和树脂上的His-Sept8能将GST-PRAK pull-down下来,而相同条件下对单独的GST蛋白没有作用,说明Sept8在离体条件下能与PRAK结合。 在在体结合研究中,将HA-PRAK和FLAG-Sept8质粒共转染HEK293细胞中,将细胞裂解并进行免疫共沉淀。分别用抗HA抗体偶agorose和抗FLAG抗体偶合琼脂微粒分别从两方面进行的免疫共沉淀实验,结果表明：Sept8和PRAK在细胞内存在相互作用,而NaAsO2刺激可以进一步增强他们之间的相互作用。上述结果提示,Sept8和PRAK之间的结合具有刺激依赖性。由于NaAsO2刺激能强烈激活p38通路,因而,PRAK与Sept8蛋白之间相互作用可能与p38通路的激活有关。 由于Sept8和PRAK之间的饿相互作用在受到NaAsO2刺激后结合增强,我们采用免疫共沉淀进一步分析了Sept8和PRAK之间相互结合与NaAsO2刺激的时间依赖性结合的时间过程。设立了200μmol／LNaAsO2刺激0 min、10 min、15 min、30 min、60 min、90 min、120 min和240 min组,结果发现,它们之间的相互作用在受到刺激10 min后结合强度明显增加,至240 min则结合减弱。 为了进一步分析调控PRAK与Sept8相互作用的上游激酶,我们分别采用了p38 MAPK激酶抑制剂SB203580、ERK1/2激酶抑制剂PD98059和JNK激酶抑制剂SP600125预处理细胞,再采用免疫共沉淀分析NaAsO2处理对PRAK与Sept8相互作用的影响,结果显示PD98059与SP600125对PRAK与Sept8的相互结合都没有影响。而SB203580预处理30 min后,NaAsO2再刺激60 min,Sept8和PRAK两者的结合增强。微管解聚剂Nocodazole不能阻断Sept8与PRAK的结合。利用PRAK的无活性突变体PRAK(182A)、活性突变体PRAK(182D)(?)口失去ATP结合活性的突变体PRAK(KM)分别与Sept8进行免疫共沉淀的结果表明：PRAK(182A)和PRAK(KM)在未受到刺激的情况下,能与Sept8结合,而在受到刺激后,PRAK (KM)结合反而显著减弱；PRAK(182D)与Sept8的结合模式则与野生型PRAK类似。这些结果都提示我们,Sept8与PRAK的结合似乎与p38通路的激活有着密不可分的联系。 最后,我们采用免疫荧光试验分析了内源性的PRAK与Sept8在NIH/3T3细胞中的共定位情况,结果显示内源性的PRAK和Sept8在细胞未受到刺激时同时分布于细胞浆与细胞核内,局部存在共定位,而刺激可显著增加他们之间的共定位,且形成了许多颗粒样的结构,其生物学意义还有待进一步的研究来揭示。 通过上述研究,可以得出以下结论：1.Sept8与PRAK在离体和在体情况下均能结合。2.Sept8与PRAK的结合具有刺激反应性,NaAsO2应激刺激能促进两者结合,提示两者的结合可能在细胞对应激刺激的反应中有着重要的作用。 3.ERK1／2激酶抑制剂PD98059和JNK激酶抑制剂SP600125及微管解聚剂Nocodazole都不能阻断Sept8与PRAK的结合,而p38激酶抑制剂SB203580可以影响两者的结合。 4.PRAK的无活性突变体与失去ATP结合活性的突变体未受刺激时能与Sept8结合,失去ATP结合活性的突变体刺激后并不能进一步提高它们之间的相互作用。提示这些突变体干扰了正常情况下PRAK与Sept8的结合。 6.正常情况下内源性的Sept8与PRAK在NIH/3T3细胞中存在共定位。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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