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Science:再次颠覆教科书观点--PKA全酶发挥蛋白激酶活性不需要调节亚基解离

2017-06-30 佚名 免疫细胞研究bioworld

教科书就是拿来推翻的,最新的SCIENCE发表的关于PKA结构的文章(1)再次推翻了教科书中关于PKA结构的描述。在教材第六版《细胞的分子生物学》(Molecular Biology of the Cell, 6th)的图15-26中明确说明,PKA全酶是一个由两个催化亚基(C)和两个调节亚基(R)组成的,cAMP与调节亚基结合后,调节亚基离开催化亚基,解放后者蛋白激酶活性。然而在最新的研究文章中

教科书就是拿来推翻的,最新的SCIENCE发表的关于PKA结构的文章(1)再次推翻了教科书中关于PKA结构的描述。在教材第六版《细胞的分子生物学》(Molecular Biology of the Cell, 6th)的图15-26中明确说明,PKA全酶是一个由两个催化亚基(C)和两个调节亚基(R)组成的,cAMP与调节亚基结合后,调节亚基离开催化亚基,解放后者蛋白激酶活性。然而在最新的研究文章中发现在生理浓度cAMP作用下,PKA发挥催化活性不需要调节亚基与催化亚基解离。

新文首先用了单粒子电镜(Single-particle electron microscopy)技术观察到PKA锚定蛋白AKAP (A kinase anchoring protein)存在下,AKAP和PKA全酶五聚体(AKAP,两个催化亚基,两个调节亚基)形成的三叶状结构(图2)。锚定蛋白AKAP为一叶,两个催化亚基形成另外两叶,两叶之间的距离从紧凑型的150 到舒张型的250。五聚体的结构还可以通过非变性质谱(Native Mass Spectrometry)和pull-down方法观察到。更重要的是在cAMP与调节亚基结合情况下,仍可以显着地观察到催化亚基与调节亚基结合在一起(> 50%),而不是传统概念中的两者解离。最后在细胞内,利用免疫共沉淀、荧光共振能量转移(FRET)和临近连接技术(proximity ligation assay, PLA)观察到在β肾上腺素受体信号作用下PKA发生活化,但没有发生明显的PKA全酶解离。新文用详尽的蛋白与蛋白相互作用的研究证明细胞内PKA活化,PKA全酶不发生传统认为的解离。

为了进一步证明不解离的PKA全酶具有蛋白激酶活性,研究者非常巧妙地把PKA的调节亚基与催化亚基融合表达,使之共价连接不能发生解离。在PKA调节亚基(PRKAR2A,PRKAR2B),催化亚基(PRKACA)三基因敲除的细胞中表达融合的PKA能够正常发挥PKA激酶活性。另外,利用雷帕霉素作用下,FRB结构域能够与FK506结合结构域(FKBP)结合的特性,FRB结构域融合的PKA催化亚基和FKBP结构域融合的调节亚基在雷帕霉素作用下可以形成稳定复合物。甚至在高于生理浓度的cAMP作用下(加cAMP水解酶抑制剂),雷帕霉素仍能锁定PKA的催化亚基与调节亚基,而这一复合物具有PKA催化活性。这两实验非常明确地说明了PKA调节亚基与催化亚基的解离不是其发挥催化活性所必需。

新文让读者眼睛一亮的同时,也引起我们的思考。1,新文的想法可能源于AKAP的性质,AKAP能够与PKA的调节亚基结合,多数也能与PKA底物结合,形成信号微域(signaling microdomain)。cAMP与调节亚基结合,使催化亚基磷酸化底物,因此与底物一起结合AKAP的调节亚基解离(推开)催化亚基可能不符合信号微域的作用。2,要想打破传统的观念并不容易,新文基本上穷尽了研究蛋白蛋白相互作用的方法,还引入了相对较新的单粒子电镜技术。3,新文也留下了进一步研究方向,那就是cAMP作用下PKA全酶不解离,那在结构上发生了什么样的变化使之获得活性?在单粒子电镜下观察到多种全酶的结构,如果这些异质性结构是生理性的,又具有什么样的功能?

【作者简介】
John D Scott 博士 (http://faculty.washington.edu/scottjdw/)
Scott博士是华盛顿大学医学院教授,HHMI研究员。其实验室专注研究PKA锚定蛋白APAK赋予PKA信号的时空特异性。从1985在PNAS上发表关于PKA的抑制性作用蛋白开始,1991发表第一篇通讯作者的文章,发现PKA锚定蛋白上的两亲螺旋与PKA调节亚基相互作用,一直到最新的打破教科书的新发现,30多年来一直围绕PKA开展研究。

原始出处:
Smith, F. D., et al.Local protein kinase A action proceeds through intact holoenzymes.Science. 2017 Jun 23;356(6344):1288-1293. doi: 10.1126/science.aaj1669.

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    2018-02-08 wetgdt
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    2017-07-01 爆笑小医
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    2017-07-01 redcrab
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