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Binding of Akt PH domain to its regulatory domain
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 22
- DOI:
- 10.3321/j.issn:1000-2790.2002.22.003
- Key Word:
- 蛋白激酶类;蛋白质构象
Abstract： 目的从人T细胞中克隆Akt的PH结构域及其调节区基因并进行6-his和GST融合表达,研究二者的体外结合情况,为进一步研究Akt的调节区在其分子激活中的作用打下基础. 方法以RT-PCR的方法从人T细胞中扩增目的基因片段,测序证明序列正确. 再将正确的目的基因片段定向克隆到表达载体pRSET-A 和pGEX-4T-1中. 以IPTG诱导,获得Akt的PH结构域及其调节区与6-his和GST的融合表达. 表达的PH结构域融合蛋白经谷胱甘肽耦联的琼脂糖珠纯化,以pull-down法检测该PH结构域与6-his调节区的结合. 结果 Akt的PH结构域和调节区基因序列完全正确,得到与GST和6-his的融合表达,表达产物有较高可溶性. 结论 SDS-PAGE检测到 PH结构域可在体外与调节区特异地结合.
- [1]邓艳春,药立波,王吉村,等.一种胶质瘤相关的酸性成纤维细胞生长因子mRNA同源物[J].2001,(6).
- [2]季少平,药立波,韩月恒,等.重组信号分子GRF的PH结构域抑制蛋白激酶C活性[J].2001,(1).
- [3]Woodgett JR.,Coffer PJ.,Jin J..Protein kinase B (c-Akt): A multifunctional mediator of phosphatidylinositol 3-kinase activation [Review][J].1998,335(Pt.1).
- [4]P J, Coffer,J R, Woodgett.Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families.[J].1991,201.
- [5]P, Chomczynski,N, Sacchi.Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.[J].1987,162(1).
- [6]P F,Jones.Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily.[J].1991,88.
- [7]S, Zimmermann,K, Moelling.Phosphorylation and regulation of Raf by Akt (protein kinase B).[J].1999,286(5445).
- [8]Dario R.AIessi,Barry Caudwell,Brian A.Hemmings,etc.Mechanism of activation of protein kinase B by insulin and IGF-1[J].1996,15(23).
- [9]Masato Hirata,Hiroaki Konishi,Yoshio Misumi,etc.Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein[J].1997,1359(3).
- [10]T, Pawson.Protein modules and signalling networks.[J].1995,373(6515).
- [11]B M, Marte,J, Downward.PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond.[J].1997,22(9).
- [12]Miller WE,Hall RA,Ringstad N,etc.Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.[J].1999,96(22).
- [13]DeFea K,Li J,Roth RA.Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway.[J].1999,274(14).
- [14]K, Touhara.Binding of multiple ligands to pleckstrin homology domain regulates membrane translocation and enzyme activity of beta-adrenergic receptor kinase.[J].1997,417(2).