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拟南芥PKS5激酶点突变体外表达与磷酸化测试(PDF)

《广西植物》[ISSN:1000-3142/CN:45-1134/Q]

期数:
2015年03期
页码:
408-413
栏目:
遗传与育种
出版日期:
2015-05-20

文章信息/Info

Title:
In vitro expression and determination of phosphorylation activity of point mutants of the PKS5 kinase in Arabidopsis
文章编号:
1000-3142(2015)03-0408-06
作者:
赵菲佚12 焦成瑾1 陈 荃1 马伟超1 安建平1 呼丽萍12
1. 天水师范学院 生命科学与化学学院, 甘肃 天水 741000; 2. 甘肃大樱桃工程技术中心, 甘肃 天水 741000
Author(s):
ZHAO Fei-Yi12 JIAO Cheng-Jin1 CHEN Quan1 MA Wei-Chao1 AN Jian-Ping1 HU Li-Ping1 2
1. School of Life Sciences and Chemistry, Tianshui Normal Universiry, Tianshui 741000, China; 2. Gansu Engineering and Research Center for Sweet Cherry, Tianshui 741000, China
关键词:
PKS5 点突变 蛋白表达 磷酸化活性
Keywords:
PKS5 point mutant protein expression phosphorylation activity
分类号:
Q948.112
DOI:
10.11931/guihaia.gxzw201306008
文献标识码:
A
摘要:
PKS5(protein kinase SOS2-like 5)虽为拟南芥(Arabidopsis thaliana)中介导植物响应外界高pH的蛋白激酶,但其关键功能结构域尚未被确定。该研究用PCR对PKS5不同位置点突变形式进行克隆,并在原核系统中进行表达,得到PKS5不同的点突变蛋白; 使用激酶通用底物MBP(myelin basic protein)及PKS5体内特异底物AHA2(A. thaliana isoform of the PM H+-ATPase,拟南芥质膜质子泵等位形式之一)对PKS5点突变蛋白磷酸化活性进行了测试。结果表明:点突变PKS5-2失去了激酶活性,PKS5-4、PKS5-5、PKS5-9自磷酸化与MBP磷酸化活性与PKS5相比无差异; 而与PKS5相比,点突变PKS5-6和PKS5-7自磷酸化及对AHA2的磷酸化活性升高,且PKS5-7活性高于PKS5-6。说明PKS5特定位置点突变改变PKS5的自磷酸化及底物磷酸化活性水平,不同位置的点突变对其磷酸化活性的影响存在差异。研究结果可为确定PKS5功能结构域及体内作用机理提供依据。
Abstract:
In Arabidopsis,PKS5(protein kinase SOS2-like 5), a serine-threonine kinase, involves in the response to the external high pH stress based on the study of its loss-of-function mutant. Whereas, the fine functions of the domains resided in PKS5 are not currently well determined. We report here the dissection of domains of PKS5 in the activity of phosphorylation against MBP(myelin basic protein)and AHA2(one of the Arabidopsis thaliana isoform of PM H+-ATPases), which is the specific substrate of PKS5 in vivo, using the assay of phosphorlation in vitro via expressing the distinct PKS5 mutant versions in bacteria using the PKS5 cloning from plants employing PCR approach. The results showed that the point mutated PKS5-2 lost its activity, PKS5-4, PKS5-5 and PKS5-9 displayed no difference in autophosphorylation and the MBP phosphorylation. Moreover,autophosphorylation and the AHA2 phosphorylation of the point mutated PKS5-6 and PKS5-7 increased compared with PKS5 and the PKS5-7 activity was higher than PKS5-6. Taken together, the specific point mutations in PKS5 altered its activity of autophosphorylation and the substrate phosphoylation of BMP and AHA2. The effects due to the alteration of mutations resided in PKS5 on the activity of phosphorylation were comparable within the various PKS5 mutated protein versions. The results of this study would provide the basis for pinpointing the functional domains of PKS5 and the mechanism of functions of PKS5 in planta.

参考文献/References

Albrecht V,Ritz O,Linder S,et al. 2001. The NAF domain defines a novel protein-protein interaction module conserved in Ca2+ regulated kinases[J]. Embo J,20(5):1 051-1 063
Anja TF,Yan G,Tracey AC,et al. 2007. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+-ATPase by preventing interaction with 14-3-3 protein[J]. Plant Cell,19(5):1 617-1 634
Changgen X,Huixin L,Xingwang D,et al. 2009. Roles of SCaBP8 in salt stress response[J]. Plant Sign Behav,4(10):956-958
Cheong YH,Kim KN,Pandey GK,et al. 2003. CBL1,a calcium sensor that differentially regulates salt,drought,and cold responses in Arabidopsis[J]. Plant Cell,15(8):1 833-1 845
Deming G,Zizhong G,Jiankang Z. 2002. Expression,activation and biochemical properties of a novel Arabidopsis protein kinase[J]. Plant Physiol,129(1):225-234
Deming G,Changqing Z,Xiuyin C,et al. 2002. Constitutive activation and transgenic evaluation of the function of an Arabidopsis PKS protein kinase[J]. J Biol Chem,227(44):42 088-42 096
Detlef W,Jane G. 2002. Arabidopsis:A Laboratory Manual[M]. New York:Cold Spring Harbor Laboratory Press:168-169
Hardie DG,Carling D,Carlson M. 1998. The AMP-activated/SNF1 protein kinase subfamily:metabolic sensors of the eukaryotic cell[J]. Ann Rev Biochem,67(1):821-855
Hrabak EM,Chan CW,Gribskov M,et al. 2003. The Arabidopsis CDPK-SnRK superfamily of protein kinases[J]. Plant Physiol,132(2):666-680
Jen S,Li Z,Jang JC. 1999. Sugars as signaling molecules[J]. Curr Opin Plant Biol,2:410-418
Qin YZ(秦玉芝),Li X(李旭),Guo M(郭明),et al. 2008. Regulation of salt and ABA responses by CIPK14,a calcium sensor interacting protein kinase in Arabidopsis(钙传感蛋白互作激酶CIPK14参与拟南芥盐和ABA 胁迫应答调节)[J]. Sci Chin Ser C:Life Sci(中国科学C辑:生命科学),38(5):446-457
Qin YZ(秦玉芝),Guo M(郭明),Li X(李旭),et al. 2010. Stress responsive gene CIPK14 is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis(胁迫相关基因CIPK14在PHYA介导抑制拟南芥远红光黄化苗转绿过程中的作用)[J]. Sci Chin:Life Sci(中国科学:生命科学),40(10):970-977
Uner K,Stefan W,Dragica B,et al. 2004. Calcium sensors and their interacting protein kinases:genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J]. Plant Physiol,134(1):43-58
Xie C,Zhou X,Deng X,et al. 2010. PKS5,a SNF1-related kinase,interacts with and phosphorylates NPR1,and modulates expression of WRKY38 and WRKY62[J]. J Genet Genom,37(6):359-369
Yan G,Ursula H,Manabu I,et al. 2001. Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance[J]. Plant Cell,13(6):1 383-1 399
Yong QY,Yun XQ,Chang XX,et al. 2010. The Arabidopsis chaperone J3 regulates the plasma membrane H+-ATPase through interaction with the PKS5 kinase[J]. Plant Cell,22(4):1 313-1 332

备注/Memo

备注/Memo:
收稿日期: 2014-08-30修回日期: 2014-12-22
基金项目: 国家自然科学基金(31160060,31260568); 天水师范学院中青年教师科研项目(TSB1016)。
作者简介: 赵菲佚(1972-),男,甘肃天水人,博士,副教授,研究方向为植物抗逆分子生物学,(E-mail)tspaulzhao@163.com。
更新日期/Last Update: 2015-05-20