拟南芥PKS5激酶点突变体外表达与磷酸化测试

PKS5(protein kinase SOS2-like 5)虽为拟南芥(Arabidopsis thaliana)中介导植物响应外界高p H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。

In vitro expression and determination of phosphorylation activity of point mutants of the PKS5 kinase in Arabidopsis

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.