突变型nm23-H1基因的磷酸化活性研究  

The Study on the Phosphorylation Activity of Mutant nm23-H1

作　　者：杨雪琴[1,2] 马力[1] 朱大兴[1] 王东[2] 王阁[2] 周清华[1] 

机构地区：[1]天津医科大学总医院天津市肺癌研究所,天津市肺癌转移与肿瘤微环境重点实验室,天津300052 [2]第三军医大学大坪医院野战外科研究所肿瘤中心,重庆400042

出　　处：《中国肺癌杂志》2009年第3期193-197,共5页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金重点项目(No.30430300);国家自然科学基金(No.30070333);国家“十一五”科技攻关项目(No.2006BAI02A01);高等学校博士学科点专项基金(No.20040610060)资助~~

摘　　要：背景与目的磷酸化活性是nm23-H1重要的生物学活性,本研究旨在探讨不同位点的突变对nm23-H1磷酸化活性的影响。方法以野生型nm23-H1研究对照,采用放射自显影的方法检测nm23-H1野生型(WT)和4种突变型(P96S,H118F,S120G和S44A)原核表达蛋白的自身丝氨酸与自身组氨酸磷酸化活性,应用反相高效液相色谱技术(RP-HPLC)检测上述蛋白的NDPK激酶活性。结果野生型和突变型nm23-H1蛋白的自身丝氨酸与自身组氨酸磷酸化活性由大到小依次为P96S、WT、S44A、S120G和H118F,而NDPK活性由大到小依次为:WT、S120G、P96S、S44A和H118F;经线性相关分析,自身丝氨酸与组氨酸磷酸化活性呈正相关(r=0.983,P<0.01),但NDPK活性与自身丝氨酸和自身组氨酸活性均无相关性(分别为r=0.458,P>0.05;r=0.482,P>0.05)。结论不同位点的突变对nm23-H1自身丝氨酸、自身组氨酸及NDPK激酶活性影响不同,NDPK活性与自身磷酸化活性并不完全关联。其中118位组氨酸是nm23-H1激酶活性的关键氨基酸;96位脯氨酸可能与nm23-H1的磷酸转移能力有关;而120位丝氨酸可能为自身丝氨酸和组氨酸磷酸化位点;44位丝氨酸可能为另一具有NDPK激酶的氨基酸。Background and objective The Phosphorylation is the key activity of nm23-H1. The aim of this study is to explore the effect of different amino acid mutation on the phosphorylation status of nm23-H1. Methods The wild type nm23-H1 was as the control of this study. Autoradiography was used for detecting the serine and histidine autophosphorylation of wild type （WT） and mutant nm23-H1 （P96S, H118F, S120G and S44A）; RP-HPLC was used for detecting the NDPK activity of above proteins. Results The autophosphorylation activities of serine and histidine from high to low were P96S, WT, S44A, S120G and H118F, respectively, while the NDPK activities from high to low were WT, S120G, P96S, S44A, H118F. A highly positive correlation was found between serine and histidine autophosphorylation activity of above proteins （r=0.985, P〈0.01）, but no significant correlation was found between NDPK and serine or histidine autophosphorylation activity （r=0.458, P〉0.05, and r=0.482, P〉0.05, respectively）. Conclusion Site mutation of nm23-H1 can affect the phosphorylation activity. H118 site was the key amino acid of kinase activity, P96 site maybe related to phosphotransferring, S120 was the site of histidine autophosphorylation and sefine autophosphorylation, while the S44 site may be another amino acid which possessed NDPK activity.

关 键 词：NM23-H1 突变 磷酸化活性 

分 类 号：R73-3[医药卫生—肿瘤]