酪氨酸蛋白激酶Fyn突变体FynY531F与FynK299M活性的鉴定

Activity Identification of Fyn Tyrosine Kinase Mutants FynY531F and FynK299M

Fyn是Src酪氨酸蛋白激酶家族(Src family of protein tyrosine kinases;SrcPTKs)中的一员,在神经元的增殖、分化和存活中起着重要作用.本文利用分子克隆手段构建Fyn突变体FynY531F(酪氨酸突变为苯丙氨酸)和FynK299M(赖氨酸突变为甲硫氨酸)的真核表达载体,并通过检测突变体对NMDA受体亚基NR2A的磷酸化作用以鉴定其激酶活性.根据GenBank提供的大鼠Fyn的cDNA序列(NM-012755),采用RT-PCR扩增野生型Fyn(wFyn)目的基因,应用定点突变技术扩增活化型Fyn(FynY531 F)目的基因,并用重叠延伸PCR定点突变技术扩增失活型Fyn(FynK299 M)目的基因,分别克隆入真核表达载体pcDNA3.1(+).免疫印迹分析显示,wFyn、FynY531F及FynK299M重组体均能在COS-7细胞表达.将构建的3个重组体分别与NMDA受体亚基NR2A的表达质粒共转染COS-7细胞.结果显示,活化型Fyn能够使NR2A酪氨酸磷酸化,而失活型Fyn则没有表现出激酶活性.结果表明,本文成功构建了野生型、活化型及失活型Fyn的真核表达载体,为进一步揭示Fyn在中枢神经系统中的病理生理意义奠定了基础.

Fyn, a member of Src family protein tyrosine kinases （SrcPTKs）, plays an important role in neuron proliferation, differentiation and survival. Fyn mutants of FynY531 F （conversion of tyrosine to phenylalanine） and FynK299M （conversion of lysine to methionine） were constructed by molecular cloning technology, and the kinase activities of wild type and mutant form of Fyn were examined by testing tyrosine phosphorylation state of N-methyl-D-aspartate（NMDA） receptor subunit NR2A. According to the cDNA sequence of Fyn （GenBank, NM_ 012755） ,we obtained the fragments of wild-type Fyrt（wFyn） by RT-PCR from extracted rat RNA, the active Fyn （FynY531 F） by site-specific mutagenesis and inactive Fyn （FynK299M） by overlap extension PCR and site-specific mutagenesis, and cloned these fragments into the eukaryotie expression vector peDNA3.1 （＋）respectively. The immunoblotting analysis indicated that the recombinants of wFyn, FynY531F, FynK299M were successfully expressed in COS-7 ceils. Subsequently, COS-7 ceils were transfected with NMDA subunit NR2A expression plasmid together with a plasmid encoding wFyn, FynY531F or conditions of central nervous system（CNS）.