催乳素在Jurkat细胞中信号传导分子的蛋白质组分析

Proteomics analysis for the signal transduction under stimulation of prolactin on Jurkat cells

目的:利用蛋白质组学分析催乳素(PRL)在人T淋巴白血病细胞株JurkatD1.1细胞中所激活的信号传导分子。方法:应用重组人催乳素(rhPRL)刺激JurkatD1.1细胞。利用磷酸化金属亲和层析法(PMAC)和免疫沉淀法(IP)富集磷酸化蛋白,单向凝胶电泳(1 DE)或者双向凝胶电泳(2 DE)分离磷酸化蛋白。分析不同组的凝胶,获取有差异的蛋白质条带和斑点。质谱分析并与蛋白质数据库进行匹配鉴定。结果:PMAC法获取的磷酸化蛋白用1 DE分离,胶扫描分析发现对照组和rhPRL刺激组之间存在五条明显差别的条带,其中三条条带在rhPRL刺激组,两条在对照组。质谱分析并与数据库匹配,成功鉴定刺激组中一条条带的蛋白质,为热休克蛋白90(Hsp90)。IP法获取的磷酸化蛋白,经过2 DE分离,胶扫描分析后,挖取rhPRL刺激组凝胶的九个蛋白质点。质谱分析并与数据库匹配,成功鉴定三个斑点的蛋白质分别是核内受体辅助抑制因子2变异体、半乳糖-1-磷酸尿苷酰转移酶和锌指蛋白ZIM3。结论:催乳素上调磷酸化热休克蛋白90(Hsp90),Hsp90可能参与催乳素的信号传导。催乳素信号分子调节目的基因的表达可能有核内受体辅助抑制因子2变异体和锌指蛋白ZIM3的参与。

Objective： The experiment was designed to apply proteomics to analyze the signal transduction molecules in T lymphocyte strain Jurkat D1. 1 cells with the stimulation of prolactin.Methods：Jttrkat D1. 1 cells were cultured in the presence of recombinant human prolactin（rhPRL）. Phosphated metal affinity chromography （PMAC） and immunoprecipitation （IP） assay were applied to enrich phosphoproteins from the total cell lystates which were resolved by one-dimensional electrophoresis （1 DE） or 2 DE.The different bands and different spots in the gels between control group and rhPRL-treated group were excised,digested,and analyzed by MALDI-TOF mass spectrometry.Results： Three major protein bands were observed specifically in the rhPRL-treated group and other two major protein bands were observed specifically in the control group. The bands were excised and analyzed by MALDI-TOF mass spectrometry. Protein band a in rhPRL-treated group was successfully identified as hot shock protein 90 （hsp90）. Nine different protein spots which were observed in the 2 DE gels between the two groups were excised and analyzed by MALDI-TOF mass spectrometry. In the rhPRL-treated group the protein spot 4 was identified as nuclear receptor co-repressor 2 variant （NcoR 2 variant） ;The protein spot 5 was identified as galactose-1-phosphate uridyl transfemse; And the protein spot 6 was identified as zinc finger ZIM3. Conclusion： Hot shock protein 90 participates the signal transduction pathways of prolactin. Nuclear receptor corepresser 2 variant and zinc finger ZIM3 may play a role in the process of prolactin modulating target genes＇ expression.