Cao Heping, Deterding Leesa J, Blackshear Perry J
U. S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana, United States of America.
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2014 Jul 10;9(7):e100977. doi: 10.1371/journal.pone.0100977. eCollection 2014.
Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83-103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.
锌指蛋白36（Tristetraprolin，TTP/ZFP36）可结合某些促炎细胞因子的信使核糖核酸（mRNA）并使其不稳定。TTP基因缺陷的小鼠由于促炎细胞因子过度产生而出现严重的炎症综合征。TTP的表达可由多种因素诱导，包括胰岛素以及肉桂和绿茶的提取物。TTP在体内高度磷酸化，是几种蛋白激酶的底物。在人类TTP中已鉴定出多个磷酸化位点，但难以区分主要和次要磷酸化位点。本研究旨在通过磷酸肽图谱分析和质谱法（MS）获取有关TTP磷酸化的更多信息。野生型和定点突变的TTP蛋白在转染的人类细胞中表达，随后用[32P] - 正磷酸盐进行体内放射性标记。带有组氨酸标签的TTP蛋白用镍 - 氮三乙酸（Ni - NTA）亲和珠纯化，并用胰蛋白酶和赖氨酰内肽酶消化。消化后的肽段通过高效液相色谱在C18柱上分离。野生型和所有突变型TTP蛋白均定位于细胞质中，在体内广泛磷酸化，并且能够与含ARE的RNA探针结合。具有S90和S93突变的突变型TTP导致一个主要磷酸肽峰消失。具有S197突变的突变型TTP导致另一个主要磷酸肽峰比野生型更早洗脱。S186、S296和T271处的其他突变对磷酸肽图谱几乎没有影响。质谱分析确定S90和S93突变蛋白中缺失的肽段为LGPELSPSPTSPTATSTTPSR（对应于人类TTP的氨基酸残基83 - 103）。质谱还鉴定出一个与第一个锌指区域相关的主要磷酸肽。这些分析表明，包含S90和S93的胰蛋白酶肽段是人类TTP中的主要磷酸肽。
