Analyses for protein kinase D2 substrate using 2D-DIGE and Q-Trap mass-spectrometer.

虽然一直认为T淋巴细胞（T细胞）严格识别同源抗原肽，但我们发现在小鼠L细胞表面过表达的一种氨基酸残基取代的类似肽/HLA-DR4复合物成功地刺激了同源的人类T细胞克隆并诱导了强烈的增殖反应。这一现象表明，与自身蛋白中的非自身抗原肽非常相似的氨基酸序列可能刺激不必要的T细胞活化，从而导致自身免疫性疾病。为了找出参与T细胞活化过程的分子，我们使用了一组抑制T细胞反应的抑制剂。结果表明，在类似肽/HLA-DR4刺激的T细胞中观察到蛋白激酶D （PKD）的参与，PKD实际上被激活。为了寻找PKD在T细胞活化途径中的作用，我们使用Qstar Pulser i高效质谱进行了Maldi-MS/MS分析，发现T细胞中存在的PKD是D2-isotype (PKD2)，而不是PKD1。野生型PKD2在人白血病T细胞株Jurkat中的表达表明PKD2参与了TCR刺激后IL-2启动子的激活。有趣的是，在tcr刺激后，Jurkat细胞中组成活性形式PKD2的表达增强了细胞死亡，这表明PKD2的活性取决于IL-2启动子激活或细胞死亡。为了寻找PKD2可能的底物，我们使用Jurkat细胞的核提取物和放射性ATP进行了蛋白质组学分析，用于体外激酶测定。将反应混合物进行2d凝胶分析，鉴定出其中一个磷酸化（=放射性）蛋白为SET，也称为I2PP2A。为了找出激活T细胞中PKD2磷酸化SET的意义，我们研究了PKD2磷酸化SET的位点。使用各种SET突变体进行的体外激酶试验显示，PKD2磷酸化SET的一个位点是Ser 171。该Ser残基与一致的PKD磷酸化基序Leu-X-Lys/Arg-X-X-Ser一致。已知SET是ERKs去磷酸化的蛋白磷酸酶2A （PP2A）的特异性抑制剂，Jurkat细胞中Ser 171对Glu突变体的表达抑制ERK磷酸化，而Ser 171对Ala突变体的表达抑制ERK磷酸化，提示PKD2可能通过SET磷酸化调节PP2A活性。少

Although it has been believed that T lymphocytes (T cells) strictly recognize their cognate antigenic peptides, we found that one amino acid residue-substituted analogue peptide/HLA-DR4 complexes over-expressed on the surface of mouse L cells successfully stimulated the cognate human T cell clone and induced strong proliferative response. This phenomenon suggests that an amino acid sequence with close similarity to the non-self antigenic peptide in a self protein may stimulate unwanted T cell activation that may cause autoimmune diseases. To find out molecules involved in the T cell activation process, we used a panel of inhibitors that suppressed the T cell responses. The results suggested the involvement of protein kinase D (PKD) and actually, activation of PKD was observed in the T cells stimulated with the analogue peptide/HLA-DR4.To seek for the role of PKD in the T cell activation pathway, we carried out Maldi-MS/MS analyses using the Qstar Pulser i high performance mass-spectrom … More eter and found that the PKD present in the T cells was D2-isotype (PKD2), not PKD1. Expression of wild- type PKD2 in the human leukemic T cell line Jurkat revealed that PKD2 was involved in the IL-2 promoter activation after TCR- stimulation. Interestingly, expression of constitutively-active form of PKD2 in Jurkat cells enhanced cell death after TCR-stimulation, suggesting that depending on the PKD2 activity, PKD2 contributes either IL-2 promoter activation or cell death. To search for the possible substrates of PKD2, we performed proteomic analyses using the nuclear extract of Jurkat cells and radioactive ATP for in vitro kinase assay. The reaction mixture was subjected to 2D-gel analysis and one of the phosphorylated (=radioactive) proteins was identified to be SET, also called I2PP2A.To find out the significance of SET phosphorylation by PKD2 in activated T cells, we studied the phosphorylation sites of SET by PKD2. The in vitro kinase assay using various SET mutants revealed that one of the phosphorylation sites of SET by PKD2 is Ser 171. The Ser residue was in accordant with the consensus PKD phosphorylation motif of Leu-X-Lys/Arg-X-X-Ser. SET is known to be the specific inhibitor for protein phosphatase 2A (PP2A) that dephosphorylates ERKs and expression of Ser 171 to Glu mutant of SET in Jurkat cells suppressed ERK phosphorylation compared with the expression of Ser 171 to Ala mutant of SET in Jurkat cells, suggesting that PKD2 might regulate PP2A activity through SET phosphorylation. Less