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)严格识别其同源抗原肽，但我们发现在小鼠L细胞表面过表达的一个氨基酸残基取代的类似肽/HLADR4复合体成功地刺激了同源人T细胞克隆并诱导了强烈的增殖反应。这一现象表明，与自身蛋白质中的非自身抗原肽非常相似的氨基酸序列可能会刺激不必要的T细胞激活，从而可能导致自身免疫性疾病。为了找出参与T细胞激活过程的分子，我们使用了一组抑制T细胞反应的抑制剂。为探讨蛋白激酶D在T细胞活化途径中的作用，我们用QStar Pulser I高性能质谱仪…进行了MALDIMS/MS分析进一步研究发现，T细胞中存在的PKD是D2-亚型(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Jurkat细胞中表达组成活性形式的PKD2增加了TCR刺激后的细胞死亡，这表明根据PKD2的活性，PKD2促进了IL-2启动子的激活或细胞死亡。为了寻找PKD2的可能底物，我们使用Jurkat细胞的核提取液和放射性三磷酸腺苷进行了蛋白质组学分析，以进行体外激酶活性测定。反应混合物进行2D-Gel分析，其中一个被磷酸化的蛋白质被鉴定为SET，也称为I2PP2A。为了了解PKD2对激活的T细胞SET的磷酸化的意义，我们研究了PKD2对SET的磷酸化位点。用不同的SET突变体进行的体外激酶分析表明，PKD2的SET的一个磷酸化位点是Ser171。Ser残基与公认的Leu-X-Lys/Arg-X-X-Ser的PKD磷酸化基序一致。已知SET是蛋白磷酸酶2A(PP2A)的特异性抑制剂，可使ERKs去磷酸化，在Jurkat细胞中Ser 171对SET的Glu突变体的表达抑制ERK的磷酸化，而在Jurkat细胞中Ser 171对SET的Ala突变体的表达抑制ERK的磷酸化，提示PKD2可能通过SET的磷酸化来调节PP2A的活性。较少

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