Analyses for phosphoproteins in activated T lymphocytes using Maldi-MS/MS method.

点击翻译按钮获取中文摘要

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 Cμ(PKCμ) and actually, activation of PKCμ was observed in the T cells stimulated with the analogue peptide/HLA-DR4.To seek for the role of PKCμ in the T cell activation pathway, we carried out the Maldi-MS/MS analyses using the Qstar Pulser i high performance mass-sp … More ectrometer and found that the kinase present in the T cells was not PKCμ but protein kinase D2(PKD2), a closely related kinase to PKCμ. Expression of GFP-tagged PKD2 in the human leukemic T cell line Jurkat revealed that PKD2 was mainly present in the cytosol and a fraction of the kinase translocated to the nucleus after TCR stimulation. To search for the possible substrates of PKD2 in the nucleus, we performed proteomic analyses using the nuclear extract of Jurkat cells and identified three tentative substrate proteins, all of which had the putative PKCμ phsophorylation site(Leu/Ile-X-X-X-X-Ser).Also, to find out other molecules involved in the T cell activation process, we performed proteomic analyses of phosphoproteins. Also, to find out other molecules involved in the T cell activation process, we performed proteomic analyses of phosphoproteins prepared from unstimulated, stimulated with wild-type peptide/HLA-DR4 and stimulated with analgue peptide/HLA-DR4 T cells and analyzed with Qstar. More that 40 phosphoproteins were identified and some of which were specifically phosphorylated in the T cells stimulated with the analgue peptide/HLA-DR4. Less