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

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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