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研究発表を行った学会；
・第３６回日本免疫学会総会・学術集会、2006年 12月11~13日(大阪)

タイトル； Protein kinase D2, a regulator of IL-2 promoter activity and cell death, phosphorylates SET protein in Jurkat cells.
発表者；　入江　厚　氏
　　　（熊本大学　大学院医学薬学研究部　免疫識別学分野）
Abstract；
　Protein kinase D2 (PKD2) but not PKD1 is a major molecular species of PKD family protein serine kinases expressed in T cells and is involved in antigen-stimulated T cell activation. In Jurkat cells, expression of wild-type and constitutively active (CA-) PKD2 enhanced IL-2 promoter activity and cell death upon anti-CD3 stimulation, while expression of kinase-dead mutant suppressed those phenomena. While GFP-tagged PKD2 was excluded from the nucleus and localized in the cytosol in unstimulated
Jurkat cells, a part of GFP-PKD2 relocated in the nucleus after
TCR-stimulation. To further analyze the role of PKD2 in T cells, we tried to identify the substrates of PKD2 in Jurkat cells.

[Method] We prepared phosphoprotein fractions from Jurkat cells
over-expressing constitutively active CA-PKD2 and from parental Jurkat cells and compared the protein profiles by performing 2D-gel electrophoresis. The major protein spots observed only in the phosphoprotein fraction from CA-PKD2 expressing cells were excised, trypsin digested and subjected to mass-spectrometric analyses.

[Results] There were three major phosphoprotein spots observed only in the phosphoprotein fraction from CA-PKD2 expressing cells. Two of them were SET protein and pp32, which were nuclear phosphoproteins involved in the regulation of histone acetylation. Recombinant SET proteins were subjected to in vitro kinase assay and were phosphorylated by CA-PKD2 but not by the
kinase dead (KD) mutant of PKD2. The SET phosphorylation was inhibited by the addition of Go6976, a conventional PKC and PKD specific inhibitor, but not by Go6983, a pan-PKC specific inhibitor but ineffective with PKD. Using recombinant truncated mutants of SET protein, the phosphorylation sites were
identified within the C-terminal half of the SET protein.

[Conclusion] A nuclear phosphoprotein SET was identified to be a candidate substrate of PKD2. Since PKD2 relocalizes in the nucleus after TCR-stimulation, one of the functions of PKD2 might be the regulation of gene expression through phosphorylation of SET.