| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2009: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
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| Research Abstract |
Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme concentration. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein, and autophosphorylation subsequently occurs. The molecular mechanism of heme-binding, autophosphorylation, and degradation in HRI remained to be established. In the present study, 1) we demonstrate that His119/His120 and Cys411 are the axial ligands for heme in human HRI, as well as mouse HRI. 2) With the aid of mass spectroscopy we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichia coli. We further generated 30 enzymes with mutations at the phosphorylated residues and examined the catalytic activities. The activities of Y193F, T485A, and T490A mutants were significantly lower than that of wild-type protein. Accordingly, we suggest that Tyr193, Thr485, and Thr490 are essential residues in the catalysis. 3) Due to degradation of HRI in cultured cell, we show that HRI degradation is induced by 20S proteasome in vitro.
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