The role of heme-binding, phosphorylation, and degradation in heme-regulated eukaryotic initiation factor 2α kinase (HRI)

血红素调控的真核起始因子2α (eIF2α)激酶（HRI），在血红素浓度下起作用。在正常情况下，血红素与HRI结合并阻断其功能。然而，在血红素短缺期间，血红素与蛋白质分离，随后发生自磷酸化。HRI中血红素结合、自磷酸化和降解的分子机制仍有待确定。在本研究中，1)我们证明了His119/His120和Cys411是人HRI和小鼠HRI中血红素的轴向配体。2)利用质谱技术鉴定出小鼠HRI在大肠杆菌中过表达的33个磷酸化位点。我们进一步在磷酸化残基处产生了30个突变酶，并检测了催化活性。Y193F、T485A和T490A突变体的活性显著低于野生型蛋白。因此，我们认为Tyr193、Thr485和Thr490是催化过程中必不可少的残基。3)由于HRI在培养细胞中降解，我们发现20S蛋白酶体在体外诱导HRI降解。

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.