Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis
The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of...
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| Format: | Article |
| Language: | English |
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Wiley
2009-01-01
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| Series: | Advances in Hematology |
| Online Access: | http://dx.doi.org/10.1155/2009/251915 |
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| author | Kimon C. Kanelakis Jayashree Pyati Pamela C. Wagaman Jui Chang Chuang Young Yang Nigel P. Shankley |
| author_facet | Kimon C. Kanelakis Jayashree Pyati Pamela C. Wagaman Jui Chang Chuang Young Yang Nigel P. Shankley |
| author_sort | Kimon C. Kanelakis |
| collection | DOAJ |
| description | The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis. |
| format | Article |
| id | doaj-art-1550ff257a9b4840b7223c3f1e398751 |
| institution | Kabale University |
| issn | 1687-9104 1687-9112 |
| language | English |
| publishDate | 2009-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advances in Hematology |
| spelling | doaj-art-1550ff257a9b4840b7223c3f1e3987512025-02-03T01:21:27ZengWileyAdvances in Hematology1687-91041687-91122009-01-01200910.1155/2009/251915251915Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein SynthesisKimon C. Kanelakis0Jayashree Pyati1Pamela C. Wagaman2Jui Chang Chuang3Young Yang4Nigel P. Shankley5Department of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USADepartment of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USADepartment of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USADepartment of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USADepartment of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USADepartment of Internal Medicine, Johnson & Johnson Pharmaceutical Research & Development L.L.C., Merryfield Row 3210, San Diego, CA 92121, USAThe heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.http://dx.doi.org/10.1155/2009/251915 |
| spellingShingle | Kimon C. Kanelakis Jayashree Pyati Pamela C. Wagaman Jui Chang Chuang Young Yang Nigel P. Shankley Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis Advances in Hematology |
| title | Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis |
| title_full | Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis |
| title_fullStr | Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis |
| title_full_unstemmed | Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis |
| title_short | Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis |
| title_sort | functional characterization of the canine heme regulated eif2α kinase regulation of protein synthesis |
| url | http://dx.doi.org/10.1155/2009/251915 |
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