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Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2
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| Zeitschriftentitel: | Genes & Development |
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| Personen und Körperschaften: | , , , |
| In: | Genes & Development, 16, 2002, 10, S. 1271-1280 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Cold Spring Harbor Laboratory
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| Schlagwörter: |
| Zusammenfassung: | <jats:p>The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, <jats:italic>R794G</jats:italic> and <jats:italic>F842L</jats:italic>, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated by<jats:italic>R794G</jats:italic> and <jats:italic>F842L</jats:italic>, and the autophosphorylation sites remained critical for <jats:italic>GCN2</jats:italic> function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain.</jats:p> |
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| Umfang: | 1271-1280 |
| ISSN: |
1549-5477
0890-9369 |
| DOI: | 10.1101/gad.979402 |