This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qiu, H.
Right arrow Articles by Hinnebusch, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qiu, H.
Right arrow Articles by Hinnebusch, A. G.

 Previous Article  |  Next Article 

Mol Cell Biol, May 1998, p. 2697-2711, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Dimerization by Translation Initiation Factor 2 Kinase GCN2 Is Mediated by Interactions in the C-Terminal Ribosome-Binding Region and the Protein Kinase Domain

Hongfang Qiu, Minerva T. Garcia-Barrio, and Alan G. Hinnebusch*

Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892

Received 21 November 1997/Returned for modification 5 January 1998/Accepted 2 February 1998

The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.

* Corresponding author. Mailing address: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Building 6A, Room B1A-13A, Bethesda, MD 20892-2716. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail: ahinnebusch{at}nih.gov.

Mol Cell Biol, May 1998, p. 2697-2711, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Zaborske, J. M., Narasimhan, J., Jiang, L., Wek, S. A., Dittmar, K. A., Freimoser, F., Pan, T., Wek, R. C. (2009). Genome-wide Analysis of tRNA Charging and Activation of the eIF2 Kinase Gcn2p. J. Biol. Chem. 284: 25254-25267 [Abstract] [Full Text]  
  • Garriz, A., Qiu, H., Dey, M., Seo, E.-J., Dever, T. E., Hinnebusch, A. G. (2009). A Network of Hydrophobic Residues Impeding Helix {alpha}C Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the {alpha} Subunit of Translation Initiation Factor 2. Mol. Cell. Biol. 29: 1592-1607 [Abstract] [Full Text]  
  • Kim, K.-Y., Truman, A. W., Levin, D. E. (2008). Yeast Mpk1 Mitogen-Activated Protein Kinase Activates Transcription through Swi4/Swi6 by a Noncatalytic Mechanism That Requires Upstream Signal. Mol. Cell. Biol. 28: 2579-2589 [Abstract] [Full Text]  
  • Reibarkh, M., Yamamoto, Y., Singh, C. R., del Rio, F., Fahmy, A., Lee, B., Luna, R. E., Ii, M., Wagner, G., Asano, K. (2008). Eukaryotic Initiation Factor (eIF) 1 Carries Two Distinct eIF5-binding Faces Important for Multifactor Assembly and AUG Selection. J. Biol. Chem. 283: 1094-1103 [Abstract] [Full Text]  
  • Rubenstein, E. M., Schmidt, M. C. (2007). Mechanisms Regulating the Protein Kinases of Saccharomyces cerevisiae. Eukaryot Cell 6: 571-583 [Full Text]  
  • Dey, M., Cao, C., Sicheri, F., Dever, T. E. (2007). Conserved Intermolecular Salt Bridge Required for Activation of Protein Kinases PKR, GCN2, and PERK. J. Biol. Chem. 282: 6653-6660 [Abstract] [Full Text]  
  • Padyana, A. K., Qiu, H., Roll-Mecak, A., Hinnebusch, A. G., Burley, S. K. (2005). Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2{alpha} Protein Kinase GCN2. J. Biol. Chem. 280: 29289-29299 [Abstract] [Full Text]  
  • Narasimhan, J., Staschke, K. A., Wek, R. C. (2004). Dimerization Is Required for Activation of eIF2 Kinase Gcn2 in Response to Diverse Environmental Stress Conditions. J. Biol. Chem. 279: 22820-22832 [Abstract] [Full Text]  
  • Hannum, C., Kulaeva, O. I., Sun, H., Urbanowski, J. L., Wendus, A., Stillman, D. J., Rolfes, R. J. (2002). Functional Mapping of Bas2. IDENTIFICATION OF ACTIVATION AND Bas1-INTERACTION DOMAINS. J. Biol. Chem. 277: 34003-34009 [Abstract] [Full Text]  
  • Garcia-Barrio, M., Dong, J., Cherkasova, V. A., Zhang, X., Zhang, F., Ufano, S., Lai, R., Qin, J., Hinnebusch, A. G. (2002). Serine 577 Is Phosphorylated and Negatively Affects the tRNA Binding and eIF2alpha Kinase Activities of GCN2. J. Biol. Chem. 277: 30675-30683 [Abstract] [Full Text]  
  • Qiu, H., Hu, C., Dong, J., Hinnebusch, A. G. (2002). Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2. Genes Dev. 16: 1271-1280 [Abstract] [Full Text]  
  • Yang, R., Wek, S. A., Wek, R. C. (2000). Glucose Limitation Induces GCN4 Translation by Activation of Gcn2 Protein Kinase. Mol. Cell. Biol. 20: 2706-2717 [Abstract] [Full Text]  
  • Rafie-Kolpin, M., Chefalo, P. J., Hussain, Z., Hahn, J., Uma, S., Matts, R. L., Chen, J.-J. (2000). Two Heme-binding Domains of Heme-regulated Eukaryotic Initiation Factor-2alpha Kinase. N TERMINUS AND KINASE INSERTION. J. Biol. Chem. 275: 5171-5178 [Abstract] [Full Text]  
  • Sood, R., Porter, A. C., Olsen, D., Cavener, D. R., Wek, R. C. (2000). A Mammalian Homologue of GCN2 Protein Kinase Important for Translational Control by Phosphorylation of Eukaryotic Initiation Factor-2{alpha}. Genetics 154: 787-801 [Abstract] [Full Text]  
  • Donze, O., Picard, D. (1999). Hsp90 Binds and Regulates the Ligand-Inducible alpha Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2. Mol. Cell. Biol. 19: 8422-8432 [Abstract] [Full Text]  
  • Sattlegger, E., Hinnebusch, A. G., Barthelmess, I. B. (1998). cpc-3, the Neurospora crassa Homologue of Yeast GCN2, Encodes a Polypeptide with Juxtaposed eIF2alpha Kinase and Histidyl-tRNA Synthetase-related Domains Required for General Amino Acid Control. J. Biol. Chem. 273: 20404-20416 [Abstract] [Full Text]  
  • Fernandez, J., Yaman, I., Merrick, W. C., Koromilas, A., Wek, R. C., Sood, R., Hensold, J., Hatzoglou, M. (2002). Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2alpha Phosphorylation and Translation of a Small Upstream Open Reading Frame. J. Biol. Chem. 277: 2050-2058 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»