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Molecular and Cellular Biology, March 2009, p. 1592-1607, Vol. 29, No. 6
0270-7306/09/$08.00+0     doi:10.1128/MCB.01446-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Network of Hydrophobic Residues Impeding Helix C Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the Subunit of Translation Initiation Factor 2

Andrés Gárriz, Hongfang Qiu, Madhusudan Dey, Eun-Joo Seo, Thomas E. Dever, and Alan G. Hinnebusch^*

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

Received 15 September 2008/ Returned for modification 23 October 2008/ Accepted 19 December 2008

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the subunit of translation initiation factor 2 (eIF2). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix C rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2 phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of C and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of C are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

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* Corresponding author. Mailing address: NIH, Building 6A/Room B1A-13, Bethesda, MD 20892. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail: ahinnebusch{at}nih.gov

Published ahead of print on 29 December 2008.

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Molecular and Cellular Biology, March 2009, p. 1592-1607, Vol. 29, No. 6
0270-7306/09/$08.00+0     doi:10.1128/MCB.01446-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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