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Molecular and Cellular Biology, February 2003, p. 1292-1303, Vol. 23, No. 4
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.4.1292-1303.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Growth Arrest and DNA Damage-Inducible Protein GADD34 Targets Protein Phosphatase 1{alpha} to the Endoplasmic Reticulum and Promotes Dephosphorylation of the {alpha} Subunit of Eukaryotic Translation Initiation Factor 2

Matthew H. Brush, Douglas C. Weiser, and Shirish Shenolikar*

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Received 9 September 2002/ Returned for modification 8 October 2002/ Accepted 22 November 2002

The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the {alpha} subunit of eukaryotic translation initiation factor 2, (eIF-2{alpha}). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2{alpha} phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2{alpha} phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2{alpha} phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2{alpha} dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the {alpha} isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2{alpha} phosphatase that regulates protein translation in mammalian cells.

* Corresponding author. Mailing address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, LSRC C315, Research Dr., Durham, NC 27710. Phone: (919) 681-6178/9. Fax: (919) 681-9567. E-mail: sheno001{at}mc.duke.edu.

Molecular and Cellular Biology, February 2003, p. 1292-1303, Vol. 23, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.4.1292-1303.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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