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Molecular and Cellular Biology, May 2008, p. 3489-3501, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.01847-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Integration of Protein Kinases mTOR and Extracellular Signal-Regulated Kinase 5 in Regulating Nucleocytoplasmic Localization of NFATc4

Teddy T. C. Yang,^1, Raymond Y. L. Yu,^1, Anissa Agadir,² Guo-Jian Gao,² Roberto Campos-Gonzalez,² Cathy Tournier,³ and Chi-Wing Chow^1*

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461,¹ BD Biosciences-Pharmingen, San Diego, California 92130,² Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom³

Received 10 October 2007/ Returned for modification 9 November 2007/ Accepted 6 March 2008

The target of rapamycin (TOR) signaling regulates the nucleocytoplasmic shuttling of transcription factors in yeast. Whether the mammalian counterpart of TOR (mTOR) also regulates nucleocytoplasmic shuttling is not known. Using a phospho-specific monoclonal antibody, we demonstrate that mTOR phosphorylates Ser^168,170 of endogenous NFATc4, which are conserved gate-keeping Ser residues that control NFAT subcellular distribution. The mTOR acts as a basal kinase during the resting state to maintain NFATc4 in the cytosol. Inactivation and nuclear export of NFATc4 are mediated by rephosphorylation of Ser^168,170, which can be a nuclear event. Kinetic analyses demonstrate that rephosphorylation of Ser^168,170 of endogenous NFATc4 is mediated by mTOR and, surprisingly, by extracellular signal-regulated kinase 5 (ERK5) mitogen-activated protein kinase as well. Ablation of ERK5 in the Erk5^–/– cells ascertains defects in NFATc4 rephosphorylation and nucleocytoplasmic shuttling. In addition, phosphorylation of NFATc4 by ERK5 primes subsequent phosphorylation mediated by CK1. These results demonstrate that distinct protein kinases are integrated to phosphorylate the gate-keeping residues Ser^168,170 of NFATc4, to regulate subcellular distribution. These data also expand the repertoire of physiological substrates of mTOR and ERK5.

Published ahead of print on 17 March 2008.

These authors contributed equally to this work.