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Molecular and Cellular Biology, January 2005, p. 854-864, Vol. 25, No. 2
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.2.854-864.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

Sandrine Marchetti ,^, Clotilde Gimond,^* Jean-Claude Chambard, Thomas Touboul, Danièle Roux, Jacques Pouysségur, and Gilles Pagès

Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre Antoine Lacassagne, Nice, France

Received 30 April 2004/ Returned for modification 3 June 2004/ Accepted 28 October 2004

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

S.M. and C.G. contributed equally to this work.

Present address: INSERM U526, Physiopathologie de la Survie et de la Mort Cellulaires et Infections Virales, 06107 Nice, France.

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