👤 Céline Tárrega

MAPK (MAP kinase) phosphatase 3 (DUSP6/MKP3) is a cytosolic MKP (MAPK phosphatase) that regulates negatively ERK1/2 downstream to growth factor or apoptotic signaling. Transcription of DUSP6 gene is activated through the ERK1/2 pathway, which constitutes a feedback regulatory loop of ERK1/2 activation. However, the regulation of the function of the DUSP6/MKP3 protein is poorly known. MKP3 possesses a linker region between its N-terminal MAPK-binding domain and its C-terminal catalytic domain, which is conserved in the related MKPs DUSP7/MKPX and DUSP9/MKP4. In MKP3, the interdomain linker region contains a secondary ERK1/2 binding motif and an active nuclear export sequence. Here, we report that MKP3 protein levels are decreased in cells upon apoptotic stimulation in a caspase-dependent manner, and we identify a novel MKP3 regulatory mechanism mediated by the pro-apoptotic protease caspase-3, which involves the MKP3 interdomain linker region. Active caspase-3 targeted the linker region of MKP3 at several residues, rendering N-terminal and C-terminal MKP3 fragments that contain specific arrangements of nuclear export sequence and ERK1/2 interaction motifs. MKP3 caspase-3-generated fragments displayed differential properties to regulate ERK1/2 nuclear/cytosolic localization and activity. Our results indicate that caspase-3 cleavage of MKP3 down-regulates MKP3 full length and renders active MKP3 fragments, which may participate in novel regulatory pathways controlling the subcellular localization and activation of ERK1/2 during apoptosis. Show less

Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases (MKPs), in a cell type- and stimuli-dependent manner. MCF-7 human breast carcinoma cells treated with the phorbol 12-myristate 13-acetate (PMA) suffer growth arrest and show morphological alterations, which depend on the activation of the ERK1/2 MAP kinases. MKP3/DUSP6 and DUSP5 MAP kinase phosphatases, two negative regulators of ERK1/2, were specifically up-regulated in MCF-7 and SKBR3 cells in response to PMA. MKP3 and DUSP5 up-regulation required the prolonged activation of the ERK1/2 pathway, and correlated with the shutdown of this route. MKP3 induction relied on the activation of the Ets2 transcription factor, whereas DUSP5 induction depended on the activation of c-Jun. Diminishing the expression of MKP3 and DUSP5 raised the activation of ERK1/2, and accelerated growth arrest of PMA-treated MCF-7 cells. Conversely, MCF-7 cell lines expressing high levels of MKP3 or DUSP5 did not undergo PMA-triggered growth arrest, displayed a migratory phenotype, and formed colonies in soft agar. We propose that the differential up-regulation of MKP3 by Ets2 and of DUSP5 by c-Jun may converge in similar functional roles for these MAP kinase phosphatases in the growth arrest versus proliferation decisions of breast cancer cells. Show less

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors. Show less