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Regulation of Atypical MAP Kinases ERK3 and ERK4 by the Phosphatase DUSP2

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Regulation of Atypical MAP Kinases ERK3 and ERK4 by the Phosphatase DUSP2

Maria Perander et al. Sci Rep.

Abstract

The atypical MAP kinases ERK3 and ERK4 are activated by phosphorylation of a serine residue lying within the activation loop signature sequence S-E-G. However, the regulation of ERK3 and ERK4 phosphorylation and activity is poorly understood. Here we report that the inducible nuclear dual-specificity MAP kinase phosphatase (MKP) DUSP2, a known regulator of the ERK and p38 MAPKs, is unique amongst the MKP family in being able to bind to both ERK3 and ERK4. This interaction is mediated by a conserved common docking (CD) domain within the carboxyl-terminal domains of ERK3 and ERK4 and the conserved kinase interaction motif (KIM) located within the non-catalytic amino terminus of DUSP2. This interaction is direct and results in the dephosphorylation of ERK3 and ERK4 and the stabilization of DUSP2. In the case of ERK4 its ability to stabilize DUSP2 requires its kinase activity. Finally, we demonstrate that expression of DUSP2 inhibits ERK3 and ERK4-mediated activation of its downstream substrate MK5. We conclude that the activity of DUSP2 is not restricted to the classical MAPK pathways and that DUSP2 can also regulate the atypical ERK3/4-MK5 signalling pathway in mammalian cells.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Yeast 2-hybrid assays reveal a specific interaction between DUSP2 and the atypical MAPKs ERK3 and ERK4.
(a) ERK3 and ERK4 interact specifically with DUSP2 in yeast. Expression vectors (pGADT7) encoding GAL4 activation domain fusion proteins of ERK3, ERK4 or ERK2 were transformed into the yeast strain Y187. Selected transformants were mated with transformants of yeast strain PJ69-2A containing expression vectors (pGBKT7) encoding different members of the MAP kinase phosphatase family fused to the GAL4 DNA-binding domain. Yeast diploids expressing both DNA-binding domain and activation domain fusions were selected on synthetic drop out (SD) medium deficient for leucine and tryptophan. Protein-protein interactions were assessed by growth on SD medium lacking leucine, tryptophan, alanine and histidine. (b) DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner independently of its catalytic activity. Yeast strain Y187 expressing GAL4 activation domain fusion proteins of ERK3, ERK4 or ERK2 were mated with PJ69-2A expressing wild-type DUSP2, a DUSP2 mutant lacking a functional KIM (DUSP2KIM) or a catalytically inactive DUSP2 mutant (DUSP2CS). Protein-protein interactions were determined as described in a. (c) ERK3 and ERK4 interact with DUSP2 via their CD domains. Semi-quantitative analysis of yeast two-hybrid interactions based on the level of induction of the b-galactosidase reporter gene were performed for the interactions between DUSP2 and wild-type ERK3, ERK4 and ERK2, or versions of the respective MAP kinases carrying a point mutation within the CD domain. The interaction between the ERK-specific phosphatase MKP-3 and ERK2 is included as a positive control. Assays were performed in triplicate and means are presented with associated errors.
Figure 2
Figure 2. DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo.
Two micrograms of either ERK3 (a) or ERK4 (b) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 (a) or anti-ERK4 (b) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. (c) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). (d) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). (e) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure 3
Figure 3. The catalytic activity of DUSP2 is increased by ERK2 but not by either ERK3 or ERK4 in vitro.
Time dependent hydrolysis of p-Nitrophenylphosphate (p-NPP) by either recombinant wild-type murine DUSP2, catalytically inactive DUSP2 (DUSP2CS), or DUSP2 with a KIM mutation (DUSP2KIM) either in the absence or presence of recombinant ERK2, ERK3, or ERK4 as indicated was monitored by measuring the change in the optical density (O.D.) at 405 nm. Results from two independent experiments are shown. Standard deviations are calculated from the mean values of triplicate samples.
Figure 4
Figure 4. DUSP2 dephosphorylates Serine 189 and 186 in the activation loop of ERK3 and ERK4 respectively in vivo.
(a) HeLa cells were co-transfected with expression vectors encoding a kinase-dead mutant of ERK4 fused to green fluorescent protein (GFP-ERK4D168A) and either myc-tagged DUSP2, MKP-1, or MKP-3. After 24 h, whole cell extracts were prepared and GFP-ERK4D168A was immunoprecipitated using an anti-GFP antibody. The phosphorylation status of Serine 186 within the activation loop in immunoprecipitated ERK4 D168A was then analysed by Western blotting using an anti-phospho S186 antibody. The levels of GFP-ERK4D168A in immunoprecipitates and of the three MKPs in whole cell extracts were analysed by Western blotting using antibodies against ERK4 and myc, respectively. (b) HeLa cells were co-transfected with vectors encoding either myc-tagged ERK4 or ERK4D320N together with either HA-tagged DUSP2, DUSP2KIM, or DUSP2CS. After 24 h, whole cell extracts were prepared and either ERK4 or ERK4D320N were immunoprecipitated using an anti-myc antibody. The phosphorylation status of Serine 186 was analysed as described in A. Co-immunoprecipitated DUSP2 was visualized using an anti-HA antibody. The levels of overexpressed ERK4- and DUSP2 proteins were analysed by Western blotting of whole cell extracts using anti-myc and anti–HA antibodies, respectively. (c) NCI-H1299 cells were co-transfected with expression vectors encoding a Flag-tagged kinase-dead mutant of ERK3 (ERK3D171A) and either an empty expression vector or plasmids encoding wild-type DUSP2, a catalytically inactive DUSP2 mutant (DUSP2CS) or a kinase interaction motif-deficient mutant (DUSP2KIM) respectively. After 24 h, cell extracts were prepared and ERK3 was immunoprecipitated using M2-FLAG conjugated agarose. The Phosphorylation status of Serine 189 within the activation loop of immunoprecipitated ERK3 was analysed by Western-blotting using a specific anti-phospho Serine 189 ERK3 antibody. Levels of ERK3 protein in the input lysates and immunoprecipitates were analysed by Western-blotting using an anti-FLAG antibody. The expression of myc-tagged DUSP2 proteins in cell lysates and immunoprecipitates were analysed by Western-blotting using an anti-myc or anti-DUSP2 antibody respectively. All experiments were performed 3 times and representative images are shown. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure 5
Figure 5. The ability of ERK3 and ERK4 to interact with DUSP2 is dependent on the common docking (CD) motif, but does not require an intact FRIEDE motif.
(a) NCI-H1299 cells were co-transfected with expression vectors encoding either FLAG-tagged wild-type ERK3 (ERK3wt), a common-docking site ERK3 mutant (ERK3D324N), or a FRIEDE ERK3 mutant (ERK3I334K) together with an expression vector encoding an myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS). After 24 h, cells were lysed and FLAG-tagged ERK3 proteins were immunoprecipitated using an anti-FLAG antibody. Any co-immunoprecipitated DUSP2 was detected by Western-blotting using a DUSP2 specific antibody (upper panel). The expression levels of ERK3 and DUSP2 proteins in the cell lysates were analysed by Western-blotting using an anti-FLAG or anti-DUSP2 specific antibody respectively (lower panels). (b) NCI-H1299 cells were co-transfected with expression vectors encoding either myc-tagged wild-type ERK4 (ERK4wt), a common-docking site ERK4 mutant (ERK4D320N), or an ERK4 mutant in which the FRIEDE motif was rendered non-functional FRIEDE (ERK4I330K) together with an expression vector encoding HA-tagged catalytically inactive DUSP2 (DUSP2CS). After 24 h, cells were lysed and myc-tagged ERK3 proteins were immunoprecipitated using an anti-myc antibody. Any co-immunoprecipitated DUSP2 was detected by Western-blotting using a DUSP2 specific antibody (upper panel). The expression levels of ERK4 and DUSP2 proteins in the cell lysates were analysed by Western-blotting using an anti-myc or anti-DUSP2 specific antibody respectively (lower panels). Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure 6
Figure 6. Coexpression of ERK3 or ERK4 results in cytoplasmatic relocalisation of DUSP2.
(a) HEK293 cells were transfected with expression plasmids encoding myc-tagged catalytically inactive DUSP2 mutant (DUSP2CS), EGFP-tagged ERK3, ERK4 or a combination of DUSP2CS and ERK3 or DUSP2CS and ERK4. After 24 h the cells were fixed, EGFP fluorescence was visualized directly, and myc-tagged DUSP2 was visualized by staining with an anti-myc antibody (9E10) and Alexa-594 anti mouse antibody. (b). HEK293 cells were cotransfected with expression plasmid encoding either myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2 (DUSP2CS-KIM) and EGFP-tagged ERK3 or myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2(DUSP2CS-KIM) and EGFP-tagged ERK4. The cell was fixed and protein visualized as in A. (c) HEK 293 cells were co-transfected with the indicated expression vectors, and EGFP-ERK3, EGFP-ERK4, myc-DUSP2CS and myc-DUSP2CS-KIM were visualized as above. In all, more than 100 cells expressing DUSP2CS or DUSP2CS-KIM alone or together with either EGFP-ERK3 or EGFP-ERK4 from three independent transfections were counted, and the distribution of DUSP2 protein was scored in percentages. The results are presented as the percentages of cells in which myc-DUSP2 was predominantly cytosolic (C > N), and the mean values with the associated SD are shown. In all experiments, 10 different fields of cells were examined, and the representative images are as shown in Fig.6a and b.
Figure 7
Figure 7. ERK4 stabilizes the DUSP2 protein in vivo.
(a,b) HeLa cells were co-transfected with expression vectors encoding either DUSP2-HA (a) or DUSP2KIM-HA (b) together with either an empty expression vector or a plasmid encoding GFP-ERK4. After 24 h, cells were treated with 10 μM cycloheximide (CHX) for the indicated time periods before cell harvesting and lysis. The levels of DUSP2-HA or DUSP2KIM-HA in whole cell extracts were analysed by Western blotting using an anti-HA antibody. Levels of GFP-ERK4 and endogenous actin in the cell extracts were analysed by Western blotting using antibodies against GFP and actin, respectively. Unprocessed original scans of the blots are shown in Supplementary Fig. 1 (c) The intensities of the signals corresponding to the DUSP2 and DUSP2KIM bands in both a and b, were then quantified using the Odyssey infrared imaging System. The band intensities at the indicated time points are then expressed graphically as a percentage of the intensity at time zero. (d) A kinase-dead mutant of ERK4 stabilizes DUSP2 less efficiently than wild-type ERK4. Experiments were performed as described in (a–c). (e) Co-expression of classical MAP kinases does not lead to stabilization of the DUSP2 protein. HeLa cells were co-transfected with an expression vector encoding HA-tagged wild type DUSP2 together with expression vectors encoding either myc- tagged ERK4, ERK2, p38α or JNK1. Cycloheximide time course experiments, followed by Western blot analyses and quantification were performed exactly as described in a-c. The average of three independent experiments is shown including standard deviation. The experiments (a–d) were performed three times with identical results and the results of a single representative experiment are shown.
Figure 8
Figure 8. Expression of DUSP2 inhibits the ERK3 and ERK4-mediated phosphorylation of MK5.
NCI-H1299 cells were co-transfected with expression plasmids encoding either myc-tagged ERK4 (a and b) or myc-tagged ERK3 (C and D) together with an expression vector encoding an EGFP-MK5 fusion protein and either an empty expression vector or plasmids encoding either wild-type DUSP2 or a KIM mutant of DUSP2 as indicated. After 24 h, the cells were lysed and MK5 phosphorylated at Thr-182 was detected by Western-blotting using a phospho-specific anti-Thr182 MK5 antibody (a and c). The expression of MK5, ERK3, ERK4 and either wild-type DUSP2 or the DUSP2 KIM mutant were verified by Western blotting of the cell lysates using appropriate antibodies (bottom panels in a and c). Unprocessed original scans of the blots are shown in Supplementary Fig. 1. The data in a and c and two additional replicate experiments were then quantified using the Odyssey infrared imaging System. The relative intensity of the p-Thr182 MK5 bands are calculated using the band intensity from cells transfected with EGFP-MK5 alone as the reference and mean values are presented with associated errors (b and d).

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