Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

Abstract

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.

FIGURE 1.

Dephosphorylation of phosphorylated MAPK by MKP3. A, time courses of p38α/pTpY and ERK2/pTpY dephosphorylation by MKP3. The assay system contained 175 nm MKP3 and 4 μm ERK2/pTpY (curve 1) or 4 μm p38α/pTpY (curve 2). B, time-dependent dephosphorylation of p38α/pTpY upon treatment with different phosphatases. The assay system contained 0.75 μm p38α/pTpY. The reaction was initiated by the addition of 400 nm MKP3 and then followed by the addition of 177 nm HePTP and 200 nm MKP5 (as indicated) to the reaction mixture. Western blot analysis of dephosphorylation of p38α/pTpY (C) and p38γ/pTpY (D) by different phosphatases in vitro. p38α/pTpY or p38γ/pTpY were treated with MKP5, MKP3, HePTP, and PP2Cα individually in buffer (50 mm MOPS, pH 7.0, 100 mm NaCl, 10 mm Mg²⁺) at 25 °C for 30 min, followed by Western blot using the indicated antibodies. The lower panel of the SDS-polyacrylamide gel (Coomassie-stained) shows the amounts of purified p38α, p38γ, and phosphatases used in the experiment. The proteins p38γ and PP2Cα were unable to be separated on SDS-PAGE because they have the same molecular weight.

FIGURE 2.

Effects of N-terminal domain on substrate specificity and ERK2-induced activation of MKP3. A, time-dependent dephosphorylation of p38α/pTpY upon treatment with different phosphatases. The initial mixture contains 0.9 μm bisphosphorylated p38α/pTpY. The reaction was initiated by the addition of 1.1 μm MKP3/ΔN151 and then followed by 177 nm HePTP and 200 nm MKP5. B, titration of the binding of MKP3 to ERK2. The assay system contained 10 mm pNPP and 5 μm MKP3. Increasing amounts of ERK2 were added to a solution in which the concentration of MKP3 was at least 10 times higher than the dissociation constant, and the intersection of the slope of the increase in the absorbance at 410 nm with the maximum value gives the stoichiometry. The activation of MKP3 by ERK2 is saturable, and maximal activation resulted from an MKP3/ERK2 ratio of 1:1, suggesting a 1:1 binding stoichiometry between MKP3 and ERK2. C, the time courses of p38α/pTpY dephosphorylation by MKP3 in the absence and presence of ERK2. The reaction mixture contained 3 μm p38α/pTpY and 177 nm MKP3 plus 177 nm ERK2 (curve 1) or 177 nm MKP3 only (curve 2). D, Western blot analysis of dephosphorylation of p38α/pTpY by different phosphatases in vitro. p38α/pTpY were treated with MKP3, MKP3 plus ERK2, MKP3/ΔN151, HePTP, MKP5, and PP2Cα individually in buffer (50 mm MOPS, pH 7.0, 100 mm NaCl, 10 mm Mg²⁺) at 25 °C for 30 min, followed by Western blot using the indicated antibodies. The lower panel of the SDS-polyacrylamide gel (Coomassie-stained) shows the amounts of purified p38α/pTpY and other proteins used in the experiment.

FIGURE 3.

Direct binding between p38α/pTpY and MKP3. A, gel filtration analysis of MKP3 and p38α/pTpY alone or an equimolar mixture of MKP3 and p38α/pTpY, in which p38α/pTpY was dephosphorylated to be p38α/pT by MKP3 during incubation. B, gel filtration analysis of the p38α/pTpY, mutant MKP3 (C293S), and ERK2 alone or an equimolar mixture of two of the three proteins as indicated and an equimolar mixture of the three proteins. Gel filtration chromatography experiments were performed with a Superdex 200 gel filtration column mounted on an AKTA FPLC system (Amersham Biosciences). All proteins were in 50 mm MOPS buffer, pH 7.0, 100 mm NaCl, 0.1 mm EDTA. C, in vivo association and dephosphorylation effects of MKP3, HePTP, and MKP5 on overexpressed p38α. 293T cells were transfected with expression vectors for Myc-phosphatase (MKP3, MKP3C293S, HePTP, and MKP5) and FLAG-p38α. After 48 h, cells were treated for 30 min with 0.5 m sorbitol. Complexes were precipitated with anti-FLAG followed by Western blot (WB) with the indicated antibodies. IP, immunoprecipitation; TCL, total cell lysate. The heavy chain of the anti-FLAG antibody, which was used for the immunoprecipitation, is marked by an asterisk.

FIGURE 4.

The KIM sequence of MKP3 is specific for ERK2 binding. Gel filtration profiles for the interaction of the N-terminal domain of MKP3 (residues 1–154) with p38α (A), p38α/pTpY (B), and ERK2 (C), respectively. Gel filtration chromatography experiments were performed with a Superdex 200 gel filtration column mounted on an AKTA FPLC system (Amersham Biosciences). All proteins were in 50 mm MOPS buffer, pH 7.0, 100 mm NaCl, and 0.1 mm EDTA. ITC data are shown for the interaction of KIM peptide of MKP3 with p38α (D), p38α/pTpY (E), and ERK2 (F), respectively. From curve fitting of ITC binding isotherms, the K_d for the binding of the peptide to ERK2 was determined to be 65.8 ± 15.4 μm.

FIGURE 5.

Comparison with other tyrosine-specific phosphatases. A, the time courses of p38α/pTpY dephosphorylation reaction by different phosphatases. The absorption at 360 nm was recorded following the addition of 110 nm PTP-SL (curve 1), HePTP (curve 2), MKP3 plus ERK2 (curve 3), MKP3 (curve 4), and STEP (curve 5). B, effect of ERK2 on the HePTP-catalyzed dephosphorylation of p38α/pTpY. ERK2 concentrations were 0 μm (curve 1), 1.75 μm (curve 2), 3.50 μm (curve 3), and 5.25 μm (curve 4), respectively. The concentration of HePTP was 38 nm.

FIGURE 6.

Overall view of p38α/pTpY and comparison with p38α. A, ribbon diagram of p38α/pTpY. The p38α/pTpY is colored green (N-lobe) and blue (C-lobe), with its hinge region and activation segment in red. The 2F_o − F_c electron density map for Thr(P)¹⁸⁰ and Tyr(P)¹⁸², shown on the right, is contoured at 1.0 σ. B, superimposition of p38α/pTpY and p38α (Protein Data Bank code 1p38) with C-lobe as reference. p38α/pTpY is colored green, with its hinge region and activation segment in red. The p38α is colored orange, with its hinge region and activation segment in blue. Shown is a close-up view of the activation segment and its neighboring structures in p38α (C) and p38α/pTpY (D) (color-coded as in Fig. 5B). The water molecule is shown as a red sphere, and hydrogen bonds are shown as yellow dashed lines. Shown is a close-up view of the ATP-binding site in p38α (E) and p38α/pTpY (F).

FIGURE 7.

Molecular basis of MKP3 substrate specificity. A, superimposition of ERK2/pTpY (magenta, Protein Data Bank code 2ERK) and p38γ/pTpY (yellow, Protein Data Bank code 1CM8) onto p38α/pTpY (green) with the C-lobe as a reference. B, close-up view of the activation segment of ERK2/pTpY, p38γ/pTpY, and p38α/pTpY. Shown are electrostatic surface potential representations (red, negative charge; blue, positive charge; gray, hydrophobic) of p38α (C; Protein Data Bank code 1p38), p38α/pTpY (D), ERK2/pTpY (E; Protein Data Bank code 2ERK), and p38γ/pTpY (F; Protein Data Bank code 1cm8). Shown are gel filtration profiles for the interaction of MKP3C293S/ΔN151 with p38α/pTpY (G), ERK2/pTpY (H), and p38γ/pTpY (I), respectively. Gel filtration chromatography experiments were performed with a Superdex 200 gel filtration column mounted on an AKTA FPLC system (Amersham Biosciences). All proteins were in 50 mm MOPS buffer, pH 7.0, 100 mm NaCl, and 0.1 mm EDTA.