Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness

Abstract

ERK3 is an atypical mitogen-activated protein kinase (MAPK) that has recently gained interest for its role in promoting cancer cell migration and invasion. However, the molecular regulation of ERK3 functions in cancer cells is largely unknown. ERK3 has a single phospho-acceptor site (Ser¹⁸⁹) in its activation motif rather than the TXY conserved in conventional MAPKs such as ERK1/2. Although dual phosphorylation of the TXY motif is known to be critical for the activation of conventional MAPKs, the role of Ser¹⁸⁹ phosphorylation in ERK3 activity and its function in cancer cells remain elusive. In this study, we revealed that activation loop phosphorylation is important for ERK3 in promoting cancer cell invasiveness, as the S189A mutation greatly decreased the ability of ERK3 to promote migration and invasion of lung cancer cells. Interestingly, a catalytically inactive ERK3 mutant was still capable of increasing migration and invasion, although to a lesser extent compared with WT ERK3, suggesting that ERK3 promotes cancer cell invasiveness by both kinase-dependent and kinase-independent mechanisms. To elucidate how the S189A mutation reduces the invasiveness-promoting ability of ERK3, we tested its effect on the kinase activity of ERK3 toward steroid receptor coactivator 3 (SRC3), a recently identified substrate of ERK3 critical for cancer cell invasiveness. Compared with ERK3, ERK3-S189A exhibited a dramatic decrease in kinase activity toward SRC3 and a concomitantly reduced ability to stimulate matrix metalloproteinase expression. Taken together, our study unravels the importance of Ser¹⁸⁹ phosphorylation for intramolecular regulation of ERK3 kinase activity and invasiveness-promoting ability in lung cancer cells.

Keywords: activation loop phosphorylation; cell invasion; cell migration; extracellular signal–regulated kinase (ERK); lung cancer; mitogen-activated protein kinase (MAPK).

Figure 1.

Activation loop phosphorylation is important for the migration-promoting ability of ERK3 in lung cancer cells. A and B, A549 cells were transiently transduced with lentiviruses expressing an empty vector or HA-tagged WT or mutant ERK3 (S189A, S189D, or KD). The overexpression of ERK3 was verified by Western blotting using an anti-ERK3 antibody (A), and migration was assessed by Transwell cell migration assay (B). C and D, A549 cells were stably transduced with lentiviruses expressing an empty vector, myc-ERK3, myc-ERK3-S189A, or myc-ERK3-KD. Overexpression of myc-ERK3 proteins in stable cell lines was verified by Western blotting using anti-ERK3 antibody (C). Phospho-ERK3 (Ser¹⁸⁹) was analyzed by immunoprecipitation of ERK3 using an ERK3 Ab, followed by Western blotting using anti-phospho-ERK3 (Ser¹⁸⁹) antibody. In both the ERK3 and pERK3 (Ser¹⁸⁹) immunoblots, the lower bands represent endogenous ERK3 (or endogenous phospho-ERK3-Ser¹⁸⁹), whereas the higher bands represent the overexpressed myc₆-tagged ERK3 (or phospho-myc₆-ERK3-Ser¹⁸⁹) proteins. Cell migration ability was assessed by Transwell migration assay (D). E and F, H1299 cells were transiently transfected with an empty vector or HA-tagged WT or mutant ERK3, as indicated. Two days post-transfection, the cells were lysed and analyzed by Western blotting using an anti-ERK3 antibody and an anti-phospho-ERK3 (Ser¹⁸⁹) antibody (E). The migration ability of the cells was determined by Transwell migration assay (F). The quantitated migration ability is presented as the number of migrated cells per field. Values in the bar graphs represent mean ± S.E. (n ≥ 6 fields). **, p < 0.001 (significantly different compared with empty vector); *, p < 0.05 (significantly different compared with empty vector); #, p < 0.001 (significantly different compared with ERK3); one-way ANOVA. Representative images of migrated cells stained with crystal violet are shown below each bar graph. Scale bars, 100 μm.

Figure 2.

Activation loop phosphorylation is important for the migration-promoting ability of ERK3 when re-expressed in H1299 cells with depletion of endogenous ERK3. A, generation of H1299 cells with stable expression of shCtrl) or shERK3. Knockdown of ERK3 was verified by Western blot analysis using an anti-ERK3 Ab. B, Transwell migration assay of stable H1299-shCtrl and H1299-shERK3 cells. Quantitated migration ability is presented as the number of migrated cells per field. Values in the bar graph represent mean ± S.E. (n = 6 fields). *, p < 0.001 (significantly different compared with shCtrl); Student's t test. C and D, H1299-shERK3 cells were transiently transfected with an empty vector or HA-tagged WT or mutant ERK3, as indicated. Two days post-transfection, cells were lysed and analyzed by Western blotting using an anti-ERK3 antibody and an anti-phospho-ERK3 (Ser¹⁸⁹) antibody (C). Cell migration ability was determined by Transwell migration assay and is presented as the number of migrated cells per field (D). Values in the bar graph represent mean ± S.E. (n ≥ 6 fields). **, p < 0.0001 (significantly different compared with empty vector); *, p < 0.05 (significantly different compared with empty vector); #, p < 0.0001 (significantly different compared with ERK3); one-way ANOVA. For all migration assays, representative images of migrated cells stained with crystal violet are shown below the bar graphs. Scale bars, 100 μm.

Figure 3.

Activation loop phosphorylation is important for the invasion-promoting ability of ERK3 in lung cancer cells. A, Transwell Matrigel invasion assay of A549 cells stably expressing an empty vector, WT, or mutant myc-ERK3 as described in Fig. 1C. B, Transwell Matrigel invasion assay of H1299 cells with transient overexpression of an empty vector or WT or mutant HA-ERK3 as described in Fig. 1E. Quantitated invasion ability is presented as the number of invaded cells per field. Values in the bar graphs represent mean ± S.E. (n ≥ 6 fields). **, p < 0.0001 (significantly different compared with empty vector); *, p < 0.05 (significantly different compared with empty vector); #, p < 0.05 (significantly different compared with ERK3); one-way ANOVA. Representative images of invaded cells stained with crystal violet are shown below the bar graphs. Scale bars, 100 μm.

Figure 4.

Overexpression of WT or mutant ERK3 does not affect lung cancer cell proliferation. A, an MTS cell proliferation assay was performed for A549 cells with stable overexpression of an empty vector or WT or mutant myc-ERK3 as described in Fig. 1C. Cell viability at different time points (days) was measured and expressed as A₄₉₀. B, an MTS cell proliferation assay was performed for H1299 cells with stable overexpression of an empty vector, WT, or mutant HA-ERK3 as indicated. Cell viability is expressed as A₄₉₀ normalized to values of day 1 for each condition. C,ERK3 overexpression and ERK3-Ser¹⁸⁹ phosphorylation in the generated H1299 stable cells were verified by Western blotting. Values represent mean ± S.D. (n ≥ 3). Statistical analysis was conducted to compare A₄₉₀ of the different cell lines at each time point by two-way ANOVA. No significant difference was detected between the cell lines at p < 0.05.

Figure 5.

The S189A mutation does not affect the in vitro kinase activity of recombinant ERK3 protein expressed in bacteria. A, purification of recombinant His-ERK3(1–340) proteins expressed in bacteria. The proteins were purified using Ni-NTA resin and analyzed by SDS-PAGE and subsequent Coomassie Blue staining. The molecular mass of protein markers is indicated. B, His-ERK3(1–340) proteins expressed in bacteria were subjected to Western blot analysis using either an anti-ERK3 mAb that targets an N-terminal epitope in ERK3 (top panel) or an anti-phospho-ERK3 (S189) antibody (bottom panel). C, Western blot analysis of His-ERK3(1–340) protein expressed in bacteria and HA-ERK3(1–340) expressed in 293T cells using anti-ERK3 and anti-phospho-ERK3 (Ser¹⁸⁹) antibodies. D, in vitro kinase assay of bacterially expressed ERK3(1–340) proteins. 1 μg of each purified protein was incubated with 2 μg of recombinant MBP and [γ-³²P]ATP for different times (minutes). The samples were analyzed by SDS-PAGE, followed by Coomassie staining (above) and autoradiography (below). Quantification of MBP phosphorylation by WT or mutant ERK3(1–340) proteins is shown below the autoradiograph. For the purpose of comparison, the normalized phosphorylation level of MBP by WT ERK3(1–340) at 10 min was arbitrarily set as 1.0. Quantification of MBP phosphorylation after kinase reaction for 40 min (40′) is presented on the right. The MBP phosphorylation level by WT ERK3(1–340) was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of three independent experiments. *, p < 0.05; N.S., not significantly different by one-way ANOVA. E, purification of recombinant His-ERK3-GST proteins expressed in bacteria. The proteins were purified using GSH beads, followed by elution with reduced GSH. The eluted proteins were analyzed by SDS-PAGE, followed by Coomassie Blue staining. F, Western blot analysis of the purified His-ERK3-GST proteins using an anti-ERK3 antibody that targets an N-terminal epitope in ERK3. G, in vitro kinase assay of bacterially expressed recombinant His-ERK3-GST proteins. 350 ng of purified ERK3 protein (WT or mutant) was incubated with [γ-³²P]ATP in kinase assay buffer for measuring autophosphorylation. Protein samples were analyzed by SDS-PAGE and Coomassie staining (top) and autoradiography (bottom). Quantification of ERK3 phosphorylation is shown below the autoradiograph; the normalized phosphorylation level of WT ERK3 was arbitrarily set as 1.0.

Figure 6.

The S189A mutation decreases the in vitro kinase activity of ERK3 protein expressed and immunoprecipitated from 293T cells. A, purification of WT or mutant ERK3 proteins by immunoprecipitation from 293T cells. 293T cells were transfected with the HA-ERK3, HA-ERK3-S189A, or HA-ERK3-KD plasmid, followed by immunoprecipitation of exogenously expressed ERK3 proteins using HA Ab–conjugated agarose beads and elution of the proteins with HA peptide. The purified proteins (300 ng each) were analyzed by SDS-PAGE, followed by Coomassie Blue staining. B, Western blot analysis of proteins purified from mammalian cells using anti-ERK3 and anti-phsopho-ERK3 (Ser¹⁸⁹) antibodies. C and D, in vitro kinase assay for WT or mutant HA-ERK3 proteins immunoprecipitated from mammalian cells using GST-SRC3-CID (C) or MBP (D) as substrates. The assays were performed by incubating 100 ng of WT or mutant ERK3, as indicated, together with 1 μg of recombinant protein substrate in the presence of [γ-³²P]ATP. Total protein levels of substrates in the reactions are shown by Coomassie staining (left panels). ERK3 proteins are barely seen in the Coomassie-stained gels because of their small amounts (100 ng each). Phosphorylation of the substrates was detected by autoradiography (center panels). Quantification of substrate phosphorylation by WT or mutant ERK3 proteins is shown in the right panels. For the purpose of comparison, the normalized phosphorylation level of substrates by WT ERK3 was arbitrarily set as 1.0. The bar graphs represent the mean ± S.E. of three independent experiments. *, p < 0.05 by one-way ANOVA. E, Western blot analysis of His-ERK3-GST recombinant protein expressed in bacteria and HA-ERK3 protein expressed in 293T cells using anti-phospho-ERK3 (Ser¹⁸⁹) and anti-ERK3 antibodies. F, activation loop phosphorylation and kinase activity are not required for interaction of ERK3 with its substrate SRC3 in cells. 293T cells were co-transfected with SRC3 and an HA tag–expressing empty vector, WT HA-ERK3, or mutant HA-ERK3 plasmids, as indicated. Two days post-transfection, cells were lysed, and the interactions of ERK3 with SRC3 and MK5 were analyzed by co-immunoprecipitation using HA Ab–conjugated beads, followed by Western blotting using antibodies against ERK3, SRC3, and MK5.

Figure 7.

The S189 mutation decreases the ability of ERK3 to up-regulate the expression of MMP10 and MMP9 genes. A and B, quantitative RT-PCR analysis of MMP10 (A) and MMP9 (B) gene expression in A549 cells with stable expression of an empty vector, ERK3, ERK3-S189A, or ERK3-KD as described in Fig. 1C. Values in the bar graphs represent mean ± S.D. (n = 3). **, p < 0.0001 (significantly different compared with empty vector); *, p < 0.05 (significantly different compared with empty vector); #, p < 0.005 (significantly different compared with ERK3); one-way ANOVA.