Dual-specificity phosphatase 5 controls the localized inhibition, propagation, and transforming potential of ERK signaling

Abstract

Deregulated extracellular signal-regulated kinase (ERK) signaling drives cancer growth. Normally, ERK activity is self-limiting by the rapid inactivation of upstream kinases and delayed induction of dual-specificity MAP kinase phosphatases (MKPs/DUSPs). However, interactions between these feedback mechanisms are unclear. Here we show that, although the MKP DUSP5 both inactivates and anchors ERK in the nucleus, it paradoxically increases and prolongs cytoplasmic ERK activity. The latter effect is caused, at least in part, by the relief of ERK-mediated RAF inhibition. The importance of this spatiotemporal interaction between these distinct feedback mechanisms is illustrated by the fact that expression of oncogenic BRAF^V600E, a feedback-insensitive mutant RAF kinase, reprograms DUSP5 into a cell-wide ERK inhibitor that facilitates cell proliferation and transformation. In contrast, DUSP5 deletion causes BRAF^V600E-induced ERK hyperactivation and cellular senescence. Thus, feedback interactions within the ERK pathway can regulate cell proliferation and transformation, and suggest oncogene-specific roles for DUSP5 in controlling ERK signaling and cell fate.

Keywords: DUSP; ERK; MAPK; MKP; signaling.

Fig. 1.

DUSP5 propagates cytoplasmic ERK signaling. Serum-starved primary MEFs from WT or Dusp5 KO mice were infected with either 0.3–3.0 pfu/nL Ad ERK-responsive EGR1 promoter-driven DUSP5-Myc (DUSP5) or a KIM mutant of DUSP5-Myc (DUSP5^R53/54A) and stimulated with 20% (vol/vol) FBS for times indicated. (A) Representative confocal images of p-ERK and ERK are shown. (Scale bar: 60 μm.) (B) HCM was used to quantify >10⁴ cells per condition per experiment for levels of Myc tag, nuclear (Nuc), or cytoplasmic (Cyt) p-ERK intensity, or N:C of total ERK fluorescence. Data are normalized mean arbitrary fluorescence unit (AFU) values ± SEM, n = 4–8, *P < 0.05, **P < 0.01 comparing WT vs. KO using two-way repeated-measures ANOVA and Bonferroni posttest. Note: KO data are identical in Upper and Lower plots. (C) Western blots of whole-cell lysates are shown for total ERK, p-ERK, β-tubulin, and DUSP5. A representative blot is shown from n = 3 similar experiments; blot quantification is shown in Fig. S1C.

Fig. S1.

DUSP5 increases cytoplasmic ERK responses. Primary WT or Dusp5 KO MEFs were infected with either empty Ad or 0.1–3.0 pfu/nL Ad ERK-dependent EGR1 promoter-driven DUSP5-Myc or a KIM mutant (DUSP5^R53/54A-Myc) before stimulation with 20% (vol/vol) FBS as indicated. (A) Representative images are shown for p-ERK, Myc-tag, and DAPI staining from confocal microscopy experiments comparing rescue using 0.3 and 3 pfu/nL Ad DUSP5. (Scale bar: 60 μm.) (B) Representative images and quantification in plots are shown for a single HCM experiment comparing the full range of DUSP5 or DUSP5^R53/54A rescue conditions from 0.1–3.0 pfu/nL Ad concentrations. Note: KO data are identical in Upper and Lower plots. Data are shown as normalized population averages of AFU ± SD, n = 2–4. (Scale bar: 100 μm.) (C) Primary WT or Dusp5 KO MEFs were infected with either empty Ad or 0.3–3.0 pfu/nL Ad DUSP5-Myc before 20% (vol/vol) FBS stimulus and Western blotting as described in Fig. 1C. Data shown are normalized p-ERK levels measured in whole-cell lysates ± SEM, n = 3, *P < 0.05 comparing KO vs. KO + 3.0 pfu/nL DUSP5 using two-way repeated-measures ANOVA and Bonferroni posttest.

Fig. S2.

DUSP5 regulation of ERK is dose dependent, cell autonomous, and reliant upon the kinase interaction motif. Dusp5 KO MEFs were infected with empty Ad, Ad EGR1 promoter-driven DUSP5-Myc, or a KIM mutant (DUSP5^R53/54A-Myc) using indicated pfu/nL titers and stimulated for 240 min with 20% (vol/vol) FBS before HCM. Single-cell data from a representative of n = 4 experiments is shown. (Left) Frequency histograms of Cyt p-ERK, ERK N:C ratio, and whole-cell Myc levels. (Right) Scatterplots of either ERK N:C or Myc intensity vs. Cyt p-ERK intensity are shown comparing cells infected with 3 pfu/nL empty Ad (red), DUSP5-Myc (blue, Upper), or Ad DUSP5^R53/54A-Myc (blue, Lower). All values are in raw AFU per cell.

Fig. S3.

DUSP5 regulates cytoplasmic ERK signaling in response to phorbol ester and NGF stimuli, and does not influence serum-induced AKT signaling. (A) Primary WT and Dusp5 KO MEFs were infected with empty Ad, Ad EGR1 promoter-driven DUSP5-Myc, or DUSP5^R53/54A-Myc using indicated pfu/nL titers before stimulation with 10 ng/mL TPA and HCM. Three fields per well per fluorophore were acquired from cells. (Upper) Representative images of ∼10% of a single field. (Scale bar: 150 μm.) (Lower) Graphs represent population averaged normalized AFU for whole-cell Myc, Nuc, and Cyt p-ERK intensity, or N:C ratio of total ERK intensity derived from a representative experiment ± SD, n = 2–4. (B) PC12 cells were infected with empty Ad (Ctrl) or indicated titers of Ad DUSP5-Myc before serum starvation and stimulation with 3 nM NGF and analysis using HCM. (Left) Representative images of cells are shown from HCM experiments following 360-min NGF treatment. (Scale bar: 50 μm.) (Right) Graphs show normalized population mean AFU values for whole-cell Myc levels and Cyt p-ERK intensity derived from a representative experiment ± SD, n = 2. (C) Primary WT and Dusp5 KO MEFs were infected with empty Ad, Ad EGR1 promoter-driven DUSP5-Myc, or DUSP5^R53/54A-Myc using indicated pfu/nL titers before stimulation with 20% (vol/vol) FBS for up to 360 min and were costained for p-ERK and p-Ser473 AKT before HCM. Representative images are shown for 360 min FBS stimulus; plots show normalized population means of whole-cell p-ERK and p-AKT levels in AFU ± SEM, n = 4–5. (Scale bar: 50 μm.) **P < 0.01 using two-way repeated-measures ANOVA and Bonferroni posttest, comparing KO and KO + 3 pfu/nL Ad DUSP5-Myc conditions.

Fig. 2.

DUSP5 propagates ERK signaling by increasing RAF and MEK activity. WT and Dusp5 KO primary MEFs were infected with 0.3–3.0 pfu/nL Ad EGR1 promoter-driven DUSP5-Myc (Ad DUSP5) as indicated. (A) MEFs were stimulated for 360 min with 20% (vol/vol) FBS. Western blots of whole-cell lysates were probed for p-sites on ERK (TEY activation loop), MEK (Ser217/221), CRAF (Ser338), and p90RSK (Thr-359/Ser363) as well as total kinase levels and β-tubulin. Normalized blot quantification is shown ± SEM, n = 4. *P < 0.05, **P < 0.01 comparing KO vs. all columns using one-way ANOVA and Dunnett's posttest. (B) MEFs were stimulated either with 20% (vol/vol) FBS for 360 min or 50 ng/mL EGF for 5 min before lysis and pull-down of GTP-RAS using the RAS-binding domain (RBD) of CRAF. Input levels of total RAS in whole-cell lysates and levels of GTP-RAS in the pull-down were measured by Western blotting. Normalized mean blot quantification of GTP-RAS is shown ± SEM, n = 3. (C) MEFs were stimulated with 20% (vol/vol) FBS for 120 min before addition of either DMSO vehicle or 5 μM PD0325901 MEK inhibitor for times indicated. Levels of Myc-tag and whole-cell levels of p-ERK were measured using HCM. Data shown are normalized population AFU values, n = 3 ± SEM.

Fig. S4.

DUSP5 increases cytoplasmic MEK and ERK phosphorylation in fractionated lysates. Primary Dusp5 knockout (KO) MEFs were infected with 3 pfu/nL empty Ad or Ad EGR1 promoter-driven DUSP5-Myc before stimulation with 20% (vol/vol) FBS for 120 or 360 min, separation into nuclear and cytoplasmic fractions, and Western blotting. Representative blots are shown above quantified normalized band intensities of Cyt p-ERK and p-MEK in the plots. *P < 0.05, **P < 0.01 from n = 4 experiments using one-way ANOVA and Dunnett's posttest comparing indicated columns at 360-min stimulus.

Fig. 3.

The DUSP5 nuclear localization signal is required for propagation of cytoplasmic p-ERK. Primary Dusp5 KO MEFs were infected with 0.3–3 pfu/nL Ad EGR1 promoter-driven DUSP5-Myc (Ad DUSP5), a nuclear localization sequence mutant of DUSP5-Myc or DUSP6-Myc (Ad DUSP5^NLS and Ad DUSP6, respectively), and stimulated with 20% (vol/vol) FBS. (A) Representative HCM images show cells infected with 3 pfu/nL Ad DUSP-Myc before stimulation and staining for both p-ERK and Myc. Less than 1% imaged area per condition per experiment is shown. (Scale bar: 100 μm.) (B) Scatterplots show single-cell comparisons of nuclear vs. cytoplasmic levels of Myc tag (Top), ERK N:C ratio vs. whole-cell p-ERK (Middle) or whole-cell Myc-tag vs. whole-cell p-ERK levels (Bottom) from a single representative experiment of n = 4 comparing cells infected with 3 pfu/nL Ad DUSP and stimulated for 180 min with FBS. All nonratio values are shown in raw AFU per cell. (C) Plots show average Nuc p-ERK, Cyt p-ERK, and whole-cell Myc levels in cells stimulated with FBS. Data shown are mean, normalized AFU levels ± SEM, n = 3–5. Note: KO data are identical in each row and are shown in each plot to clarify the effects of Ad DUSP-Myc expression in the Dusp5-deleted cells.

Fig. 4.

DUSP5 propagates ERK signaling by relieving upstream kinase inhibition. (A) Conceptual model network structures comprising ERK activation by an upstream kinase (K), feedback inhibition and sequestration of ERK by DUSP5, and nonlinear feedback inhibition of K by ERK. K* and ERK* denote activated forms of K and ERK, respectively. The plots show model predictions of ERK* concentration vs. time in arbitrary units (AU) under conditions where there is no, low, or high levels of DUSP5 synthesis and in which negative feedback between ERK and K is intact (Left) or completely disabled (Right). (B) WT and Dusp5 KO MEFs were infected with either empty Ad, or Ad expressing HA-ΔCRAF-ER, alongside either 0.3 or 3 pfu/nL of Ad EGR1 promoter-driven DUSP5-Myc. Cells were stimulated with either 20% (vol/vol) FBS (Left) or 0.1 µM 4HT (Right) before immunostaining for p-ERK and Myc followed by HCM analysis. Normalized population mean AFU values for whole-cell p-ERK and Myc intensity are shown ± SEM, n = 4–7, *P < 0.05, **P < 0.01 comparing KO vs. KO + 3.0 pfu/nL Ad DUSP5-Myc using two-way repeated-measures ANOVA and Bonferroni posttest. (C) Representative HCM images of p-ERK staining are shown from experiments described in B, comparing Ad HA-ΔCRAF-ER–infected MEFs after 360-min stimulus with 0.1 µM 4HT. (Scale bar: 100 μm.)

Fig. S5.

The nonlinearity of upstream negative feedback, but not ERK nuclear shuttling, determines the regulatory effects of DUSP5 in silico. (A) A network topology scheme (Left) represents a minimal interaction network comprising K (an upstream activator of ERK), ERK, and DUSP5. K* and ERK* represent activated versions of K and ERK, respectively. A full description of equations and a table describing reactions numbered as presented in the network scheme is included in SI Materials and Methods. The only reaction that is not modeled using mass action kinetics is the negative feedback between ERK* and K*, which is modeled using a Hill function (reaction no. 6 in Table S1). (Right) Plots show ERK* output kinetics under conditions in which different values of Hill coefficient (which alter cooperativity described in the Hill function) were used to model the feedback between ERK* and K* (n = 1, 2, or 4) in the presence of high levels of DUSP5 synthesis (High DUSP5) or no DUSP5 synthesis (No DUSP5). The plots only replicate our biological data, where high levels of DUSP5 cause a rebound in ERK signaling (Figs. 1 and 4), when the level of cooperativity between ERK* and K* is also high (Hill coefficient, n = 4). (B) An expanded network topology scheme is shown (Left), incorporating nuclear shuttling of ERK as well as cytoplasmic synthesis and nuclear import of DUSP5. Plots of ERK* output from model simulations in the compartmentalized model are shown (Right), in which DUSP5 synthesis levels were varied, and ERK nuclear shuttling rates (α in reaction no. 9, Table S2) were set at 0.5, 1.0, or 2.0 events per arbitrary unit of time. These estimates of shuttling rate (relative to the time course of DUSP5 effects) are consistent with published studies showing that rates of active and inactive ERK nuclear shuttling are very rapid (57). The rate of ERK nuclear shuttling in our model failed to appreciably influence the effects of DUSP5 on ERK* signal propagation even when varied to these extents. Because nuclear shuttling is unlikely to influence predictions on these much longer timescales, we therefore decided to use the simplified, reduced model in Fig. S5A in all further predictive simulations.

Fig. 5.

DUSP5 degradation controls the amplitude and duration of ERK signaling. (A) The cartoons show a “normal” network structure (Left) and one in which DUSP5 degradation is abrogated (Right). The plot compares model simulations for conditions where DUSP5 is synthesized and degraded (DUSP5), where DUSP5 is absent (No DUSP5) or synthesized but not degraded (Non-deg DUSP5). Comparisons of activated ERK (ERK*) concentrations vs. time are shown in AUs. Note: the break in the x axis is to enable direct comparison with the wet-laboratory data shown in B, in which only prolonged phases (>120 min) of FBS stimulus were measured. (B) Dusp5 KO MEFs were infected with 3 pfu/nL of Ad expressing EGR1 promoter-driven DUSP5-Myc or DUSP5^R53/54A-Myc before 20% FBS stimulus for 120 min and treatment with either DMSO vehicle, 10 μM MG132 proteasome inhibitor, or 5 μM PD0325901 MEK inhibitor (PD). Plots show HCM readouts for whole-cell p-ERK, ERK N:C ratio and whole-cell Myc intensity and are shown as normalized population mean AFU ± SEM, n = 3. *P < 0.05, **P < 0.01 using two-way ANOVA and Bonferroni posttest comparing DMSO and MG132-treated cells. (C) Representative images of KO+Ad DUSP5 cells are shown from HCM experiments described in B. (Scale bar: 100 μm.)

Fig. 6.

BRAF^V600E expression combined with Dusp5 deletion causes ERK hyperactivation and proliferative arrest. (A) Primary WT and Dusp5 KO MEFs were infected with empty Ad, Ad HA-HRAS^Q61L, or Ad HA-BRAF^V600E alongside 0.3–3 pfu/nL of Ad expressing EGR1 promoter-driven DUSP5-Myc before stimulation with 20% (vol/vol) FBS. Plots show time courses comparing whole-cell p-ERK levels using HCM. Normalized population mean AFU values are shown ± SEM, n = 4. *P < 0.05, **P < 0.01 comparing KO vs. KO + 3.0 pfu/nL Ad DUSP5-Myc using two-way repeated-measures ANOVA and Bonferroni posttest. (B) Immortalized WT and Dusp5 KO MEFs were infected with retrovirus control, HRAS^Q61L, or BRAF^V600E vectors for 2 wk before assessment of transformed foci using crystal violet staining. Representative plate scans are shown and plots show average focus formation per well ± SEM, n = 4. **P < 0.01 comparing control and oncogene-expressing cells using two-way repeated-measures ANOVA and Bonferroni posttest. (C) Primary WT and Dusp5 KO MEFs were infected with either empty Ad, Ad HA-HRAS^Q61L, or Ad HA-BRAF^V600E and treated with increasing doses of PD0325901 MEK inhibitor for 24 h before a 2-h fluorescent EdU pulse label, immunostaining for p-ERK, and analysis using HCM. Plots show Nuc p-ERK levels and the percentage of EdU-positive cells ± SEM, n = 3. **P < 0.01 using two-way repeated-measures ANOVA and Bonferroni posttest comparing WT and KO. Representative images are shown beneath. (Scale bar: 200 μm.) (D) WT and Dusp5 KO primary MEFs were infected with Ad HA-BRAF^V600E (Lower), Ad HA-HRAS^Q61L (Upper), and 0.3 pfu/nL Ad DUSP5-Myc and treated with 0.1 µM PD0325901 (PD) as indicated for 24 h before 2-h fluorescent EdU pulse-labeling of S-phase cells and counterstaining for p-ERK and HA-tag. The plots show HA intensity vs. Nuc p-ERK in single cells; contour lines show equal cell density; and the heat map reflects the percentage of S-phase–positive cells within HA and p-ERK bins. Data plotted are in raw AFU and population statistics, including mean p-ERK levels, p-ERK SD, and overall percentage of S-phase–positive cells are shown within individual plots.

Fig. S6.

Single-cell expression ranges of adenovirus-delivered HA-tagged oncogenes in MEFs. Primary WT and Dusp5 KO MEFs were infected with 3 pfu/nL empty Ad, Ad HA-HRAS^Q61L, or Ad HA-BRAF^V600E for 24 h. Cells were stained for HA-tag before HCM. Four fields per well per fluorophore were acquired from cells. Frequency histograms of whole-cell HA-oncogene stain intensity using raw AFU values per cell from a representative experiment from n = 4 are shown.

Fig. S7.

DUSP5 facilitates BRAF^V600E-driven cell proliferation. WT and Dusp5 KO MEFs were infected with 3 pfu/nL of empty Ad, Ad HA-HRAS^Q61L, or Ad HA-BRAF^V600E and either treated with 100 nM PD0325901 MEK inhibitor (PD) or coinfected using 0.3 pfu/nL Ad DUSP5-Myc for 24 h before 2-h pulse labeling with fluorescent EdU for assessment of proliferative index using HCM. Plots show the percentage of EdU-positive cells (% S-phase) in each condition ± SEM, n = 3–5. **P < 0.01 using one-way ANOVA and Dunnett's posttest comparing all columns to KO.

Fig. 7.

Dusp5 deletion accelerates BRAF^V600E-driven senescence. (A) WT and Dusp5 KO primary and immortalized MEFs were infected with empty Ad, Ad HA-HRAS^Q61L, Ad HA-BRAF^V600E, and Ad EGR1 promoter-driven DUSP5-Myc (Ad DUSP5) and treated with 0.1 µM PD0325901 (PD) MEK inhibitor as indicated. Representative images show SA β-gal staining of MEFs after 72 h. (Scale bar: 125 μm.) The quantification of SA β-gal staining is shown for primary MEFs after 72 h ± SEM, n = 3. **P < 0.01 using two-way repeated-measures ANOVA and Bonferroni posttest. Western blots compare indicated protein levels in primary MEFs 24 h after oncogene expression. (B) The plots show quantitative PCR comparisons of indicated mRNA levels from primary MEFs treated as in A for 24 h. *P < 0.05, **P < 0.01 using two-way repeated-measures ANOVA and Bonferroni posttest, ±SEM, n = 3. (C) Model depicting cellular consequences of combined BRAF^V600Eexpression and DUSP5 deletion.