Deubiquitinating Enzyme USP20 Regulates Extracellular Signal-Regulated Kinase 3 Stability and Biological Activity

Abstract

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose regulatory mechanisms and biological functions remain superficially understood. Contrary to most protein kinases, ERK3 is a highly unstable protein that is subject to dynamic regulation by the ubiquitin-proteasome system. However, the effectors that control ERK3 ubiquitination and degradation are unknown. In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator. USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells. The overexpression of USP20 results in the stabilization and accumulation of the ERK3 protein, whereas USP20 depletion reduces the levels of ERK3. We found that the expression levels of ERK3 correlate with those of USP20 in various cellular contexts. Importantly, we show that USP20 regulates actin cytoskeleton organization and cell migration in a manner dependent on ERK3 expression. Our results identify USP20 as a bona fide regulator of ERK3 stability and physiological functions.

Keywords: ERK3; MAPKs; deubiquitinating enzymes.

FIG 1

The DUB USP20 regulates ERK3 protein levels. (A) HT-29 cells were transfected in duplicate with individual SMARTpool siRNAs targeting 99 human DUBs. After 72 h, cell extracts were analyzed by immunoblotting with anti-ERK3 antibody and antitubulin, which was used as loading control. The blots were quantified by densitometry, and the mean expression value from two independent experiments was calculated. Results are expressed as the mean log₂ fold change relative to the value for control siRNAs. Error bars indicate standard deviations. (B) 293T cells were transfected with increasing amounts of pCMV6-XL4-USP20 as indicated. After 48 h, cell lysates were analyzed by immunoblotting with anti-ERK3 antibody and anti-HSC70 as a loading control. (Top) Representative blot; (bottom) quantification of ERK3 expression levels performed in three independent experiments. Results are expressed as means ± standard deviations. (C) 293T cells were transfected with 2 μg of pCMV6-XL4-USP20. RNA was extracted 48 h after transfection, and the ERK3 mRNA level was quantified by real-time qPCR. RU, relative units. (D) 293T cells were cotransfected with pcDNA3-Myc₆-ERK3 and increasing amounts of pCMV6-XL4-USP20 as indicated. The expression of ERK3 was analyzed by immunoblotting as described above for panel B. (E) HeLa cells were transfected with SMARTpool USP20 or nontarget (NT) siRNAs. After 48 h, the cells were exposed to pH 6.4 acidic medium or treated with 0.5 mM CoCl₂ for 3 h. The expression of ERK3 was analyzed by immunoblotting as described above for panel B. Statistical significance was determined by two-way analysis of variance with a Bonferroni posttest using GraphPad Prism software version 5. (F) HeLa cells were transfected with SMARTpool USP20 siRNAs in the absence or presence of the pCMV6-XL4-USP20 plasmid. After 48 h, the cells were exposed to pH 6.4 acidic medium for 3 h, and ERK3 expression was analyzed by immunoblotting. Statistical significance was determined by one-way analysis of variance with a Bonferroni posttest. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 2

USP13 cooperates with USP20 to regulate ERK3 protein expression. HeLa cells were transfected with the indicated SMARTpool siRNAs. After 48 h, cells were exposed to pH 6.4 acidic medium for 3 h, and lysates were analyzed by immunoblotting with anti-ERK3 antibody. (Top) Representative immunoblot; (bottom) quantification of ERK3 levels performed in three independent experiments. Results are expressed as means ± standard deviations. Statistical significance was determined by one-way analysis of variance with a Bonferroni posttest. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant.

FIG 3

USP20 stabilizes ERK3. (A) 293T cells were transiently transfected with an empty vector (Ctl) or pCMV6-XL4-USP20. After 48 h, the cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated times. Endogenous ERK3 levels were analyzed by immunoblotting. (B and C) HeLa cells were transfected with SMARTpool USP20 siRNAs. After 48 h, the cells were treated with 0.5 mM CoCl₂ (B) or switched to pH 6.4 culture medium (C) for 3 h. The half-life of endogenous ERK3 was measured by cycloheximide chase experiments. (Left) Representative immunoblot; (right) quantification of immunoblotting data performed in three independent experiments. Results are expressed as means ± standard deviations of relative ERK3 expression levels normalized to values for the 0-min time point. Degradation curves were fitted to a one-phase decay model.

FIG 4

USP20 physically interacts with ERK3. (A) Lysates of exponentially proliferating 293T cells were subjected to immunoprecipitation (IP) with anti-USP20 or anti-HA antibody, which was used as a control. Immunoprecipitated proteins were analyzed by immunoblotting with the indicated antibodies. (B) 293T cells were transfected with full-length ERK3 or the truncated ERK3Δ(1–365) mutant comprising only the kinase domain. Cell lysates were immunoprecipitated with anti-USP20 or anti-HA antibody and analyzed by immunoblotting.

FIG 5

USP20 deubiquitinates ERK3 in vivo and in vitro. (A) 293T cells were transfected with pcDNA3-HA-ERK3-GST, pMT123-HA-Ubiquitin, and pCMV6-XL4-USP20 as indicated. After 48 h, the cells were lysed, and ERK3 was pulled down by using glutathione-agarose beads. Ubiquitination was analyzed by immunoblotting with anti-HA antibody. (B) Cells were processed as described above for panel A, and purified HA-ERK3-GST was incubated with purified recombinant USP20 in deubiquitination buffer for 2 h. Ubiquitination of ERK3 was assessed by immunoblotting with anti-HA antibody. Ub, ubiquitin.

FIG 6

USP20 regulates the ubiquitination and stability of a lysine-less ERK3 mutant. (A) 293T cells were transfected with the WT or the lysine-less (0K) ERK3Δ(1–365) construct. After 48 h, the cells were treated with 100 μg/ml cycloheximide for the indicated times. Endogenous ERK3 levels were analyzed by immunoblotting. (Top) Representative immunoblots; (bottom) quantification of immunoblotting data performed in three independent experiments. Results are expressed as means ± standard deviations of relative ERK3 expression levels normalized to values for the 0-min time point. Degradation curves were fitted to a linear curve model. (B) 293T cells were transfected with pcDNA3-HA-ERK3Δ-His₆ (WT or 0K), pMT123-HA-Ubiquitin, and pCMV6-XL4-USP20, as indicated. After 48 h, the cells were lysed, and ERK3 was pulled down with Ni-NTA–agarose beads. Ubiquitination was analyzed by immunoblotting with anti-HA antibody.

FIG 7

USP20 controls ERK3 protein levels during myogenic differentiation. (A) C2C12 myoblasts were cultured in growth medium (GM) or switched to differentiation medium (DM) for 4 days. The expression of ERK3 and USP20 was analyzed by immunoblotting. (B) C2C12 cells were transfected with an empty vector (Ctl) or pCMV6-XL4-USP20 and cultured as described above. The expression of ERK3 and USP20 was analyzed by immunoblotting. (C) C2C12 cells were transfected with an empty vector (Ctl) or pCMV6-XL4-USP20. After 48 h, the levels of ERK3 mRNA were quantified by real-time qPCR. Results are expressed as means ± standard deviations (n = 3).

FIG 8

USP20 regulates actin cytoskeleton dynamics and migration via ERK3. (A) HeLa cells were transfected with the indicated constructs in the absence or presence of SMARTpool ERK3 siRNAs. After 48 h, the cells were fixed, and actin filaments were stained with rhodamine-conjugated phalloidin. Actin cytoskeleton organization was visualized by epifluorescence microscopy. (B) The cellular adhesive area was quantified and is presented as a ratio of transfected (GFP-positive [GFP+])/untransfected (GFP-negative [GFP−]) cells. A minimum of 50 cells were counted, and the bar graph represents the means ± standard deviations of data from at least three independent experiments. (C and D) HeLa cells were infected with lentiviral vectors encoding GFP-ERK3 or USP20, and populations of transduced cells were selected with blasticidin. USP20-overexpressing cells were transfected with nontarget or SMARTpool ERK3 siRNAs. After 24 h, the cell lysates were analyzed for ERK3 expression by immunoblotting (C) and cells were plated at 90% confluence (D). Twenty-four hours after seeding, the confluent monolayer of cells was scraped with a sterile P200 tip. Cell migration was assessed by measuring the surface of the wound area 24 h later by phase-contrast microscopy. The bar graph represents the means ± standard deviations of data from at least three independent experiments. Statistical significance was determined by an unpaired t test. **, P < 0.01; *, P < 0.05.

FIG 9

The USP20-ERK3 axis regulates the migration of breast cancer cells. (A) The expression of ERK3 and USP20 was analyzed by immunoblotting with a panel of immortalized and transformed breast epithelial cell lines. Protein expression was quantified by densitometry, normalized by Ponceau S staining, and expressed as mean centered expression levels. (B) MCF7, MCF10A, and T47D cells were transfected with SMARTpool USP20 siRNAs. After 48 h, the cells were lysed, and ERK3 expression was analyzed by immunoblotting. (Left) Representative immunoblot; (right) densitometric quantification of immunoblotting data from three independent experiments. Results are expressed as means ± standard deviations. (C) MCF7 cells were infected with pLenti6-GFP or pLenti6-GFP-ERK3 in the absence or presence of SMARTpool USP20 siRNAs as indicated. After 24 h, the cells were plated at 90% confluence. Twenty-four hours after seeding, the cell monolayer was scratched, and cellular migration was analyzed by measuring the surface of the wound area after 24 and 48 h of recovery. Statistical significance was determined by two-way ANOVA with a Bonferroni posttest. (D) MCF10A cells stably infected with pLenti6-GFP or pLenti6-GFP-ERK3 were infected with two distinct shRNAs targeting USP20. After selection, wound-healing assays were performed as described above for panel C. Statistical significance was determined by one-way ANOVA with a Bonferroni posttest. **, P < 0.01; ****, P < 0.0001.