The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane Trafficking and Activation but Leaves K-Ras Unaffected

Abstract

The proto-oncogenic Ras isoforms (H, N, and K) have a C-terminal CAAX motif and undergo the same post-translational processing steps, although they traffic to the plasma membrane through different routes. Previously, we have shown that overexpression of the deubiquitinating enzyme USP17 inhibits H-Ras localization to the plasma membrane. Now we report that whereas H-Ras and N-Ras were unable to localize to the plasma membrane in the presence of USP17, K-Ras4b localization was unaffected. EGF stimulation was unable to induce N-Ras membrane localization in USP17-expressing cells. In addition, N-Ras activity and downstream signaling through the MAPK MEK/ERK and PI3K/JNK pathways were blunted. However, we still detected abundant N-Ras localization at the ER and Golgi in USP17-expressing cells. Collectively, our data showed that the deubiquitinating enzyme USP17 blocks EGF-induced N-Ras membrane trafficking and activation, but left K-Ras unaffected.

Full text

The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane
Trafficking and Activation but Leaves K-Ras Unaffected□
S
Received for publication, November 3, 2009, and in revised form, January 29, 2010 Published, JBC Papers in Press, February 10, 2010, DOI 10.1074/jbc.M109.081448
Michelle de la Vega
‡
, James F. Burrows
‡§
, Cheryl McFarlane
‡
, Ureshnie Govender
‡
, Christopher J. Scott
§
,
and James A. Johnston
‡1
From the
‡
Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, and
§
Molecular Therapeutics,
School of Pharmacy, Queen’s University, Lisburn Road, Belfast BT9 7BL, Northern Ireland
The proto-oncogenic Ras isoforms (H, N, and K) have a C-ter-
minal CAAX motif and undergo the same post-translational
processing steps, although they traffic to the plasma membrane
through different routes. Previously, we have shown that over-
expression of the deubiquitinating enzyme USP17 inhibits
H-Ras localization to the plasma membrane. Now we report that
whereas H-Ras and N-Ras were unable to localize to the plasma
membrane in the presence of USP17, K-Ras4b localization was
unaffected. EGF stimulation was unable to induce N-Ras mem-
brane localization in USP17-expressing cells. In addition, N-Ras
activity and downstream signaling through the MAPK MEK/
ERK and PI3K/JNK pathways were blunted. However, we still
detected abundant N-Ras localization at the ER and Golgi in
USP17-expressing cells. Collectively, our data showed that the
deubiquitinating enzyme USP17 blocks EGF-induced N-Ras
membrane trafficking and activation, but left K-Ras unaffected.
The Ras family of GTPases are signaling proteins involved in
cell proliferation, differentiation, and apoptosis. Ras proteins
act as molecular switches at the plasma membrane (PM)
2
con-
veying signals from the external environment to the interior of
the cell. For Ras to be functional it cycles from its inactive GDP-
bound to the active GTP-bound state. The active Ras then binds
to downstream effectors initiating signaling cascades. For ex-
ample, binding to the effector Raf results in mitogen-activated
protein kinase (MAPK) signaling and activation of ERK and
JNK. Regulation of GTPase activity is critical, and constitutively
active Ras mutants frequently contribute to the development of
aggressive cancers (1).
Additionally, Ras can signal from the ER and Golgi without
prior trafficking to the PM (2–5). Whereas PM activation of Ras
is thought to be rapid and transient, ER/Golgi activation is
delayed and sustained (2, 6). Differences in signals from the
varying locations may be due in part to the differential localiza-
tion of scaffolds. Indeed, KSR1 has been shown to act preferen-
tially on ERK from the PM, whereas Sef-1 regulates ERK at the
ER and Golgi (7, 8). Ras has also been shown to signal from the
endosomes where H-Ras and N-Ras can be localized as a result
of their ubiquitination (9).
To achieve membrane localization Ras must go through a
series of post-translational modifications, with processing of
the C-terminal CAAX box being a central event. The CAAX box
cysteine is initially isoprenylated by farnesyl transferase (FTase)
or geranylgeranyl transferase type I (GGTaseI) (K-Ras and
sometimes N-Ras) (10). Prenylation is essential for trafficking
Ras to the ER, where the intramembrane protease Ras-convert-
ing enzyme 1 (RCE1) cleaves the AAX and isoprenylcysteine
methyltransferase (ICMT) methylates the prenylated cysteine
(11, 12). However, the subsequent processing varies for differ-
ent isoforms: H-Ras and N-Ras are further palmitoylated at the
Golgi whereas K-Ras4b contains a polybasic stretch of lysines
that behaves as a membrane targeting signal and does not traffic
through the Golgi (13).
Ubiquitination and deubiquitination of proteins is an impor-
tant regulatory mechanism altering the fate of the modified
protein and disregulation of this process can have profound
effects. To date, five families of deubiquitinating enzymes have
been identified: ubiquitin-specific proteases (USPs), ubiquitin
C-terminal hydrolases (UCHs), ovarian tumor proteases
(OTUs), Josephins, and JAB1/MPN/MOV34 metallo-proteases
(JAMMs) (14, 15). The USP family are cysteine proteases iden-
tified by histidine and cysteine boxes within their catalytic
domain (15). USP17 is an immediate early-gene being cytokine
induced and is highly expressed in many cancers (16, 17). We
have previously shown that expression of USP17 blocks plasma
membrane localization and activation of H-Ras at least in part
by inhibition of its post-translational processing through mod-
ulation of RCE1 (18). Furthermore, the MAPK pathway was
disrupted resulting in delayed cell growth.
Because N-Ras and K-Ras are frequently mutated in cancers,
we asked whether USP17 expression would also result in the
mislocalization of these isoforms and what effect there would
be on downstream effectors. Using confocal microscopy we
show that USP17 inhibits H-Ras and N-Ras, but not K-Ras4b
membrane localization. This is true for both wild-type Ras and
oncogenic mutants. However, Ras activation and MAPK signal-
ing is decreased, but still present, in cells overexpressing
USP17. We propose that this is due to H-Ras and N-Ras being
localized at the ER and Golgi where Ras can still be activated
and signal to the MAPK pathway. These findings suggest that
USP17 regulates differential Ras isoform signaling from various
intracellular platforms.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S4.
1
To whom correspondence should be addressed: Rm. 330, Whitla Medical
Bldg., 97 Lisburn Rd., Belfast BT9 7BL, Northern Ireland. Tel.:
44-2890972260; Fax: 44-2890325839; E-mail: jim.johnston@qub.ac.uk.
2
The abbreviations used are: PM, plasma membrane; MAPK, mitogen-acti-
vated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun
N-terminal kinase; ER, endoplasmic reticulum; ANOVA, analysis of variance;
USP, ubiquitin-specific proteases; EGF, epithelial growth factor; BFA,
Brefeldin A; GFP, green fluorescent protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 16, pp. 12028 –12036, April 16, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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EXPERIMENTAL PROCEDURES
Plasmids—pDQ-EV (His), pDQ-USP17 (His), and pDQ-
USP17CS (His) were kind gifts from Dr. Derek Quinn (Queen’s
University, Belfast). pEGFP-C3-HRas, pEGFP-C3-HRasG12V,
pEGFP-C3-NRas, pEGFP-C3-NRasG12V, pEGFP-C3-KRas4b,
and pEGFP-C3-KRas4bG12V were kind gifts from Dr. Ian Prior
(Liverpool, UK). pSUPER-USP17shRNA (sequence GCAG-
GAAGATGCCCATGAA) was a kind gift from Prof. Rene Ber-
nards (The Netherlands Cancer Institute, Amsterdam, The
Netherlands), and scrambled shRNA was purchased from Ori-
Gene Technologies.
Cell Culture and DNA Transfections—HeLa cells (American
Type Culture Collection (ATCC)) were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf
serum, 1% penicillin (10,000 units/ml)/streptomycin (10,000
␮
g/ml), and 1% L-glutamine (200 mM), grown at 37 °C in 5%
CO
2
humidified incubator. Cells were transfected with
FuGENE
TM
6 transfection reagent (Roche) according to the
manufacturer’s instructions. Cells were seeded between 0.5 ⫻
10
6
and 5.0 ⫻10
6
cells for protein experiments or 0.20 ⫻10
5
on
LabTek II, CC2-treated 4 chamber slides (Nalge Nunc) for
microscopy experiments. The cells were transfected with 3
␮
g
of plasmid DNA for protein experiments and biological assays
or 0.25
␮
g of plasmid DNA for confocal microscopy experi-
ments. For those experiments with EGF stimulation, cells were
rested for 12 h in Dulbecco’s modified Eagles medium without
serum to minimize Ras activation. Cells were then stimulated
with 100 ng/ml EGF (R&D) for the indicated times in the fig-
ures. Brefeldin A (Sigma) was used at 5
␮
g/ml for 2 h after
serum-starving cells for 12 h.
Cell Lysis and Immunoblotting—Whole cell lysates and
immunoprecipitations were generated and separated prior to
immunoblotting as previously described (15). The following
primary antibodies were used: anti-USP17 (Fusion Antibodies),
anti-
␥
-tubulin (Sigma), mouse Pan-Ras (Calbiochem), as well
as
␤
-actin, pERK, ERK, pJNK, and JNK (all from Cell Signaling).
FIGURE 1. USP17 inhibits H-Ras and N-Ras plasma membrane localization, but not K-Ras localization. HeLa cells were transfected with GFP-HRas (A),
GFP-NRas (B), and GFP-KRas (C) along with USP17 or USP17CS. Cells were stained with a USP17 monoclonal antibody to confirm transfection (sup-
plemental Figs. S1–S3) and imaged with confocal microscopy. Arrows indicate plasma membrane-localized Ras, and arrowheads perinuclear Ras localization.
Scale bars are 20
␮
m. Quantification (n⫽3) of plasma membrane localization for each Ras isoform is shown in the fourth panel. ** indicates p⬍0.01 versus
control.
USP17 Alters N-Ras Localization
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Densitometry was performed using ImageJ software (NIH), and
error bars represent standard error from different gel expo-
sures. ANOVA statistics were carried out to compare Ras activ-
ity using Prism GraphPad.
Ras-RBD Pulldown Assay—HeLa cells were transfected with
the indicated plasmids and stimulated with EGF as previously
described. Ras RBD pulldown assays were carried out as previ-
ously described (18).
Confocal Microscopy—HeLa cells were seeded, fixed, and
stained as previously described (18). Antibodies and costains
used were as follows: mouse anti-USP17 (Fusion Antibodies),
rabbit anti-calnexin (AbCam), rabbit anti-GM130 (AbCam),
donkey anti-mouse or rabbit Cy5 (Jackson ImmunoResearch),
donkey anti-mouse or rabbit TRITC (Jackson ImmunoRe-
search). Slides were viewed on a Leica Sp2 Confocal Micro-
scope and images analyzed using Leica LAS AF software. The
images presented in the same figures were captured using stan-
dardized setting and exposure times. Cell counts were from at
least 100 cells in greater than three experiments unless other-
wise noted. ANOVA statistics compares PM localization.
RNA Extraction and RT-PCR—RNA extractions and RT-
PCR was carried out as previously described (17).
RESULTS
USP17 Regulates Ras Localization—To characterize whether
USP17 expression would affect the different Ras isoforms, we
examined membrane localization at steady-state conditions in
HeLa cells (Fig. 1). In the majority of control cells transfected
with the Ras isoforms, H-Ras, N-Ras, and K-Ras were localized
to the plasma membrane (Fig. 1, A–C,first panels, respectively;
arrows). However, in the presence of USP17, H-Ras and N-Ras
were mislocalized from the PM (Fig. 1, Aand B,second panels,
arrowheads). Quantification revealed a significant reduction in
membrane localization in the presence of USP17 (Fig. 1, Aand
B,fourth panels). The majority of these cells exhibited clear
cytosolic distribution with highly concentrated pools of Ras
FIGURE 2. USP17 inhibits oncogenic mutant H-Ras and N-Ras plasma membrane localization but not K-Ras localization. HeLa cells were trans-
fected with GFP-HRasV12 (A), GFP-NRasV12 (B), and GFP-KRasV12 (C) along with control, USP17, or USP17CS. Cells were stained with USP17 monoclonal
antibody to confirm transfection (not shown) and imaged with by confocal microscopy. Arrows indicate plasma membrane localized Ras, and arrow-
heads perinuclear localization. Scale bars are 20
␮
m. Quantification (n⫽3) of plasma membrane localization is shown for each Ras isoform in the fourth
panel. ** indicates p⬍0.01 versus control.
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most evident in the perinuclear region. In sharp contrast, K-Ras
was still localized to the plasma membrane in USP17-express-
ing cells (Fig. 1C,second and fourth panels). USP17CS (a cata-
lytically inactive mutant of USP17)-transfected cells had similar
Ras localization to control cells. To confirm USP17 and
USP17CS expression, cells were stained with an antibody
against USP17 (supplemental Figs. S1–S3). The experiments
were repeated more than three times and at least 100 cells were
blindly scored for membrane distribution by two individuals
(Fig. 1, histograms,panel 4). These observations with H-Ras and
N-Ras confirm our previous report that showed H-Ras was mis-
localized in the presence of USP17 (18). We also previously
demonstrated that depletion of USP17 with a shRNA results in
constitutively active H-Ras following serum stimulation (18).
Additionally, we now demonstrated that in USP17-depleted
cells N-Ras was also constitutively activated whereas K-Ras was
not affected (supplemental Fig. S4). This suggests that USP17 is
differentially regulating the Ras isoforms and plays a role prior
to its PM localization.
Oncogenic Ras has been reported in ⬃30% of cancers and
inhibition of Ras localization is an appealing therapeutic
approach (19). It was important therefore to examine whether
USP17 would also block the PM distribution in cells expressing
constitutively active Ras, in which codon 12 (valine) was
mutated to glycine. Similar to the results with wild-type Ras, the
oncogenic Ras isoforms were predominantly localized to the
plasma membrane in control and USP17CS-transfected cells
(Fig. 2, A–C;first,third, and fourth panels). Again, USP17 inhib-
ited oncogenic H-Ras and N-Ras localization to the plasma
membrane but not the localization of mutant K-Ras (Fig. 2,
second and fourth panels). USP17 and USP17CS expression was
confirmed by staining with an antibody as in Fig. 1 (data not
shown). These experiments were repeated more than three
times and quantification revealed the difference in PM localiza-
tion in USP17-expressing cells was
statistically significant for H-Ras
and N-Ras but not K-Ras (Fig. 2,
fourth panel). Again, a predominant
pool of perinuclear Ras was evident
in USP17 cells. Taken together,
USP17 could inhibit plasma mem-
brane localization of both wild-type
and oncogenic H-Ras and N-Ras but
not K-Ras.
EGF Stimulated Ras Activation in
USP17 Cells—Previous data dem-
onstrated that USP17 could inhibit
H-Ras and N-Ras PM localization
under steady-state conditions. In
order for Ras to be fully activated,
an external stimulus is required.
Therefore, it was important to
examine ligand-activated Ras. EGF
stimulation results in Ras binding to
its effectors, for example Raf, initiat-
ing downstream signaling cascades
(20). This is most commonly associ-
ated with Ras plasma membrane
localization. Because USP17 inhibited H-Ras and N-Ras local-
ization to the plasma membrane, it was expected that Ras acti-
vation would also be decreased. Therefore, we examined the
activity of N-Ras in the presence of USP17 using a Raf pulldown
assay.
Transiently transfected HeLa cells were incubated in growth
medium lacking serum for 12 h, then stimulated with EGF (100
ng/ml) for 5 or 10 min. Fig. 3Ashowed that EGF stimulated
N-Ras activation, peaked 5 min after treatment, which was con-
sistent with plasma membrane activation demonstrated previ-
ously (6). In the presence of USP17, N-Ras was still active but to
a much lesser degree than that observed in control cells. Omer-
ovic et al. (21) have demonstrated that in HeLa cells, N-Ras
accounts for ⬃50% of the total endogenous Ras protein and so
the effect of USP17 on the endogenous Ras protein was also
examined. As with overexpressed N-Ras, the endogenous pro-
tein was also activated in the presence of USP17, though mark-
edly less than observed in control cells (Fig. 3B). Although
USP17CS has a slight decrease in activation compared with
control cells, the increase in activation following stimulation is
still statistically significant. Densitometry of both the overex-
pressed N-Ras and endogenous Ras established that after 5 min
EGF stimulation Ras activation in the presence of USP17 was
nearly half that of control cells (Fig. 3, right panels). These
results demonstrated that Ras activation was blunted in the
presence of USP17.
USP17 Decreases N-Ras Localization to the PM but Not
Perinuclear Structures—Because activation of Ras by EGF was
markedly decreased in USP17-expressing cells, we assessed if
Ras was being localized to intracellular compartments where
activation may occur. Therefore, cellular localization of N-Ras
following EGF stimulation was examined. Cells were grown in
serum-free medium for 12 h, thereby markedly decreasing
membrane localization of Ras (Fig. 4). As expected, the plasma
FIGURE 3. USP17 blunts N-Ras activation. A, HeLa cells were transfected with GFP-NRas along with control
(empty vector), USP17, or USP17CS. B, HeLa cells were transfected with control (empty vector), USP17, or
USP17CS. Cells were serum-starved then stimulated with 100 ng/ml EGF for 0, 5, or 10 min. To visualize active
Ras, Raf pulldown assays (upper panels) and cell lysates (lower two panels) were analyzed by SDS-PAGE and
blotted for pan-Ras or USP17. Densitometry of Ras activity is shown on the right. Statistics compare 0 to 5 min
EGF treatment. *** indicates p⬍0.001; **, p⬍0.01; and ns for not significant.
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membrane pool of N-Ras was rapidly induced by EGF stimula-
tion for control and USP17CS-transfected cells (Fig. 4, Aand C,
respectively, arrows). This experiment was repeated in dupli-
cate, and cell counts illustrate that 2 min after EGF stimulation,
the percentage of membrane localized N-Ras increased from
⬃10% to 70% for both control and USP17CS-transfected cells
(Fig. 4D). However, this was not observed in USP17-transfected
cells, where only a slight increase in PM localization was meas-
ured after EGF stimulation (Fig. 4, Band D). Ras localization at
the intracellular organelles was also examined and quantified,
but no change in distribution was observed in USP17-trans-
fected cells (Fig. 4, arrowheads, quantification not shown). Data
suggested that PM-localized Ras activation was rapid, whereas
the ER/Golgi pools were stable and sustained. This is similar to
reports by Ibiza et al. (22) that showed that a proportion of
N-Ras was localized to the Golgi in cells under steady-state
conditions.
Ras Colocalizes with the ER and Golgi in USP17 Cells—Given
that Ras activation can occur at the ER and Golgi, we sought to
determine whether N-Ras was still localized to the ER and Golgi
in the presence of USP17. This may
account for the N-Ras activation
seen in Fig. 3. In control cells, N-Ras
was distributed both at the plasma
membrane and in the cytosol, which
colocalized mostly with the ER
marker calnexin. (Fig. 5A,arrow).
Although N-Ras was not visualized
at the plasma membrane in USP17-
transfected cells, there was again a
pool of internal N-Ras, which colo-
calized with the ER marker (Fig. 5B).
We then examined the localization
of N-Ras with the cis-Golgi marker
GM130 and observed a similar
trend, where N-Ras colocalized with
the Golgi in both control and
USP17-transfected cells (Fig. 5, C
and D). Whereas intracellular pools
of N-Ras predominantly co-local-
ized with the ER and Golgi, there are
likely additional cellular organelles
that the isoforms localize to. This
experiment was repeated more than
three times, and consistently dem-
onstrated that although the plasma
membrane pool of N-Ras was dis-
rupted in USP17-expressing cells,
the ER and Golgi pools were still
intact. Interestingly, although K-
Ras is modified at the ER, it does not
stably localize to intracellular struc-
tures (23). We also demonstrate
that K-Ras does not co-localize with
calnexin, and so does not stably
interact with the ER (Fig. 5, Eand F).
USP17 Diminishes but Does Not
Abolish Ras Signaling—In the pres-
ence of USP17, we demonstrated that N-Ras was relocalized
from the PM to the ER and Golgi, and that Ras activation by
EGF stimulation was blunted. This suggested that expression of
USP17 would inhibit downstream Ras signaling after stimulation.
We, therefore, sought to determine whether USP17 inhibited
MAPK activation. Because EGF-induced Ras activation at the
plasma membrane occurs rapidly, whereas activation at endo-
membranes occurs later at 40– 60 min (2), we stimulated cells
with EGF over a 60-min time course. We detected pERK at 5
min after EGF (100 ng/ml) stimulation, with levels peaking at 30
min (Fig. 6, A,top panels, and B). Although phosphorylation of
ERK1/2 was detected in the presence of USP17, the levels were
decreased compared with controls. Furthermore, there was a
marked decrease in p44 pERK1, but only a minor decrease in
p42 pERK2. The increase in pERK1 at 30 min of EGF stimula-
tion was statistically significant in control cells but not USP17-
expressing cells. Given that p42 ERK1 is predominant at the
endomembranes (such as the ER and Golgi), whereas p44 ERK1
is not, suggests USP17 differentially regulated the ERK
isoforms.
FIGURE 4. USP17 inhibits EGF-stimulated N-Ras localization to the PM. HeLa cells were transfected with
GFP-NRas along with control (empty vector) (A), USP17 (B), or USP17CS (C). Cells were serum-starved then
stimulated with EGF (100 ng/ml) for 0, 2, 5, or 10 min. Ras membrane distribution was visualized by confocal
microscopy and quantification for PM Ras scored in duplicate experiments (D). Scale bars are 20
␮
m.
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In addition, JNK can be phosphorylated in response to active
Ras at endomembranes via PI3K activation (5). Following EGF
stimulation, JNK phosphorylation increased, peaking at 30 min
(Fig. 6, A,middle panels and C). Similar to the results with pERK,
detectable pJNK levels were decreased in the presence of USP17
but not completely inhibited. Densitometry illustrated that pJNK
expression was approximately half in USP17-transfected cells ver-
sus control and USP17CS-transfected cells (Fig. 6C). This modest
increase of total pJNK following 30 min of EGF stimulation was
significant. These results clearly indicated that MAPK activation
was markedly reduced in the presence of USP17.
EGF Stimulates Ras Membrane Localization and Activity
after Golgi Disruption—H-Ras and N-Ras predominantly traf-
fic through the Golgi to the PM, but an alternative trafficking
route has been suggested (25). Because plasma membrane
localization of N-Ras was clearly reduced in USP17-expressing
cells, we hypothesized that USP17 would also inhibit N-Ras
transport to the PM via this alternate route. Brefeldin A
(BFA) reversibly disrupts Golgi structure by interfering with
COP I complex formation and so prevents vesicular traffick-
ing (26). H-Ras and N-Ras trafficking to the PM via the Golgi
have been demonstrated to be inhibited by BFA treatment
(23, 28). Therefore, N-Ras localization in the presence of
USP17 following BFA treatment and EGF stimulation was
examined (Fig. 7, A–D).
In the presence of BFA treatment (5
␮
g/ml, 2 h), N-Ras was
unable to traffic to the plasma membrane, similar to what has
been observed in USP17-expressing cells. As expected, EGF
stimulation alone (100 ng/ml 5 min) resulted in N-Ras localiza-
tion to the plasma membrane in control cells, but not USP17-
transfected cells as previously shown in Fig. 4 (Fig. 7, A–D).
Furthermore, BFA treatment (5
␮
g/ml 2h) followed by EGF
(100 ng/ml 5 min) stimulation resulted in increased cell mem-
brane association in control cells but not USP17-expressing
cells. These data illustrated that N-Ras membrane localization
by EGF stimulation overcame Golgi disruption by BFA treat-
ment. This suggested that when the Golgi was disrupted, N-Ras
could traffic to the membrane via an alternate route, which
could still be modulated by USP17. Furthermore, we saw that
K-Ras localization was not affected by the presence of USP17 or
disruption of the Golgi (data not shown).
To examine Ras activity, untransfected control or trans-
fected cells were serum starved for 12 h, treated with BFA (5
␮
g/ml) for 2 h, then stimulated with EGF (100 ng/ml) for 5 min.
As expected, Ras activity was increased following EGF treat-
ment in the absence of BFA in control and USP17CS-trans-
fected cells (Fig. 7E). However, Ras was not activated in USP17-
expressing cells upon EGF stimulation alone or following the
combination of BFA and EGF treatment. Thus, this confirms
that when PM transport through the Golgi is inhibited and
N-Ras is trafficked to the PM through an alternative route,
ligand-induced N-Ras activation still occurred. However, the
presence of USP17 also completely disrupts this.
DISCUSSION
Results presented in this study indicated that USP17 can
inhibit H-Ras and N-Ras localization to the plasma membrane,
but not K-Ras. Furthermore, USP17 is induced rapidly in
response to EGF (data not shown) and its presence inhibits
membrane localization blunting both N-Ras activation and
EGF-induced MAPK signaling. N-Ras has been shown to signal
from internal cellular compartments such as the ER and Golgi
and the results presented here confirm this, because mislocal-
ized N-Ras is still activated in response to EGF. Indeed, in con-
trol cells disruption of the Golgi with BFA resulted in Ras mis-
localization and decreased activity, which could be overcome
with EGF stimulation. However, in USP17-expressing cells
N-Ras was still mislocalized from the PM suggesting that
USP17 inhibits both the conventional transport route through
the Golgi and the alternative route when the Golgi is inhibited.
These results may have important implications in cancer
FIGURE 5. N-Ras colocalizes with the ER and Golgi. HeLa cells were trans-
fected with GFP-NRas along with control (empty vector) (Aand C) or USP17 (B
and D) or GFP-KRas with control and USP17 (Eand F). Cells were stained with
primary antibodies: calnexin for the ER (A,B,E, and F) or GM130 for the Golgi
(Cand D) and pseudo-colored blue. Representative images were captured by
confocal microscopy. Scale bars are 20
␮
m.
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because USP17 can inhibit specific Ras isoform localization and
activation even in the presence of growth factors.
Ras mutations are found in ⬃30% of all cancers with some
cancers having much higher mutation rates (19). Therapeutic
targets have predominantly focused on the prenylation en-
zymes FTase or GGTase I. However, a major problem encoun-
tered was that cancers treated with a specific prenylation inhib-
itor, such as a FTase inhibitor, were able to overcome this by
being alternatively prenylated by the closely related enzyme
GGTase I (29 –31). Therefore, alternative approaches to inhibit
Ras localization and downstream signaling are therapeutically
attractive (32, 33).
USP17 has previously been shown to regulate H-Ras process-
ing and activation (18). Because N-Ras and K-Ras undergo sim-
ilar processing and activation we investigated the effect of
USP17 on these isoforms as well. Here, we showed that similar
to H-Ras, N-Ras was also mislocalized from the cell membrane,
whereas K-Ras was not affected by the presence of USP17. In
addition, identical H-Ras and N-Ras
mislocalization was observed with
constitutively active oncogenic
mutants (i.e. USP17 mislocalizes
H-Ras and N-Ras but not K-Ras).
K-Ras is also processed by RCE1 and
so a similar distribution should be
expected. In keeping with our find-
ings showing K-Ras at the plasma
membrane in USP17-expressing
cells, K-Ras has been demonstrated
to be membrane-localized in RCE1-
null cells (34, 35). Furthermore,
USP17 expression decreases RCE1
activity by ⬃50% (21). Additionally,
K-Ras is crucial to the cells as the
other isoforms are unable to com-
pensate for a loss of K-Ras (36). The
decreased activity of RCE1 may be
enough to preferentially cleave
K-Ras as opposed to H-Ras and
N-Ras. Yet another possibility is
that like many deubiquitinating
enzymes, USP17 may have addi-
tional targets besides RCE1, which
may differentially regulate transport
of the Ras isoforms.
A major difference between the
isoforms is that K-Ras does not need
to go through the Golgi in order to
be translocated to the PM. Instead
K-Ras traffics to the membrane via
microtubules from the ER, whereas
H-Ras and N-Ras route by vesicular
transport from the Golgi (23).
Therefore, it would not be surpris-
ing if USP17 regulated transport
and membrane localization via
another mechanism.
Our results demonstrated that
USP17 expression could inhibit MAPK signaling. Interestingly,
USP17 preferentially inhibited the phosphorylation of the p44
ERK1 isoform over p42 ERK2. Although there is as yet no definite
role of p44 ERK1, it has been visualized that the ERK isoforms may
be localized to different regions of the cells (24). Total p42 ERK2 is
localized to the cytosolic, nuclear and membrane fractions (which
include the ER and Golgi as well as PM), whereas total p44 ERK1 is
mainly found in the cytosol and nuclear fractions (24). This sug-
gests that when N-Ras is properly localized to the PM it can signal
downstream and activate both ERK1 and ERK2. However, when
mislocalized, downstream signaling is altered and only the ERK2
isoform is phosphorylated. In addition, the ERK isoforms may
have preferred scaffold proteins because ERK2, but not ERK1, was
shown to associate with KSR-2 (37). This indicates that in the pres-
ence of USP17, when Ras is localized mainly to the ER and Golgi
membranous organelles, ERK2 may be preferentially activated.
Recent reports clearly show that Ras can be active when
localized to cellular organelles such as the ER and Golgi (5–7).
FIGURE 6. USP17 blunts Ras ERK and JNK MAPK signaling. A, HeLa cells were transfected with N-Ras
along with control (empty vector), USP17, and USP17CS. Cells were serum-starved then stimulated with
EGF (100 ng/ml) for the indicated times. Cell lysates were immunoblotted with pERK, pJNK, Ras, and USP17
antibodies. pERK and pJNK blots were reprobed for total ERK and total JNK, respectively. Densitometry of
p44 pERK1 (B) and pJNK (C). Statistics compares 0 and 30 min of EGF treatment. *** indicates p⬍0.001, and
ns is not significant.
USP17 Alters N-Ras Localization
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FIGURE 7. Inhibiting Golgi structure does not disrupt N-Ras PM localization and activation following EGF stimulation. HeLa cells were transfected with N-Ras
along with control (empty vector), USP17, or USP17CS. Cells were serum-starved, treated with brefeldin A (BFA 5
␮
g/ml) for 2 h, then with EGF (100 ng/ml) for 5 min as
indicated. A–C, representative confocal images of N-Ras localization following the indicated treatment. Arrows indicate PM-localized N-Ras; scale bars are 20
␮
m.
D, quantification of membrane localization from three experiments. Raf-pulldown assays for overexpressed GFP N-Ras (upper panel) and whole cell lysates examined
(lower two panels) for Ras or USP17 (E). Histogram on the right displays activated N-Ras. *** indicates p⬍0.001, **, p⬍0.01; *, p⬍0.05; and ns stands for not significant.
USP17 Alters N-Ras Localization
APRIL 16, 2010•VOLUME 285•NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12035
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Endomembrane activation was thought to be mainly due to Ras
initially being activated at the PM, and then trafficked through
retrograde pathways to the ER and Golgi. This would result in a
delayed response in endomembrane activation compared with
PM activation. However, Bivona et al. (38) showed that in the
Jurkat T-cell line, H-Ras was activated at the Golgi following 5
min of TCR stimulation because of binding with a Golgi-spe-
cific GEF, not requiring preliminary PM localization or activa-
tion. Our observations showed that even in USP17-expressing
cells, where PM localization was markedly diminished, Ras was
activated as early as 5 min following EGF treatment. This sug-
gests that in USP17-expressing cells, Ras activation at the
ER/Golgi may occur independent of PM-activated Ras.
To examine N-Ras trafficking to the PM in more depth, cells
were treated with Brefeldin A, which disrupts Golgi structure
preventing COP I complex formation and so prevents conven-
tional vesicular trafficking (26). After BFA treatment in control
cells, N-Ras localization to the PM and activation was blunted
although restored after 5 min of EGF stimulation. Previous
work has demonstrated that when vesicular transport from the
Golgi was disrupted, H-Ras was still trafficked to the PM via an
alternative route, in which H-Ras formed mobile clusters not
derived from perinuclear structures (25, 27). Interestingly, in
USP17-expressing cells there are small intracellular concen-
trated pools of N-Ras, which may be transport clusters. There-
fore, our data support this theory that when conventional traf-
ficking routes to the plasma membrane are inhibited, there are
alternative routes for Ras membrane localization.
Targeting oncogenic mutant Ras by inhibiting its post-trans-
lational modifications may be a key area of therapeutic inter-
vention. Our data demonstrated that USP17 regulates H-Ras
and N-Ras but not K-Ras localization. These results implicate
USP17 as a novel regulator of differential trafficking of the Ras
isoforms, making it an important protein for further study in
relation to potential cancer therapy.
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USP17 Alters N-Ras Localization
12036 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 16•APRIL 16, 2010
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Christopher J. Scott and James A. Johnston
Michelle de la Vega, James F. Burrows, Cheryl McFarlane, Ureshnie Govender,
Activation but Leaves K-Ras Unaffected
The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane Trafficking and
doi: 10.1074/jbc.M109.081448 originally published online February 10, 2010
2010, 285:12028-12036.J. Biol. Chem.
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