Tài liệu Báo cáo khoa học: Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process docx

Activated Rac1, but not the tumorigenic variant Rac1b, is
ubiquitinated on Lys 147 through a JNK-regulated process
Orane Visvikis
1,2
, Patrick Lore
`
s
1,2
, Laurent Boyer
3
, Pierre Chardin
4
, Emmanuel Lemichez
3
and Ge
´
rard Gacon
1,2
1 Institut Cochin, Universite
´
Paris Descartes, CNRS (UMR8104), Paris, France
2 INSERM, U567, Paris, France
3 Faculte
´
de Me
´
decine, INSERM U627, Nice, France
4 Institut de Pharmacologie du CNRS, Sophia Antipolis, France
In all eukaryotic cells, Rho GTPases (Rho, Rac,
Cdc42) control basic cellular functions, including actin
cytoskeleton organization, vesicular trafficking, tran-

scriptional regulation and cell cycle progression, and
are therefore major intracellular regulators of cell
growth and division, and cell migration [1,2]. Consis-
tent with their key role in cell signaling, dysregulation
of Rho-dependent pathways is being found to be caus-
ally involved in the pathophysiology of a growing
number of human diseases [3]. Hence, mutations in
genes encoding Rho regulators are responsible for
human developmental defects and, particularly, several
Keywords
JNK; Rac1b; Rho GTPases; ubiquitination
Correspondence
G. Gacon, Institut Cochin, De
´
partement
Ge
´
ne
´
tique et De
´
veloppement, 24 rue du
Faubourg Saint-Jacques, 75014 Paris,
France
Fax: +33 1 44 41 24 41
Tel: +33 1 44 41 24 70
E-mail:
(Received 29 August 2007, revised 18
November 2007, accepted 27 November
2007)

doi:10.1111/j.1742-4658.2007.06209.x
Ubiquitination and proteasomal degradation have recently emerged as an
additional level of regulation of activated forms of Rho GTPases. To char-
acterize this novel regulatory pathway and to gain insight into its biological
significance, we studied the ubiquitination of two constitutively activated
forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumori-
genic splice variant Rac1b, which is defective for several downstream
signaling pathways, including JNK activation. Whereas Rac1L61 under-
goes polyubiquitination and subsequent proteasomal degradation in
HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more
resistant to proteasomal degradation than Rac1L61. Mutational analysis of
all lysine residues in Rac1 revealed that the major target site for Rac1
ubiquitination is Lys147, a solvent-accessible residue that has a similar con-
formation in Rac1b. Like Rac1L61, Rac1b was found to be largely associ-
ated with plasma membrane, a known prerequisite for Rac1 ubiquitination.
Interestingly, Rac1b ubiquitination could be stimulated by coexpression of
Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1
downstream signaling. Indeed, ubiquitination of Rac1L61 is critically
dependent on JNK activation. In conclusion: (a) Rac1b appears to be more
stable than Rac1L61 with regard to the ubiquitin–proteasome system, and
this may be of importance for the expression and tumorigenic capacity of
Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK-
activated process, which may explain the defective ubiquitination of Rac1b.
The JNK-dependent activation of Rac1 ubiquitination would create a regu-
latory loop allowing the cell to counteract excessive activation of Rac1
GTPase.
Abbreviations
CNF1, cytotoxic necrotizing factor 1; EMT, epithelial–mesenchymal transition; GST, glutathione S-transferase; HA, hemagglutinin;
UbR48, ubiquitin mutant lacking Lys48; UbR63, ubiquitin mutant lacking Lys63; UPS, ubiquitin–proteasome system.
386 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS

forms of X-linked mental retardation [4]. In addition,
a number of bacterial virulence factors and toxins have
been found to target Rho GTPases and to induce vari-
ous chemical modifications interfering with Rho func-
tion and resulting in increased invasive properties of
the pathogens [5,6]. Finally, dysregulations of Rho
GTPases have been observed in a variety of human
tumors [7]; overexpression of Rho GTPases, particu-
larly RhoC, frequently contributes to tumor progres-
sion and invasiveness [8], and Rac1b, a constitutively
active splice variant of Rac1, has been found to be
expressed in colorectal and breast tumors [9,10] and
to display oncogenic properties in cancer cell lines
[11,12].
Rho family GTPases switch between a GDP-bound
form localized in the cytosol in association with guan-
ine nucleotide dissociation inhibitors, and a GTP-
bound form localized on various membranes and
mediating interaction with specific effectors to activate
downstream signaling pathways [13]. GDP ⁄ GTP
cycling is controlled by guanine nucleotide exchange
factors and GTPase-activating proteins. An additional
level of regulation has emerged from recent studies,
including ours, showing that Rho, Rac and Cdc42,
particularly when in their GTP-bound form, can be
targeted for polyubiquitination and subsequent degra-
dation [14,15]. Ubiquitination consists of the covalent
attachment to proteins of ubiquitin, an 8 kDa polypep-
tide. It is catalyzed by three enzymes: a ubiquitin-acti-
vating enzyme (E1), a ubiquitin-conjugating enzyme

(E2), and a target-specific ubiquitin protein ligase (E3),
acting sequentially to form an isopeptide bond between
the ubiquitin C-terminus and the e-amino group of
lysines of the target protein [16]. Ubiquitin contains
seven lysine residues that can be attached to other
ubiquitins in a highly processive reaction to form
a polyubiquitin chain [17]. Classically, Lys48-linked
chains facilitate recognition of and degradation by the
26S proteasome [18], and Lys63-linked chains regulate
processes such as signal transduction and DNA repair
through proteasome-independent mechanisms, whereas
much less is known about the function of chains linked
through other lysine residues [19].
In early studies, a bacterial toxin produced by uro-
pathogenic Escherichia coli, cytotoxic necrotizing fac-
tor 1 (CNF1), which provokes permanent Rho
activation by catalyzing deamidation of Gln63 of
RhoA (Gln61 in Rac1 ⁄ Cdc42), was found to sensitize
Rho proteins to ubiquitination and proteasomal degra-
dation [14,15]. Subsequently, Smurf1, a HECT domain
E3 ligase, was shown to target RhoA for ubiquitina-
tion and degradation [20], thereby regulating epithelial
cell polarity and transforming growth factor-b-depen-
dent epithelial–mesenchymal transition (EMT) [21], as
well as tumor cell motility [22]. Consistent with a
major role of Smurf1 as a RhoA E3 ligase, CNF1-
induced ubiquitination of RhoA was found to be spe-
cifically impaired in Smurf1
) ⁄ )
cells [23]. In contrast to

RhoA, little is known about the regulation of Rac and
Cdc42 by the ubiquitin–proteasome system (UPS). In
particular, specific E3 ligases for Rac and Cdc42 have
not yet been reported. Recently however, UPS-medi-
ated degradation of activated Rac1 was shown to be
essential during the early stages of hepatocyte growth
factor-induced epithelial cell scattering [24].
To gain insight into the mechanism and biological
significance of UPS regulation of Rac1, we have stud-
ied here the ubiquitination of mutationally activated
Rac1 (Rac1L61) and its naturally occurring splice vari-
ant Rac1b. We report that, unlike Rac1L61, Rac1b
mostly escapes the ubiquitination process and is there-
fore resistant to proteasomal degradation. We show by
mutational analysis that the main target site for
Rac1L61 polyubiquitination is Lys147, a surface resi-
due that should not be hampered in Rac1b; we also
report that Rac1 ubiquitination is stimulated by JNK,
which may explain the defective ubiquitination of
Rac1b.
Results
Rac1b is much less sensitive than Rac1L61
to the UPS
As shown in Fig. 1A, the constitutively activated
mutant Rac1L61 undergoes a significant degree of
polyubiquitination in HEK293 cells. By contrast, both
Rac1WT and Rac1N17, which are predominantly
GDP-bound, are not detectably ubiquitinated in this
system. This is in full agreement with previous obser-
vations made in various epithelial and endothelial cell

types [14,24].
Using ubiquitin mutants lacking Lys48 (UbR48) or
Lys63 (UbR63), we found that ubiquitin chain synthe-
sis on Rac1L61 was markedly but not completely
inhibited by UbR48 and that, by contrast, UbR63 sup-
ported the formation of polyubiquitinated Rac1 to the
same extent as wild-type ubiquitin (Fig. 1B). This
points to a major role of Lys48 of ubiquitin in
Rac1L61 polyubiquitin chain extension. Also in agree-
ment with this view, the monoubiquitinated form of
Rac1 was found to be clearly accumulated in cells
expressing UbR48 (Fig. 1B), indicating that, in the
absence of Lys48, the chain cannot be extended. Con-
sistently, inhibition of proteasome using MG132
resulted in an increase of polyubiquitinated Rac1
O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK
FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 387
species (Fig. 1C) and accumulation of Rac1 (Fig. 1D,
right panel). Whereas most of the polyubiquitin chains
on Rac1 appear to be extended through Lys48, leading
typically to proteasome degradation, the persistence
of a significant amount of ubiquitinated Rac1 when
UbR48 was used was repeatedly observed. This could
be due to mixed chains including endogenous wild-type
and mutant K48R ubiquitin. Alternatively, a fraction
of Rac1L61 may have undergone homogeneous poly-
ubiquitination employing neither Lys48 nor Lys63
chains, or heterogeneous polyubiquitination, as
recently reported for other proteins [25].
Unlike activating point mutations of Rac1 (such as

G12V and Q61L), which have not been found to occur
in any natural circumstance, Rac1b is a constitutively
activated variant expressed in breast and colorectal
tumors. Indeed, Rac1b is an alternatively spliced vari-
ant of Rac1, containing a 19 amino acid insertion
between positions 75 and 76, immediately C-terminal
to the switch II region (residues 60–76 in Rac1), which
is critically involved in conformational changes during
GDP ⁄ GTP cycling [26]. Accordingly, Rac1b exhibits
profoundly altered properties, including enhanced
nucleotide exchange activity, impaired intrinsic
Fig. 1. Ubiquitination and proteasomal degradation of Rac1L61 and Rac1b. (A) Activated Rac1L61 undergoes ubiquitination in HEK293 cells.
Ubiquitination of Rac was assessed by transfecting HEK293 cells with a combination of expression plasmids of 6His–myc–Ub, myc–
Rac1L61, Rac1L61WT or Rac1L61N17 as indicated. Ubiquitinated proteins were collected on Co beads, and immunoblotted with Rac1-spe-
cific antibodies. Expression of transfected and endogenous proteins was monitored in total protein extracts by immunoblotting using the
indicated antibodies. (B) In ubiquitinated Rac1L61, ubiquitin moieties are linked through Lys48. HEK293 cells were transfected with a combi-
nation of expression plasmids of myc–Rac1L61 and 6His–myc–UbWT or Lys fi Arg mutants at residues 48 (R48) or 63 (R63). Overexposure
allows the detection of the monoubiquitinated form of Rac1, in the presence of UbR48. (C) Ubiquitinated forms of Rac1L61 accumulate in
the presence of the proteasomal inhibitor MG132. HEK293 cells were transfected with a combination of expression plasmids of myc–
Rac1L61 and 6His–myc–Ub as indicated, either treated or not with MG132 (20 l
M) 6 h before the ubiquitination assay. (D) Differential ubiqui-
tination of Rac1L61 and Rac1b (left panel). HEK293 cells were transfected with a combination of expression plasmids of HA–Rac1L61, HA–
Rac1bWT, HA–Rac1bL61 and 6His–myc–Ub as indicated. Proteasomal degradation of Rac1L61 and Rac1b (right panel). Proteasomal degra-
dation was assessed by determining Rac1L61, Rac1bWT and Rac1bL61 accumulation in the presence of the proteasome inhibitor MG132
(20 l
M), as indicated in Experimental procedures.
Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al.
388 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS
GTPase activity, and failure to interact with RhoGDI;
as a result, Rac1b has been found to exist predomi-

nantly in the active GTP-bound state, which can be
further stabilized by a GTPase-inactivating mutation
(Rac1bL61) [26–28]. Therefore, we examined its ability
to undergo ubiquitination and subsequent proteasomal
degradation in HEK293 cells. As shown in Fig. 1D
(left panel), ubiquitination of Rac1b is much lower
than that of constitutively activated Rac1. Introducing
the L61 mutation in Rac1b provoked a moderate
increase in Rac1b ubiquitination (see white bars in
Fig. 3A below for quantitative analysis), but did not
sensitize it significantly to proteasome-mediated degra-
dation in HEK293 cells (Fig. 1D, right panel). A simi-
lar difference between Rac1 and Rac1b was observed
in HeLa cells; in these cells, Rac1L61 was found to be
weakly ubiquitinated, whereas no ubiquitination of
Rac1b or Rac1bL61 could be detected (data not
shown).
To address the difference(s) between Rac1 and
Rac1b that might explain their differential sensitivity
to the UPS, we attempted to characterize the require-
ments for Rac1 ubiquitination, which may not be
fulfilled by Rac1b.
The main target site for Rac1 ubiquitination is
Lys147, a surface residue not hampered in Rac1b
We first reasoned that the site(s) for ubiquitin addition
in Rac1 might not be accessible in Rac1b. Therefore,
in an attempt to map the ubiquitin acceptor site(s) in
IB : Rac
IB : actin
MG132 (20 µ

M)- +
Rac1L61
Rac1L61R147
-+
Rac1L61R16
-+
Rac1L61R96
-+
6His-myc-Ub
myc-Rac1L61
+
-
-
+
+
+
Cobalt extraction
IB : Rac
IB : actin
IB : Rac
+
R147
+
R16
+
R96
A
pull-down
GST PAK
IB : myc

myc-Rac1
GST PA K GST PAK GST PAK
WT L61 L61R147L61R16
WT L61
input
myc-Rac1
L61R147L61R16
B
Lys147
C
Fig. 2. Mutation of Lys147 to Arg strongly impairs Rac1L61 ubiq-
uitination without altering Rac activation. (A) Mutation of Lys147
or Lys16 impairs Rac1L61 ubiquitination (top panel) and subse-
quent proteasomal degradation (bottom panel). HEK293 cells
were transfected with a combination of expression plasmids of
6His–myc–Ub, myc–Rac1L61 and myc–Rac1L61 with a Lys fi Arg
substitution at position 16 (R16), position 96 (R96), or posi-
tion 147 (R147), as indicated. Proteasomal degradation was
assessed by determining the accumulation of myc–Rac1L61 and
myc–Rac1L61 Lys fi Arg mutants in the presence of the protea-
some inhibitor MG132 (20 l
M), as indicated in Experimental pro-
cedures. (B) Unlike the R16 mutation, the R147 mutation does
not impair Rac1L61 activation. Myc-tagged wild-type and mutant
Rac1 were expressed in HEK293 cells (top panel), pulled down
with GST–PAK (bottom panel) and immunoblotted with antibody
to myc tag. (C) Superposition of the structures of Rac1 (Protein
Data Bank: 1MH1) and Rac1b (Protein Data Bank: 1RYH), both
bound to a GTP analog (GppNHp), using
PYMOL software. The

main differences are highlighted in green (Rac1b). The structure
of Rac1b switch I, shown as a green line at the bottom, is
clearly further away from the nucleotide (GTP, in red), explaining
a decreased affinity for nucleotide in Rac1b. The switch II region
of Rac1b (dotted green line on the right), where the 19 amino
acid insertion of Rac1b is found, is disordered in the crystal. The
mostly disordered structures of the switch II region are compati-
ble with important modifications in nucleotide binding and interac-
tion with effectors. The positions of the Lys147 residues are
almost identical. Lys147 of Rac1 is yellow, and the equivalent
residue of Rac1b is shown in orange on top. There is only a
minor change in orientation of the lysine side chain.
O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK
FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 389
activated Rac1, we performed individual substitutions
of all lysines present in the Rac1 sequence to arginine
and analyzed the mutated proteins for ubiquitination
in HEK293 cells. Most of the 18 mutations tested did
not notably affect the polyubiquitination pattern of
myc–Rac1L61 (supplementary Fig. S1); only two lysine
mutants, i.e. Rac1L61R16 and Rac1L61R147, showed
a strong decrease in polyubiquitination as compared to
control Rac1L61 or to the Rac1L61R96 ‘ubiquitina-
tion-irrelevant’ lysine mutant (Fig. 2A, top panel).
Consistent with inhibition of their polyubiquitination,
both Rac1L61R16 and Rac1L61R147 were found to
be resistant to proteasome degradation, as evidenced
by their absence of stabilization upon proteasome inhi-
bition (Fig. 2A, bottom panel).
As the rate of polyubiquitination of Rac GTPase is

critically dependent on the active GTP-bound confor-
mation, we investigated whether the K16R and K147R
substitutions might affect the strength of the L61 acti-
vating mutation. As shown in Fig. 2B, Rac1L61R16
was strongly impaired in its ability to bind to glutathi-
one S-transferase (GST)–PAK, indicating that the
active GTP-bound form of Rac1L61 is altered by the
K16R substitution. Indeed, mutations in this region of
Ras (residues 15, 16, and 17) result in reduced nucleo-
tide binding and impaired activation [29]. Consistently,
we observed that Rac1L61R16 was unable to induce
membrane ruffling in COS cells. Therefore, it is likely
that the poor ubiquitination of Rac1L61R16 is related
to a defective activated state. By contrast, GST–PAK
pulldown assays revealed that Rac1L61R147 binds the
CRIB domain of PAK with the same efficiency as
Rac1L61, suggesting a fully activated conformation
(Fig. 2B). Also consistent with a bona fide active state,
Rac1L61R147 was largely associated with plasma
membrane in HEK293 cells (supplementary Fig. S2)
and was found to activate JNK to the same extent as
Rac1L61 (data not shown); moreover, it was also
found to induce ruffles in COS cells (data not shown).
Three-dimensional structures of Rac1, either isolated
or associated with various molecular partners, indicate
that Lys147 is accessible to solvent, is located far from
the GTP-binding site (Fig. 2C) and is not involved in
contacts with interacting proteins such as effectors
[30], GTPase-activating proteins [31,32], guanine nucle-
otide dissociation inhibitors [33] or guanine nucleotide

exchange factors [34]; these structural data therefore
support our results showing that the K147R mutation
is conservative with respect to Rac1–GTP conforma-
tion and molecular interactions.
Regarding Rac1b, structural analysis has revealed
that the 19 residue insertion at position 75–76 indu-
ces significant changes in the GTP-binding and
effector-binding regions, i.e. an open switch I confor-
mation and a highly mobile switch II, but does not
interfere with the end of the a3-helix, where Lys147 of
Rac1 is located (Fig. 2C) [26].
Altogether, the above results point to Lys147 as a
major site for ubiquitin addition on activated Rac1 and
provide no evidence to suggest that the accessibility of
this site might be hampered by Rac1b-specific inser-
tion; therefore, we looked for other mechanisms that
could explain the defective ubiquitination of Rac1b.
Rac1 ubiquitination is dependent on JNK
activation
It has been shown recently in human umbilical endo-
thelial cells that preventing the interaction of activated
Rac1 with plasma membrane results in abolition of
Rac1L61 ubiquitination [35]; using the nonisoprenyla-
ble-activated Rac1L61G189 mutant, we confirmed that
plasma membrane localization is required for Rac1
ubiquitination to occur in HEK293 cells as well (sup-
plementary Fig. S3). We also observed that Rac1b and
Rac1bL61 are mainly associated with plasma mem-
brane in HEK293 cells (supplementary Fig. S2).
Indeed, it has been previously shown that Rac1b fails

to interact with RhoGDI, resulting in persistent inter-
action with plasma membrane in HT29 and MDCK
cells [27,28]. Therefore, the absence of Rac1b ubiquiti-
nation is unlikely to be related to a defective mem-
brane interaction.
While being persistently activated and associated
with plasma membrane, Rac1b has an impaired ability
to activate several Rac1 downstream signaling path-
ways [27,28]. To evaluate the possible effects of acti-
vating these pathways on Rac1b ubiquitination, we
coexpressed Rac1L61 and Rac1b in HEK293 cells and
analyzed the resulting ubiquitination pattern. Whereas
Rac1L61 ubiquitination was not modified by Rac1b
(data not shown), Rac1b ubiquitination was found to
be partially restored in the presence of Rac1L61
(Fig. 3A, left panel); quantitative analysis of the data
from three experiments demonstrated a 2.7-fold
increase in the relative rate of Rac1b ubiquitination
in the presence of Rac1L61 (Fig. 3A, right panel),
indicating that the stimulation of Rac1-dependent
pathways may, in some way, activate the Rac1 ubiqui-
tination machinery.
As Rac1b has been shown to display reduced capac-
ity to bind POSH [28] and to activate PAK and JNK
[27], we addressed the role of these specific pathways
in regulating Rac1 ubiquitination. Expression of
constitutively activated PAK in HEK293 cells had no
effect on Rac1 ubiquitination (not shown); likewise,
Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al.
390 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS

100% 16%
HA-Rac ubiquitination / HA-Rac expression (A.U)
0%
20%
40%
60%
80%
100%
HA-Rac1L61 HA-Rac1bWT HA-Rac1bL61
HA-Rac mono-expression
HA-Rac coexpressed with
myc-Rac1L61
6His-myc-Ub
HA-Rac1L61
HA-Rac1b
myc-Rac1L61
+
–
–
–
+
+
–
–
+
–
WT
–
+
–

L61
–
+
+
–
+
+
–
WT
+
+
–
L61
+
Cobalt extraction
IB : HA
IB : β-tubulin
IB : myc
IB : HA
–
IB: Rac
IB: P-JNK
IB: JNK
Cobalt extraction
IB: Rac
6His-myc-Ub
myc-Rac1
myc-Rac1b
+
–

–
+
L61
–
+
WT
–
+
–
WT
+
L61
IB: β-tubulin
6His-myc-Ub
HA-Rac1L61
HA-POSH
+
–
–
–
+
–
+
+
–
+
+
+
Cobalt extraction
IB: Rac

IB: Rac
IB: HA
IB: actin
Rac Ubiquitination / Rac Expression (A.U.)
100% 63% 68% 50% 36%
0%
20%
40%
60%
80%
100%
–
–
2 h
10 µ
M
4 h
10 µ
M
2 h
20 µ
M
4 h
20 µ
M
SP600125
treatment
6His-myc-Ub
myc-Rac1L61
SP600125 (µ

M)
incubation (h)
+
–
–
–
+
+
–
–
+
+
10
2
+
+
10
4
+
+
20
2
+
+
20
4
Cobalt Extraction
IB: Rac
IB: Rac
IB: β–tubulin

IB: P–JNK
IB: JNK
A
D
B
C
37%100% 43% 58%
Fig. 3. Rac1 ubiquitination is regulated by JNK. (A) Activation of Rac1 downstream signaling partially restores Rac1b ubiquitination. HEK293
cells were transfected with a combination of expression plasmids of 6His–myc–Ub, HA–Rac1L61, HA–Rac1bWT, HA–Rac1bL61 and myc–
Rac1L61 as indicated. Ubiquitinated proteins were immunoblotted with an antibody to HA. The graph represents ubiquitinated HA–Rac1b lev-
els, normalized to total HA–Rac1b expression, relative to Rac1L61 controls considered as 100%. The data are representative of three inde-
pendent experiments. (B) POSH does not display E3 ligase activity towards Rac1L61. HEK293 cells were transfected with a combination of
expression plasmids of 6His–myc–Ub, HA–Rac1L61 and HA–POSH. Ubiquitinated and total proteins were analyzed as described in Experi-
mental procedures. (C) Unlike Rac1L61, Rac1b does not activate JNK. HEK293 cells were transfected with a combination of expression
plasmids of 6His–myc–Ub, myc–Rac1L61, myc–Rac1WT, myc–Rac1bWT and myc–Rac1bL61 as indicated. Ubiquitinated proteins were
immunoblotted with Rac1-specific antibody. JNK activation level was monitored in total protein extracts by anti-phospho-JNK (P-JNK)
immunoblotting. (D) JNK inhibition decreases Rac1L61 ubiquitination. HEK293 cells were transfected with a combination of expression
plasmids of 6His–myc–Ub and myc–Rac1L61. Twenty hours after transfection, cells were treated with the JNK-specific inhibitor SP600125
at 10 or 20 l
M for 2 or 4 h, and then subjected to the ubiquitination assay. Ubiquitinated proteins were immunoblotted with Rac1-specific
antibody. JNK activation level was monitored in total protein extracts by P-JNK immunoblotting. The graph represents ubiquitinated Rac
levels normalized to total Rac expression relative to the control in the untreated condition, considered as 100%. The data are representative
of two independent experiments.
O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK
FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 391
expression of POSH did not appreciably modify Rac1
ubiquitination (Fig. 3B). Therefore, having confirmed
that, unlike Rac1L61, Rac1b was unable to activate
JNK in HEK293 cells (Fig. 3C), we investigated the
possible regulation of Rac1 ubiquitination by a JNK-

dependent mechanism. Indeed, we observed that, in
response to JNK inhibition by SP600125, ubiquitina-
tion of Rac1L61 was impaired in a dose-dependent and
time-dependent fashion (Fig. 3D), indicating that JNK
activity is a critical regulator of Rac1 ubiquitination.
Discussion
We have shown that, unlike Rac1L61, the constitu-
tively active splice variant Rac1b is poorly ubiquitinat-
ed in HEK293 cells, which results in decreased
sensitivity to proteasomal degradation. This prompted
us to characterize the requirements for Rac1 ubiquiti-
nation that are not fulfilled by Rac1b.
From our mutational analysis of Rac1 lysine resi-
dues, the main ubiquitin addition site in activated Rac1
appears to be Lys147; this result probably explains why
Lys147 was found, in a previous study, among the resi-
dues required for proteasomal degradation of CNF1-
activated Rac1 [36]. Indeed, in Rac2 and Rac3, which
are resistant to proteasomal degradation, Lys147 is
either absent (Rac3), or is in a different environment as
compared to Rac1 (Rac2). Lys147 is not conserved
among Rho GTPases, and in RhoA, lysine residues in
the N-terminal region (Lys6 and Lys7) have been iden-
tified as the acceptors for ubiquitin transferred by
Smurf1 [21]. Although a homologous lysine residue,
Lys5, is present in Rac1, it is apparently not used for
ubiquitin addition. Therefore, the reason why Lys147
rather than Lys5 is the main acceptor site for ubiquitin
in Rac1 remains to be elucidated. Of note, however, is
that recent studies on the role of the Rab-like GTPase

Ypt7 in vacuole fusion in yeast have demonstrated that
this small G-protein undergoes ubiquitination and sub-
sequent degradation, and that Lys140 and Lys147 con-
stitute a major target site for ubiquitin branching in
Ypt7 [37]. Interestingly, when the three-dimensional
structures of Ypt7 and Rac1 are compared (supplemen-
tary Fig. S4), Lys147 has closely similar spatial
situations; this observation raises the hypothesis that
similar mechanisms and homologous E3 ubiquitin
ligases are involved in both cases.
On the basis of structural studies, Lys147 is a
solvent-accessible residue located far from the
GTP-binding site, is not involved in contacts with any
of the interacting proteins described so far, and is not
affected by the 19-residue insertion in Rac1b. There-
fore, the impairment of Rac1b ubiquitination is not
likely to be due to inaccessibility or to a local pertur-
bation of Lys147 (Lys166 in Rac1b), and nor does it
seem to be related to a defective interaction with
plasma membrane, as Rac1b was found to be mostly
associated with plasma membrane in HEK293 cells, as
previously described for other cell types [27,28].
Previous studies have demonstrated that the interac-
tion of Rac1 with downstream effectors is required for
ubiquitination and degradation [14,35,36]. In agree-
ment with this view, we discovered that the rate of
Rac1 ubiquitination critically depends on JNK activa-
tion. This may indicate that JNK-dependent phosphor-
ylation of Rac1 and ⁄ or of its E3 ligase is required to
permit efficient Rac1 ubiquitination. Although there is,

to our knowledge, no reported evidence for JNK-
dependent phosphorylation of Rac1, direct activation
of the ligase by JNK-dependent phosphorylation is
reminiscent of the recently described activation of the
E3 ligase Itch through a phosphorylation-induced con-
formational change [38,39]. An attractive hypothesis is
that Rac1 ubiquitination depends on a similar regula-
tory mechanism, but confirmation of this must await
the identification of Rac1 E3 ligase. To date, the only
candidate Rac1 E3 ligase has been POSH, a specific
partner of activated Rac1 containing a RING finger
motif endowed with ubiquitin ligase activity [40,41];
however, our experiments in HEK293 cells did not
show any effect of POSH on Rac1L61 ubiquitination
and therefore do not support this hypothesis.
Except for a recent report on the role of protea-
some-mediated degradation of Rac1–GTP in hepato-
cyte growth factor-induced epithelial cell scattering,
the physiological significance of UPS-mediated regula-
tion of Rac GTPase has not been documented. Our
results showing that Rac1 ubiquitination critically
depends on JNK activation suggest a novel regulatory
mechanism allowing the cell to counteract excessive or
sustained activation of Rac GTPase.
Another significant outcome of our studies regards
the tumorigenic capacities of the Rac1b variant.
Indeed, Rac1b has been detected in numerous colorec-
tal and breast tumors, and although being expressed at
a low level in tumor cells, Rac1b has proved to be crit-
ical to tumor progression [11,12]. In particular, Rac1b

was shown to mediate matrix metalloproteinase-3-
induced EMT and genomic instability in mammary
epithelial cells; interestingly, in this system, RNAi-
mediated downregulation of Rac1b expression resulted
in inhibition of EMT and cell migration [11]. There-
fore, understanding the molecular basis of the defective
ubiquitination ⁄ degradation of Rac1b appears to be an
important issue. Indeed, our data suggest that the
impairment of Rac1b ubiquitination rests, at least
Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al.
392 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS
partly, on defective JNK activation, and may provide
new ideas for restoring the sensitivity of this tumori-
genic variant to UPS.
Experimental procedures
Cell culture, reagents and transfection
HEK293 cells (ATCC reference: CRL-1573) and HeLa cells
(ATCC reference: CCL-2) were grown in DMEM (Gibco,
Invitrogen, Carlsbad, CA, USA) supplemented with 10%
fetal bovine serum (Gibco, Invitrogen), 100 lg Æ mL
)1
strep-
tomycin, 100 uÆmL
)1
penicillin and 250 ngÆmL
)1
fungizone
(Gibco, Invitrogen), in humidified atmosphere of 5% CO
2
at 37 °C. Cells were treated as indicated with proteasome

inhibitor MG132 (Sigma, St Louis, MO, USA), the protein
synthesis inhibitor cycloheximide (Sigma), and the JNK-
specific inhibitor SP600125 (Sigma). Cells were transiently
transfected using FuGENE 6 Transfection Reagent (Roche,
Basel, Switzerland), following the manufacturer’s proce-
dure.
DNA plasmids and mutagenesis
Human cDNA of Rac1 and mutants Rac1N17 and
Rac1L61, and mouse cDNA of POSH, cloned in pRK5-
myc plasmids, were obtained from A. Hall (University
College London, UK). Hemagglutinin (HA)-tagged POSH
was obtained by subcloning the cDNA into a pcDNA3–
HA plasmid using BamHI and SfiI restriction sites. pXJ–
HA–Rac1L61 and pRBG4–6His–myc–Ub plasmids have
been used previously [14]. Human cDNAs for Rac1b and
Rac1bL61, a gift from P. Jordan (Instituto Nacional de
Sau´ de ‘Dr Ricardo Jorge’, Lisbon, Portugal), were sub-
cloned into pXJ–HA plasmid using BamH1 and Xho1 restric-
tion sites, and into pRK5–myc plasmid using BamH1 and
EcoR1 restriction sites.
All of the point mutations were generated by using the
QuickChange-Site Directed Mutagenesis Kit (Stratagene,
La Jolla, CA, USA), following the manufacturer’s proce-
dure. Each of the 18 lysine mutants of the myc–Rac1L61
sequence and the ubiquitin mutants UbR48 and UbR63
were obtained by replacing the lysine codons AAA and
AAG by the arginine codons AGA and AGG respectively.
Rac1L61G189 was obtained by mutation of the cysteine
codon TGC into the glycine codon GGC. In all cases, the
absence of additional mutations was verified by sequencing

the entire coding region.
Antibodies
The primary antibodies used were the 9E10 mouse mono-
clonal antibody to myc-tag (Roche), 16B12 mouse mono-
clonal antibody to HA-tag (Roche), goat polyclonal
antibody to actin (Santa Cruz, San Diego, CA, USA),
mouse monoclonal antibody to b-tubulin (Upstate, Milli-
pore, Billerica, MA, USA), mouse monoclonal antibody to
Rac (USBiological, Swampscott, MA, USA), rabbit poly-
clonal antibody to SAPK ⁄ JNK (Cell Signaling Technology,
Danvers, MA, USA), and rabbit polyclonal antibody
to phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185; Cell Signaling
Technology). For western blotting, secondary peroxidase-
conjugated rabbit anti-mouse serum (Dako, Glostrup, Den-
mark), secondary peroxidase-conjugated swine anti-rabbit
serum (Dako) or secondary peroxidase-conjugated rabbit
anti-goat serum (Dako) were used. The secondary fluores-
cent antibody used in immunofluorescence studies was
Alexa Fluor 488-labeled goat anti-(mouse IgG) (Molecular
Probes, Eugene, OR, USA).
Western blotting
Proteins were resolved in SDS ⁄ PAGE minigels and electro-
transferred onto nitrocellulose membrane (Schleicher &
Schuell, Millipore). Membranes were probed using the indi-
cated primary antibodies and secondary peroxidase-conju-
gated antibodies followed by chemiluminescence using the
ECL detection system (Amersham, Piscataway, NJ, USA).
Quantification of protein expression and ⁄ or ubiquitination
was performed using imagej software.
Immunofluorescence

HEK293 cells were plated at low confluence on glass
18-mm-diameter poly(l-lysine)-coated (0.1 mgÆmL
)1
; Sigma)
coverslips in 12-well plates, and transfected the next day.
Twenty hours after transfection, cells were fixed in 4%
paraformaldehyde for 20 min, permeabilized with NaCl ⁄ P
i
and 0.2% Triton X-100 for 5 min, and blocked with
NaCl ⁄ P
i
and 1% BSA for 1 h. Cells were then incubated in
the same solution with primary antibody for 1 h, and this
was followed by 1 h of incubation with a fluorescent
secondary antibody. For DNA staining, diaminidophenyl-
indol (0.5 lgÆmL
)1
) was added in the last NaCl ⁄ P
i
wash.
Coverslips were mounted using Vectashield Hardset (Vector
Laboratories, Burlingame, CA, USA). Cell preparations
were observed under a Zeiss (Go
¨
ttingen, Germany)
Axiophot epifluorescence microscope; images were digitally
acquired and processed using Adobe (San Jose, CA, USA)
photoshop 7.0.
Activated Rac1 GTPase pulldown assay
HEK293 cells seeded in 100 mm Petri dishes were transfect-

ed with 5 lg of pRK5–myc–Rac1WT and mutants. The
next day, cells were lysed in 500 lL of lysis buffer [50 mm
HEPES, pH 7.5, 10 mm MgCl
2
, 150 mm NaCl, 1% Tri-
ton X-100, 0.5% NP40, and protease inhibitor cocktail
O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK
FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 393
(Amersham)]. Proteins (500 lg in 150 lL) were incubated
for 2 h at 4 °C with 15 lg of GST or GST–PAK coupled
to 20 lL of glutathione–sepharose beads (Amersham). Pel-
leted beads were washed twice with washing buffer (50 mm
Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl
2
,1mm
dithiothreitol, 0.1% Triton X-100, 0.2 mgÆmL
)1
BSA, and
protease inhibitor cocktail). Bound proteins were recovered
by boiling beads in Laemmli sample buffer 2X (SB2X,
Sigma) and analysed by western blotting.
Ubiquitination assay
HEK293 or HeLa cells seeded in 100 mm Petri dishes were
transfected with 1–1.5 lg of each plasmid encoding 6His–
myc–Ub and various GTPases. Twenty hours after transfec-
tion, cells were washed in NaCl ⁄ P
i
and lysed in 1 mL of
denaturing buffer (8 m urea, 20 mm Tris ⁄ HCl, pH 7.5,
200 mm NaCl, 10 mm imidazole, 0.1% Triton X-100, 5 mm

N-ethylmaleimide, 10 mm iodoacetic acid). Fifty microliters
of lysate were resuspended in SB2X to evaluate the total
quantity of proteins. The 6His–myc-ubiquitinated fraction
of proteins was recovered by incubating the remaining
lysate for 1 h 30 min with 30 lL of cobalt-chelated resin
(BD Talon Metal Affinity resin; BD Bioscience, Lexington,
KY, USA), previously incubated in denaturing buffer with
0.2 mgÆmL
)1
BSA (RIA grade; Sigma). Beads were then
washed four times in 1 mL of denaturing buffer, and resus-
pended in 25 lL of SB2X. Total lysates and ubiquitinated
protein fractions were resolved by SDS ⁄ PAGE and ana-
lyzed by western blotting.
Assay for proteasomal degradation
HEK293 cells seeded in 100 mm Petri dishes were transfect-
ed with 5 lg of the indicated plasmids. In order to obtain
an identical transfection rate, each pool of transfected cells
was trypsinized 8 h after transfection and seeded again into
two poly(l-lysine)-coated wells of 12-well Petri dishes.
Twenty-four hours post-transfection, protein synthesis and
proteasome activity were blocked by adding cycloheximide
(1 lgÆmL
)1
) and MG132 (20 lm). Six hours after treatment,
cells were lysed with SB2X, and the lysates were resolved
by SDS ⁄ PAGE and analyzed by western blotting.
Acknowledgements
We thank Dr Peter Jordan and Dr Maria-Carla Parrini
for plasmids. We also thank Dr Romain Gautier for the

pictures of GTPase structures, and Anne Doye for tech-
nical assistance. This work was supported by grants
from INSERM, CNRS, Universite
´
Paris Descartes, and
Association pour Recherche sur le Cancer and the
Agence Nationale pour la Recherche (ANR 05-MRAR-
033-02) to Ge
´
rard Gacon, and by grants to Emmanuel
Lemichez from the Agence Nationale pour la Recherche
(ANR, A05135AS) and from the Association pour
Recherche sur le Cancer (ARC, 3800).
References
1 Ridley AJ (2006) Rho GTPases and actin dynamics in
membrane protrusions and vesicle trafficking. Trends
Cell Biol 16, 522–529.
2 Etienne-Manneville S & Hall A (2002) Rho GTPases in
cell biology. Nature 420, 629–635.
3 Boettner B & Van Aelst L (2002) The role of Rho
GTPases in disease development. Gene 286, 155–174.
4 Chelly J & Mandel JL (2001) Monogenic causes of
X-linked mental retardation. Nat Rev Genet 2, 669–
680.
5 Boquet P & Lemichez E (2003) Bacterial virulence fac-
tors targeting Rho GTPases: parasitism or symbiosis?
Trends Cell Biol 13, 238–246.
6 Aktories K, Schmidt G & Just I (2000) Rho GTPases
as targets of bacterial protein toxins. Biol Chem 381,
421–426.

7 Sahai E & Marshall CJ (2002) RHO-GTPases and can-
cer. Nat Rev Cancer 2, 133–142.
8 Clark EA, Golub TR, Lander ES & Hynes RO (2000)
Genomic analysis of metastasis reveals an essential role
for RhoC. Nature 406, 532–535.
9 Jordan P, Brazao R, Boavida MG, Gespach C & Chas-
tre E (1999) Cloning of a novel human Rac1b splice
variant with increased expression in colorectal tumors.
Oncogene 18, 6835–6839.
10 Schnelzer A, Prechtel D, Knaus U, Dehne K, Gerhard
M, Graeff H, Harbeck N, Schmitt M & Lengyel E
(2000) Rac1 in human breast cancer: overexpression,
mutation analysis, and characterization of a new iso-
form, Rac1b. Oncogene 19, 3013–3020.
11 Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson
CM, Fata JE, Leake D, Godden EL, Albertson DG,
Nieto MA et al. (2005) Rac1b and reactive oxygen spe-
cies mediate MMP-3-induced EMT and genomic insta-
bility. Nature 436, 123–127.
12 Esufali S, Charames GS, Pethe VV, Buongiorno P &
Bapat B (2007) Activation of tumor-specific splice vari-
ant Rac1b by dishevelled promotes canonical Wnt sig-
naling and decreased adhesion of colorectal cancer cells.
Cancer Res 67, 2469–2479.
13 Takai Y, Sasaki T & Matozaki T (2001) Small GTP-
binding proteins. Physiol Rev 81, 153–208.
14 Doye A, Mettouchi A, Bossis G, Clement R,
Buisson-Touati C, Flatau G, Gagnoux L, Piechaczyk
M, Boquet P & Lemichez E (2002) CNF1 exploits
the ubiquitin–proteasome machinery to restrict Rho

GTPase activation for bacterial host cell invasion.
Cell 111, 553–564.
Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al.
394 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS
15 Lerm M, Pop M, Fritz G, Aktories K & Schmidt G
(2002) Proteasomal degradation of cytotoxic necrotizing
factor 1-activated rac. Infect Immun 70, 4053–4058.
16 Hershko A & Ciechanover A (1998) The ubiquitin sys-
tem. Annu Rev Biochem 67, 425–479.
17 Pickart CM & Fushman D (2004) Polyubiquitin chains:
polymeric protein signals. Curr Opin Chem Biol 8, 610–
616.
18 Ulrich HD (2002) Natural substrates of the proteasome
and their recognition by the ubiquitin system. Curr Top
Microbiol Immunol 268, 137–174.
19 Haglund K & Dikic I (2005) Ubiquitylation and cell
signaling. EMBO J 24, 3353–3359.
20 Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA,
Alexandrova E, Thomsen GH & Wrana JL (2003)
Regulation of cell polarity and protrusion formation by
targeting RhoA for degradation. Science 302, 1775–1779.
21 Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR,
Zhang Y & Wrana JL (2005) Regulation of the polarity
protein Par6 by TGFbeta receptors controls epithelial
cell plasticity. Science 307, 1603–1609.
22 Sahai E, Garcia-Medina R, Pouyssegur J & Vial E
(2007) Smurf1 regulates tumor cell plasticity and motil-
ity through degradation of RhoA leading to localized
inhibition of contractility. J Cell Biol 176, 35–42.
23 Boyer L, Turchi L, Desnues B, Doye A, Ponzio G,

Mege JL, Yamashita M, Zhang YE, Bertoglio J, Flatau
G et al. (2006) CNF1-induced ubiquitylation and pro-
teasome destruction of activated RhoA is impaired in
Smurf1– ⁄ – cells. Mol Biol Cell 17, 2489–2497.
24 Lynch EA, Stall J, Schmidt G, Chavrier P & D’Souza-
Schorey C (2006) Proteasome-mediated degradation of
Rac1–GTP during epithelial cell scattering. Mol Biol
Cell 17, 2236–2242.
25 Kim HT, Kim KP, Lledias F, Kisselev AF, Scaglione
KM, Skowyra D, Gygi SP & Goldberg AL (2007) Cer-
tain pairs of ubiquitin-conjugating enzymes (E2s) and
ubiquitin-protein ligases (E3s) synthesize nondegradable
forked ubiquitin chains containing all possible isopep-
tide linkages. J Biol Chem 282 , 17375–17386.
26 Fiegen D, Haeusler LC, Blumenstein L, Herbrand U,
Dvorsky R, Vetter IR & Ahmadian MR (2004) Alterna-
tive splicing of Rac1 generates Rac1b, a self-activating
GTPase. J Biol Chem 279, 4743–4749.
27 Matos P, Collard JG & Jordan P (2003) Tumor-
related alternatively spliced Rac1b is not regulated by
Rho-GDP dissociation inhibitors and exhibits selective
downstream signaling. J Biol Chem 278, 50442–50448.
28 Singh A, Karnoub AE, Palmby TR, Lengyel E, Sondek
J & Der CJ (2004) Rac1b, a tumor associated, constitu-
tively active Rac1 splice variant, promotes cellular
transformation. Oncogene 23, 9369–9380.
29 Feig LA (1999) Tools of the trade: use of dominant-
inhibitory mutants of Ras-family GTPases. Nat Cell
Biol 1, E25–E27.
30 Lapouge K, Smith SJ, Walker PA, Gamblin SJ, Smer-

don SJ & Rittinger K (2000) Structure of the TPR
domain of p67phox in complex with Rac.GTP. Mol
Cell 6, 899–907.
31 Wurtele M, Renault L, Barbieri JT, Wittinghofer A &
Wolf E (2001) Structure of the ExoS GTPase activating
domain. FEBS Lett 491, 26–29.
32 Stebbins CE & Galan JE (2000) Modulation of host
signaling by a bacterial mimic: structure of the
Salmonella effector SptP bound to Rac1. Mol Cell 6,
1449–1460.
33 Scheffzek K, Stephan I, Jensen ON, Illenberger D &
Gierschik P (2000) The Rac–RhoGDI complex and the
structural basis for the regulation of Rho proteins by
RhoGDI. Nat Struct Biol 7, 122–126.
34 Worthylake DK, Rossman KL & Sondek J (2000)
Crystal structure of Rac1 in complex with the guanine
nucleotide exchange region of Tiam1. Nature 408, 682–
688.
35 Doye A, Boyer L, Mettouchi A & Lemichez E (2006)
Ubiquitin-mediated proteasomal degradation of Rho
proteins by the CNF1 toxin. Methods Enzymol 406,
447–456.
36 Pop M, Aktories K & Schmidt G (2004) Isotype-specific
degradation of Rac activated by the cytotoxic necrotiz-
ing factor 1. J Biol Chem 279, 35840–35848.
37 Kleijnen MF, Kirkpatrick DS & Gygi SP (2007) The
ubiquitin–proteasome system regulates membrane
fusion of yeast vacuoles. EMBO J 26, 275–287.
38 Gallagher E, Gao M, Liu YC & Karin M (2006) Acti-
vation of the E3 ubiquitin ligase Itch through a phos-

phorylation-induced conformational change. Proc Natl
Acad Sci USA 103, 1717–1722.
39 Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu
YC & Karin M (2004) Jun turnover is controlled
through JNK-dependent phosphorylation of the E3
ligase Itch. Science 306, 271–275.
40 Tapon N, Nagata K, Lamarche N & Hall A (1998) A
new rac target POSH is an SH3-containing scaffold
protein involved in the JNK and NF-kappaB signalling
pathways. EMBO J 17, 1395–1404.
41 Xu Z, Kukekov NV & Greene LA (2003) POSH acts as
a scaffold for a multiprotein complex that mediates
JNK activation in apoptosis. EMBO J 22, 252–261.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Ubiquitination of Rac1L61 lysine mutants.
Fig. S2. Subcellular localization of Rac1L61 and
Rac1b in HEK293 cells.
Fig. S3. Mutation of the prenyl acceptor C189 abol-
ishes Rac1 ubiquitination.
O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK
FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 395
Fig. S4. Equivalent position of Lys147 in the struc-
tures of Rac1 (upper panel) and YPT7 (lower panel),
both bound to a GTP analog (GppNHp).
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al.
396 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS