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 d
Trang 1ubiquitinated on Lys 147 through a JNK-regulated process Orane Visvikis1,2, Patrick Lore`s1,2, Laurent Boyer3, Pierre Chardin4, Emmanuel Lemichez3
and Ge´rard Gacon1,2
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: gacon@cochin.inserm.fr
(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.
Trang 2forms 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 degradaubiquitina-tion [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 activUPS-medi-ated 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
Trang 3species (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 lM) 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 lM), as indicated in Experimental procedures.
Trang 4GTPase 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 PAK GST PAK GST PAK
WT L61 L61R16 L61R147
WT L61
input
myc-Rac1 L61R16 L61R147
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 lM), 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 posiinterac-tions 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.
Trang 5activated 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 nuclenucle-otide
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 interacconforma-tions
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,
Trang 6100% 16%
0%
20%
40%
60%
80%
100%
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
100% 63% 68% 50% 36%
0%
20%
40%
60%
80%
100%
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
37%
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 lM 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.
Trang 7expression 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 decreasubiquitinat-ed
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
Trang 8partly, 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 lgÆmL)1
strep-tomycin, 100 uÆmL)1 penicillin and 250 ngÆmL)1 fungizone
(Gibco, Invitrogen), in humidified atmosphere of 5% CO2
at 37C 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
clonal antibody to myc-tag (Roche), 16B12 mouse
mono-clonal antibody to HA-tag (Roche), goat polymono-clonal
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⁄ Pi
and 0.2% Triton X-100 for 5 min, and blocked with NaCl⁄ Piand 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⁄ Pi 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) photoshop7.0
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 MgCl2, 150 mm NaCl, 1% Tri-ton X-100, 0.5% NP40, and protease inhibitor cocktail
Trang 9(Amersham)] Proteins (500 lg in 150 lL) were incubated
for 2 h at 4C 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 MgCl2, 1 mm
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⁄ Pi 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 indicattransfect-ed 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)
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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