Ruk is ubiquitinated but not degraded by the proteasome
Fre
´
de
´
rique Verdier
1
, Taras Valovka
1
, Alexander Zhyvoloup
1,4
, Ludmila B. Drobot
5
, Vladimir Buchman
2,3
,
Mike Waterfield
1
and Ivan Gout
1,4
1
Ludwig Institute for Cancer Research, University College of London Medical School, London, UK;
2
Department of Preclinical
Veterinary Sciences, the University of Edinburgh, UK;
3
Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia;
4
Institute of Molecular Biology and Genetics, Kyiv, Ukraine;
5
Institute of Cell Biology, Lviv, Ukraine

tion; proteasomal degradation.
Ubiquitination of proteins plays a major role in the
regulation of various cellular processes. The best studied
function of ubiquitination is its role in protein degradation,
where polyubiquitinated proteins are recognized by the 26S
proteasome or, in certain cases, by the lysosomes/vacuole
and rapidly degraded. Ubiquitination of target proteins
involves a cascade of reactions catalysed by the E1, E2 and
E3 enzymes. Ubiquitin (Ub) is first activated by an
activating enzyme (E1) to form a high energy thioester
bond between Ub and E1 and is then transferred to a
conjugating enzyme (E2). The Ub protein ligases or E3s are
responsible for specific substrate recognition and for
promoting covalent Ub ligation to the target protein. Thus,
the E3s provide specificity to the Ub system [1,2].
The Cbl proteins form a family of related proteins
harboring several highly conserved domains, such as an
N-terminal variant SH2 domain, a RING finger and a
C-terminal proline-rich domain containing potential tyro-
sine phosphorylation sites. Previous studies have shown that
c-Cbl and Cbl-b
2
function as adaptor proteins by interacting
with other signaling molecules through their various pro-
tein–protein interacting motifs [3].
Biochemical and genetic studies have shown that Cbl
family proteins, including those from Drosophila and
Caenorhabditis elegans, attenuate intracellular signaling
induced by the engagement of cell surface receptors. The
mechanism underlying the negative regulation of activated

´
partement d’He
´
matologie, Institut
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Abbreviations: Ub, ubiquitin; Ruk, regulator of ubiquitous kinase;
PtdIns 3-kinase, phosphoinositol 3-kinase; HA, hemaglutinin
(Received 13 March 2002, revised 14 May 2002,
accepted 30 May 2002)
Eur. J. Biochem. 269, 3402–3408 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03031.x
We found that Ruk L binds to PtdIns-3 kinase via the P85
regulatory subunit of the enzyme and inhibits its catalytic
activity. Furthermore, Ruk L overexpression in primary
neurons induces apoptosis, which could be rescued by
coexpression of constitutively activated forms of PtdIns-3
Kinase or its downstream effector PKB/Akt [11]. In
agreement with these findings, overexpression of SETA
was shown to trigger apoptosis in astrocytes [13]. Specific
associations between SETA and proteins involved in
apoptotic processes, such as AIP1, Alg2, Sb-1 further
confirm its importance in maintaining cellular homeostasis
[13,18]. Cin85 has been also implicated in the regulation of
cytoskeletal architecture since it interacts with p130 Cas and
colocalizes with actin cytoskeleton in epithelial cells [18] and
more recently Ruk L has been involved in the regulation of
receptor-mediated endocytosis [19].
Because Ruk L contains PEST sequences that are usually
found in proteins with short half lives [20], and because it

splicing forms of Ruk (Ruk L, Ruk M and Ruk S) were
amplified by PCR using rat cDNAs as templates. Amplified
cDNA fragments were then cloned into pRc/CMV2 vector
(Invitrogen, Life Technologies) in-frame with the N-ter-
minal EE-tag epitope (MEFMPME). Generated constructs
were verified by restriction enzyme digestion and DNA
sequencing. pcDNA3/Cbl plasmid was a generous gift from
Y. Yarden (The Weizmann Institute of Science, Rehovot,
Israel). Mammalian expression vector encoding Ub–HA
was a gift from D. Bohmann (EMBL, Heidelberg,
Germany).
Cell culturing and transient transfection
Human embryonic kidney cells (HEK293) were cultured at
37 °Cand5%CO
2
in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (Life
Technologies, Inc.), 2 m
ML
-glutamine, 50 UÆmL
)1
penicil-
lin and 50 lgÆmL
)1
streptomycin. Transient transfections
were carried out by using LipofectAMINE according to the
manufacturer’s recommendations (Life Technologies, Inc.).
Twenty-four hours post-transfection cells were treated with
500 l
M

M
EDTA, 5 m
M
EGTA, 20 m
M
NaF, 20 l
M
2-glycero-phosphate, 1 m
M
sodium pyrophosphate, 1 m
M
vanadate, 10% glycerol, 1 m
M
phenylmethanesulfonyl
fluoride and a cocktail of protease inhibitors (Roche) at
4 °C for 20 min. Lysates were cleared by centrifugation for
30 min at 27 000 g and supernatants used for further
experiments. Immunoprecipitating antibodies were incuba-
ted with solubilized cell extracts for 1 h at 4 °C before the
addition of protein G–Sepharose beads, prewashed in lysis
buffer. After a 2-h incubation on the wheel, the beads were
washed three times with lysis buffer and twice with buffer
containing 0.1% Brij 98
6
. Immune complexes were removed
from beads by boiling in Laemmli sample buffer and
separated by SDS/PAGE. Resolved proteins were trans-
ferred onto a poly(vinylidene difluoride) membrane, which
was incubated for 1 h with blocking solution (5% milk in
Tris/NaCl/0.1%Tween) followed by specific antibody over-

binds the E3 ligase c-Cbl [12,18], we decided to investigate
ubiquitination of Ruk isoforms in vivo. In this experiment,
EE-tagged versions of all three isoforms of Ruk were
cotransfected into Hek293 cells together with HA-tagged
Ub. Two days after transfection, Ruk isoforms were
immunoprecipitated using anti-EE antibodies, separated
by electrophoresis and analysed by immunoblotting using
anti-HA Ig. The results presented in Fig. 1B clearly
demonstrate the appearance of multiple high molecular
mass bands detected by anti-HA antibody. No signal was
observed when Hek293 cells were transfected only with
EE-tagged isoforms of Ruk, indicating the specificity of
anti-HA Ig. Reprobing of the membrane with anti-Ruk Ig,
which is specific for all three isoforms, confirmed that equal
amounts of EE-tag fusions were coimmunoprecipitated
from transfected cells (Fig. 1B, lower panel). The appear-
ance of multiple bands on the anti-HA Western blot, clearly
demonstrate that Ruk isoforms are polyubiquitinated.
Specific interaction between Ruk isoforms and the E3
ubiquitin ligase Cbl
Specific interaction between c-Cbl and Cin85/SETA, which
corresponds to the Ruk L isoform, has been recently
reported [12,18]. These findings and the results presented
above prompted us to investigate whether all Ruk isoforms
can interact with Cbl. To assess these interactions,
EE-tagged versions of Ruk L, M or S were cotransfected
into Hek293 cells with c-Cbl or vector alone. Immune
complexes were precipitated with anti-EE Ig and analysed
by Western blotting using anti-Cbl Ig. Figure 2A clearly
demonstrates that all three isoforms of Ruk interact with

blotting of total cell lysates with anti-EE antibodies
(Fig. 3B). These data suggest that Ruk isoforms have the
potential to form homodimers and heterodimers and that
the coiled-coil domain mediates the formation of these
complexes. Moreover, specific interaction between Cbl and
two shorter isoforms of Ruk (Ruk M and Ruk S) was
found to be mediated by heterodimerization with Ruk L.
3404 F. Verdier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Ruk L is ubiquitinated but not degraded
by proteasomes
Ubiquitination, in most cases, targets modified proteins for
degradation by 26S proteasomes. In order to determine
whether ubiquitination of Ruk isoforms would induce their
degradation, a panel of proteosome- and lysosome-specific
inhibitors has been used. It is well established that lactacys-
tine and the peptide aldehyde ALLN inhibit proteasome-
mediated proteolysis, causing an accumulation of proteins
that are usually degraded by this pathway [22]. In contrast
to lactacystine, which is a highly specific proteasomal
inhibitor [23], ALLN inhibits also nonproteasomal
proteases, such as calpains and cathepsins.
Initially, we tested the stability of EE-tagged Ruk L
transiently overexpressed in Hek293 cells. Two days after
transfection, cells were treated with various inhibitors or
with the vehicle alone. Equal amounts of total cell lysates
Fig. 3. Heterodimerization of Ruk L with Ruk M and Ruk S. Hek293 cells were transfected with 1.5 lg of plasmid containing nontagged version of
Ruk L cDNA, and cotransfected with the same quantity of either EE-tagged Ruk L, EE-tagged Ruk M, or EE-tagged Ruk S cDNA. As a control,
Hek293 cells were transfected with an empty vector (NT). Cell lysates were immunoprecipitated with anti-EE antibody. The immunoprecipitates
were analysed by Western Blot using anti-Ruk antibody raised against the last 17 C-terminal amino acids and thereby able to recognize all Ruk
isoforms (Ruk L, EE-tagged Ruk L, EE-tagged Ruk M and EE-tagged Ruk S). Ruk L and EE-tagged Ruk L can be separated by SDS/PAGE,

ALLN or vehicle alone. As shown in Fig. 4B, no accumu-
lation of ubiquitinated EE-Ruk L is detected in the presence
of ALLN.
We then measured the half-life of endogenous Ruk L,
which is expressed at high level in Hek293 cells. In most
cases, the half-life of ubiquitinated proteins is short due to
their rapid degradation by the proteasome. Time-course
treatment of cells with a protein synthesis inhibitor cyclo-
heximide showed no variations in the level of endogenous
Ruk L, suggesting a long half-life for the protein (Fig. 4C).
Furthermore, we examined the effect of selected inhibitors
of protein degradation on the level of endogenous Ruk L.
As can be seen in Fig. 4D, neither ALLN nor lactacystine
induce any accumulation of endogenous Ruk L in Hek293
cells, even 6 h after treatment. These results were also
confirmed in human monocytic cell line U937 (data not
shown). To verify that proteolytic activities of proteasomes
were effectively inhibited by the use of indicated inhibitors,
we checked the expression level of b-catenin, which is
degraded via the Ub-proteasome pathway [24,25].
Re-probing of the membrane with anti-(b-catenin) Ig clearly
demonstrated the accumulation of ubiquitinated forms of
b-catenin upon ALLN and lactacystine treatment (Fig. 4D,
lower panel). No effect on the stability of Ruk L protein was
also observed when cells were treated with lysosomal
inhibitors: NH
4
Cl or chloroquine
7
(Fig. 4D). Taken together,

M
Cycloheximide (CHX) for the times indicated. As described in (A), 30 lg of proteins from the total cell lysates was separated by SDS/PAGE
electrophoresis, and the expression level of endogenous Ruk L determined by Western blot (WB) using anti-Ruk Ig (upper panel). The membrane
wasreprobedwithanti-(b-actin) Ig to confirm equal loading of protein in each lane (lower panel). (D) Hek293 cells were incubated for the time
indicated with various inhibitors or with vehicle alone as described for panel A. Cells were lysed and the quantity of proteins measured by Bradford
assay. 30 lg of proteins from the total cell lysates was separated by SDS/PAGE electrophoresis. The expression level of endogenous Ruk L was
determined by Western blot analysis using anti-Ruk Ig (upper panel). The membrane was reprobed with anti-(b-catenin) Ig (bottom panel). The
position of polyubiquitinated b-catenin species [(Ub)n-b-catenin] are indicated. The arrowheads mark the locations of unmodified b-catenin.
3406 F. Verdier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
proteins. Many ubiquitinated proteins are targeted for
degradation by 26S proteasomes, but some undergo
endocytosis, leading to proteolysis in the lysosome. New
findings show that ubiquitination is not always associated
with the degradation of modified proteins, but could be also
involved in regulation of enzymatic activities and intranu-
clear trafficking of tagged proteins [26].
The data presented in this study clearly demonstrate that
a recently identified adaptor-type protein Ruk L, also
known as Cin85 or SETA, is ubiquitinated in vivo.
Furthermore, we showed that shorter splicing variants of
Ruk, termed Ruk M and Ruk S, are also modified by
covalent attachment of ubiquitin. Ubiquitination of all three
isoforms of Ruk indicates that ubiquitin conjugation occurs
at the C-terminal region, which is common between them. It
is well established that ubiquitin is conjugated to target
proteins through lysine residues. Sequence analysis of Ruk
isoforms showed that their common C-terminal region
contains numerous lysine residues, which could be potential
sites for ubiquitination. The identification and characteri-
zation of Ruk ubiquitination sites is currently in progress.

under investigation.
The discovery of Ruk isoform ubiquitination in cells
prompted us to investigate the importance of this modifi-
cation, especially in regulating its stability. Using proteo-
somal and lysosomal inhibitors, we found that
ubiquitination of exogenously expressed and native Ruk L
does not induce its degradation via these proteolytic
pathways. If ubiquitination of Ruk L is not a signal for
its degradation, what is the role of this post-translational
modification?
Proteolysis-independent regulation by ubiquitination has
recently been reported in several systems. Ubiquitination of
a number of cell surface receptors in response to ligand
binding serves as an internalization signal [28]. Moreover,
ubiquitination-dependent processing of precursor proteins
[29] and the regulation of multienzyme complex formation
have also been described [30]. An interesting paper by Fang
et al. demonstrates that like Ruk L, the P85 regulatory
subunit of PtdIns-3 kinase is ubiquitinated, but not
degraded by the proteasome pathway [31,32]. In addition,
Cbl was shown to be the E3 ligase responsible for P85
ubiquitination and to regulate the recruitment of PtdIns-3
kinase to CD28 and T cell antigen receptor complexes,
thereby inhibiting PtdIns-3 kinase activation.
Two contradictory mechanisms have been proposed for
Cbl binding to the P85 subunit: one involves the proline-rich
domain of Cbl and the SH3 domain of P85 and while the
other implicates a phosphorylated C-terminal tyrosine
(Y731) of Cbl and an undefined domain of P85 [32,33].
We previously reported specific association between Ruk L

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