Báo cáo khoa học: The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain pdf

The E3 ubiquitin ligase Itch regulates sorting nexin 9
through an unconventional substrate recognition domain
Claudia Baumann, Cecilia K. Lindholm*, Donata Rimoldi and Fre
´
de
´
ric Le
´
vy

Ludwig Institute for Cancer Research Ltd, University of Lausanne, Switzerland
Introduction
Ubiquitin (Ub) ligases play a crucial role in regulating
intracellular protein levels in eukaryotes. The reaction
catalysed by Ub ligase activity, ubiquitylation, results
in a reversible covalent modification of substrate
proteins and constitutes a signal for proteasomal and
lysosomal degradation. This is an important mecha-
Keywords
E3 ligase; Itch; protein–protein interaction;
sorting nexin 9; ubiquitin
Correspondence
C. Baumann, Ludwig Institute for Cancer
Research Ltd, Chemin des Boveresses 155,
CH-1066 Epalinges, Switzerland
Fax: +41 21 692 59 95
Tel: +41 21 692 59 77
E-mail:
Present address
*Philip Morris International SA, Neucha
ˆ

tel,
Switzerland

Debiopharm SA, Lausanne, Switzerland
(Received 9 January 2010, revised 20 April
2010, accepted 27 April 2010)
doi:10.1111/j.1742-4658.2010.07698.x
The level of intracellular proteins is mainly regulated through modifications
by ubiquitin ligases that target them for degradation. Members of the
NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting
protein 4), possess up to four WW domains for specific association with
PY motif-containing substrates. We have identified sorting nexin 9 (SNX9),
a protein involved in endocytic processes, as a new substrate of Itch. Itch
ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using trun-
cated proteins, we found that the interaction with SNX9 is mediated by the
proline-rich domain (PRD) of Itch, a domain distinct from the conven-
tional WW recognition domain, and the SH3 domain of SNX9. Interaction
with the PRD of Itch is essential for SNX9 ubiquitylation and degradation.
Furthermore, this effect is specific for Itch, as NEDD4, a related PRD-
containing E3 ligase, does not bind SNX9. SNX18, a second member of
the SNX family containing an SH3 domain, was also found to bind to
Itch. Our results indicate that the pool of substrates of NEDD4 family E3
ubiquitin ligases extends beyond proteins containing PY motifs.
Structured digital abstract
l
MINT-7889719: SNX18 (uniprotkb:Q96RF0) physically interacts (MI:0915) with ITCH (uni-
protkb:
Q96J02)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7889571, MINT-7889619: ITCH (uniprotkb:Q96J02) physically interacts (MI:0915)with

SNX9 (uniprotkb:
Q9Y5X1)bypull down (MI:0096)
l
MINT-7889653: Melan-A (uniprotkb:Q16655) physically interacts (MI:0914)withNEDD4
(uniprotkb:
P46934) and ITCH (uniprotkb:Q96J02)bypull down (MI:0096)
l
MINT-7889591, MINT-78896 73, MINT-7890033 : SNX9 (uniprotkb:Q9Y5X1) physically
interacts (
MI:0915)withITCH (uniprotkb:Q96J02)byanti tag coimmunoprecipitation (MI:
0007)
l
MINT-7889689: SNX 9 (uniprot kb:Q9 Y5X1 ) physically i nteracts (MI:0914)withITCH (uniprotkb:
Q96J02) and Ubiquitin (uniprotkb:P62988)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7889928: Ub (uniprotkb:P62988) physically interacts (MI:0915) with SNX9 (uniprotkb:
Q9Y5X1)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7889610: SNX9 (uniprotkb:Q9Y5X1) physically interacts (MI:0915) with ITCH (uni-
protkb:
Q96J02)byanti bait coimmunoprecipitation (MI:0006)
Abbreviations
GST, glutathione S-transferase; HA, hemagglutinin; PRD, proline-rich domain; SH3, Src homology 3 domain; SNX, sorting nexin;
Ub, ubiquitin.
FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2803
nism to terminate protein activity and thereby control
numerous cellular processes. The specific selection of
substrates targeted for ubiquitylation is determined by
E3 Ub ligases (EC 6.3.2.19).
Itch, or atrophin-1 interacting protein 4 (AIP4, here-

after referred to as Itch) (UniProtKB accession num-
ber Q96J02), is a member of the NEDD4 family of E3
Ub ligases, and Itch knockout mice display severe
immunological disorders. All NEDD4 family members
have a C-terminal HECT (homologous to E6-AP
C-terminus) domain catalysing the direct transfer of
Ub onto substrates. In addition, they have an N-terminal
C2 domain for phospholipid ⁄ membrane association
and multiple WW domains for protein–protein interac-
tions. The latter are members of the group I WW
domains with specificity for ligands containing a Pro-
Pro-X-Tyr consensus sequence, the so-called PY motif
[1]. Many substrate proteins of Itch have been
identified (e.g. c-Jun, JunB, ErbB4, CXC-chemokine
receptor 4) [2]. We have previously shown that, in
melanoma cells, Itch is involved in lysosomal degrada-
tion of the melanosomal protein Melan-A (UniProtKB
accession number Q16655) [3]. Substrates of Itch
generally contain PY motifs that associate with one of
the four WW domains of the ligase, and ubiquitylation
usually targets them for degradation. Unlike other
NEDD4 family members, Itch and NEDD4 (Uni-
ProtKB accession number P46934) also possess a short
proline-rich domain (PRD).
Sorting nexin 9 (SNX9) (UniProtKB accession num-
ber Q9Y5X1) belongs to a large family of proteins
involved in endocytosis and intracellular trafficking
[4,5]. Sorting nexins are characterized by the presence
of a phosphoinositide-binding PX domain that mediates
interactions with cellular membranes. SNX9, originally

identified through its association with metalloproteas-
es [6], has since been shown to participate in clathrin-
mediated endocytosis of cell-surface receptors such as
the transferrin receptor [7]. In addition to the PX mem-
brane binding module, SNX9 has a C-terminal Bin ⁄
amphiphysin ⁄ Rvs (BAR) domain and an N-terminal
Src homology 3 (SH3) domain. The BAR domain facili-
tates homodimerization of SNX9, sensing of membrane
curvature and tubulation of membranes [8]. The SH3
domain mediates protein–protein interactions by bind-
ing to proline-rich regions present in interacting
proteins. Many interaction partners of the SH3 domain
of SNX9 have been described [7]. It has also been
shown that SNX9 undergoes tyrosine phosphorylation,
and that this modification can modulate binding of
SNX9 to other proteins [9–11]. Due to its important
role in endocytosis, intracellular SNX9 levels are
expected to be tightly regulated.
Here we identify SNX9 as a new substrate of the E3
Ub ligase Itch. We show that Itch ubiquitylates and
regulates the level of SNX9. Unlike most substrates
studied previously, we found that binding of SNX9 to
Itch occurs through the SH3 domain of SNX9 and the
PRD of Itch. We demonstrate that the PRD of Itch is
essential for SNX9 binding, ubiquitylation and degra-
dation. Taken together, these results extend the pool
of substrates of NEDD4 family E3 ligases to proteins
containing SH3 domains.
Results
Itch interacts with sorting nexin 9

We previously showed that, in melanoma cells, the Ub
ligase Itch is involved in lysosomal degradation of
the melanosomal protein Melan-A [3]. To identify addi-
tional substrates of Itch in melanoma cells, we per-
formed pulldown experiments by incubating an
immobilized GST–Itch fusion protein with cell extracts
from pigmented SK-MEL-23 melanoma cells. The
recovered associated proteins were identified by mass
spectrometry. The Itch sequence used in this experiment
(GST–Itch–PWW) contained the proline-rich domain
and four WW protein–protein interaction domains, but
lacked the phospholipid-interacting C2 domain and the
catalytic HECT domain (Fig. 1). SNX9 was identified
among the proteins that were found to associate with
GST–Itch–PWW but not with GST alone. SNX9 is a
Fig. 1. Schematic representations of the constructs used to identify
proteins associated with Itch and to analyse interactions between
Itch and SNX9. The various domains and their localization within
the proteins are indicated. BAR, Bin ⁄ amphiphysin ⁄ Rvs domain; C2,
Ca
2+
-dependent phospholipid binding domain; GST, glutathione
S-transferase; HECT, homologous to E6-AP C-terminus domain;
myc, myc tag; PRD, proline-rich domain; PX, phosphoinositide bind-
ing domain; SH3, Src homology 3 domain; WW, WW domain.
The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al.
2804 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS
cytosolic protein that contains an SH3 domain, a PX
domain and a homodimerization BAR domain. In mass
spectrometric analysis, seven unique peptides matching

the SNX9 protein sequence were detected, covering
about 17% of the entire SNX9 sequence. To confirm
the interaction between SNX9 and Itch, we repeated
the pulldown experiment using GST–Itch–PWW and
extracts of HEK293 cells expressing vesicular stomatitis
virus-G epitope (VSV)-tagged SNX9. As shown in Fig.
2A, a band of approximately 75 kDa reacting with anti-
body against the VSV tag was detectable in the lane
containing material immunoprecipitated with GST–Itch–
PWW. In parallel, an aliquot of the same cell extract was
analysed directly with the antibody against the VSV tag.
Having identified SNX9 in GST–Itch–PWW pull-
down experiments, we confirmed the interaction in
cells. We first analysed this interaction in cells exoge-
nously expressing the proteins. HEK293 cells were
chosen for this and other over-expression experiments
as SK-MEL-23 melanoma cells have a very low trans-
fection efficiency. In HEK293 cells co-transfected with
VSV-tagged SNX9 and HA-tagged Itch, Itch was
found to be associated with SNX9 (Fig. 2B). The inter-
action of Itch with SNX9 was further validated in
SK-MEL-23 melanoma cells by co-immunoprecipita-
tion of endogenous Itch and SNX9 proteins (Fig. 2C).
Itch ubiquitylates and regulates the level of SNX9
Next we tested whether SNX9 was ubiquitylated by
Itch. Plasmids encoding SNX9 and HA-tagged ubiqu-
itin (Ub
HA
) were transiently transfected into HEK293
cells. The total pool of ubiquitylated proteins was

immunoprecipitated with an antibody against the HA
tag and analysed by Western blot with antibody
against SNX9. In cells expressing SNX9 and Ub
HA
,
several bands corresponding to ubiquitylated SNX9
were detected (Fig. 3A). We confirmed that Itch
directly ubiquitylated SNX9 by performing an in vitro
ubiquitylation assay. Immobilized GST–SNX9 fusion
protein was incubated with biotinylated Ub and a pro-
teasome-depleted lysate of HEK293 cells transfected
either with a vector encoding full-length Itch or with
an empty vector. GST–SNX9 was eluted and re-immu-
noprecipitated with antibody against SNX9. Ubiquity-
lated GST–SNX9 was detected by immunoblotting
with horseradish peroxidase-conjugated streptavidin.
In the presence of over-expressed Itch, a smear of
high-molecular-mass proteins was observed, indicating
ubiquitylated forms of SNX9 (Fig. 3B). Prolonged
exposure of the membrane revealed a similar smear in
the sample containing a lysate of control-transfected
cells, probably due to endogenous Itch protein (data
not shown). No evidence of ubiquitylation was found
with GST alone. Note that analysis of SNX9 ubiquity-
lation in cells showed a few discrete bands (Fig. 3A),
while a smear of ubiquitylated SNX9 was detected in
the in vitro assay (Fig. 3B). This may be due to the
fact that, in the latter case, a vast excess of ubiquitin
was present and proteasomes were absent. The absence
of highly ubiquitylated SNX9 in cells may also be due

to factors regulating SNX9 ubiquitylation in a cellular
A
C
B
Fig. 2. Itch interacts with sorting nexin 9 (SNX9). (A) Extracts from HEK293 cells transfected with VSV-tagged SNX9 were incubated with
immobilized GST or GST–Itch–PWW. Bound material was resolved by SDS ⁄ PAGE and analysed by immunoblotting using antibodies against
the VSV tag. Cell extracts were analysed in parallel and correspond to 10% of the eluted material. (B) Digitonin-soluble extracts were
prepared from HEK293 cells transfected with HA-tagged Itch, VSV-tagged SNX9 or both. Extracts were immunoprecipitated (IP) using anti-
bodies against the VSV tag, resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibodies against the HA tag or against the
VSV tag as indicated. Cell extracts were analysed in parallel. (C) Digitonin-soluble extracts from SK-MEL-23 melanoma cells were immuno-
precipitated using polyclonal antibodies against SNX9 or control polyclonal antibodies against CD2AP (Ctrl Ab). Proteins were analysed by
immunoblotting with monoclonal antibody against Itch or polyclonal antibodies against SNX9. Cell extracts were analysed in parallel.
C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9
FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2805
context, such as co-factors and deubiquitylating
enzymes.
To investigate whether ubiquitylation of SNX9 led
to its degradation, we analysed the impact of modulat-
ing Itch levels on intracellular levels of SNX9. We
transduced pigmented SK-MEL-23 melanoma cells
with a recombinant lentivector delivering a short hair-
pin RNA sequence that efficiently silenced Itch expres-
sion. Itch silencing caused an increase in the level of
endogenous SNX9 protein (Fig. 3C). Quantitative
analysis indicated that the level of SNX9 increased
approximately twofold (2.1 ± 0.4, mean ± standard
deviation from three independent experiments) com-
pared to cells transduced with control lentivector.
Consistent with this observation, over-expression of
Itch in HEK293 cells decreased the level of SNX9

(Fig. 3D).
To demonstrate that Itch regulates the degradation
of SNX9, we performed pulse–chase experiments using
HEK293 cells over-expressing VSV-tagged SNX9 alone
or in combination with Itch. We analysed SNX9
degradation for 24 h. In the absence of exogenous
Itch, SNX9 appeared to be a very stable protein, with
a half-life of approximately 24 h (Fig. 3E). The half-
life of SNX9 was reduced in the presence of over-
expressed Itch, such that the level of labelled SNX9
protein has decreased to approximately 20% of its
initial amount after 24 h (Fig. 3E).
Altogether, these results show that the Ub ligase Itch
binds to and ubiquitylates SNX9, directly affecting
AB
CD
E
Fig. 3. Itch ubiquitylates SNX9 and targets it for degradation. (A) HEK293 cells transfected with plasmids encoding HA-tagged ubiquitin
(Ub
HA
), SNX9 or both were treated with lactacystin before lysis. Proteins immunoprecipitated (IP) with monoclonal antibody against the HA
tag and cell extracts were analysed by immunoblotting using polyclonal antibodies against SNX9. (B) In vitro ubiquitylation assay. GST–SNX9
or GST immobilized on glutathione beads were incubated with cytosolic extracts from HEK293 cells, supplemented with 2 lg biotinylated
ubiquitin (Ub-biotin) and ATP. HEK293 cells were transfected with Itch (+) or empty vector ()), as indicated. After the indicated time, the
reaction was stopped by boiling the beads for 5 min in 2% SDS. SNX9 was immunoprecipitated and analysed by immunoblotting using
horseradish peroxidase-conjugated streptavidin to reveal SNX9-bound Ub-biotin. (C) Extracts from SK-MEL-23 cells transduced with lentivec-
tors encoding short hairpin RNA specific for Itch (rec. lv ⁄ siItch) or an irrelevant target gene (rec. lv ⁄ siControl) were resolved by SDS ⁄ PAGE
and analysed by immunoblotting with the indicated antibodies. (D) Extracts from HEK293 cells expressing SNX9 alone or together with Itch
were analysed by immunoblotting with antibodies against SNX9 and Itch. Note that, in (C) and (D), the level of SNX9 is inversely proportional
to the level of Itch. (E) HEK293 cells transfected with VSV-tagged SNX9 or a combination of VSV-tagged SNX9 and myc-tagged Itch were

incubated for 30 min in medium containing
35
S-methionine ⁄ cysteine to label newly synthesized proteins (pulse). After labelling, cells were
incubated in regular medium for the indicated times (chase). Extracts were prepared and immunoprecipitated using antibody against the VSV
tag. Eluted material was resolved by SDS ⁄ PAGE. The left panel shows autoradiography of the gel. Immunoprecipitated radioactivity was
quantified using a phosphorimager and the radioactivity remaining was determined relative to time 0 (right panel).
The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al.
2806 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS
the intracellular level of the latter by promoting its
degradation.
The SH3 domain of SNX9 binds to the
proline-rich domain of Itch
Given that substrates of Itch and NEDD4 family E3
ligases generally contain PY motifs, we screened the
sequence of SNX9 for such motif but failed to detect
one. Previous studies have shown that Itch binds to the
SH3 domains of endophilin A1 and beta-p21-activated
kinase-interactive exchange factor (bPIX) via its PRD
[12,13]. We therefore produced a GST fusion product
that contained only the SH3 domain of SNX9 (Fig. 1)
and tested whether this truncated version of SNX9 was
able to interact with Itch. As shown in Fig. 4A, immo-
bilized GST–SNX9(SH3) pulled down Itch from cellu-
lar extracts of SK-MEL-23. These results indicate that
the SH3 domain of SNX9 mediates the interaction with
Itch. It is interesting to note that SNX9(SH3) associated
with Itch but not with NEDD4 (Fig. 4A). NEDD4 is a
close functional homologue of Itch that shares over
45% amino acid identity but diverges in the sequence of
its proline-rich segment. Under the same experimental

conditions, the cytoplasmic tail of the melanosomal
protein Melan-A associated with both Itch and
NEDD4, as previously shown [3].
Having found that the interaction was mediated by
the SH3 domain of SNX9, we tested whether SNX9
binds to the PRD of Itch. We co-transfected myc-
tagged full-length Itch protein or Itch lacking the PRD
(myc-tagged DPRD-Itch) together with VSV-tagged
SNX9 into HEK293 cells (Fig. 4B). Association
between SNX9 and full-length Itch was readily
observed, but the interaction between SNX9 and
DPRD-Itch was drastically reduced, confirming that
SNX9 binds to the PRD of Itch. Next, we investigated
whether ubiquitylation of SNX9 depends on its interac-
tion with the PRD of Itch. We therefore co-transfected
either full-length Itch or DPRD-Itch protein together
C
AB
D
Fig. 4. SNX9 interacts via its SH3 domain with the proline-rich domain of Itch. (A) Extracts from SK-MEL-23 cells were incubated with
immobilized GST, GST–Itch–PWW, GST–SNX9(SH3) containing only the SH3 domain of SNX9 or GST–Melan-A protein. The GST fusion con-
structs used are shown in Fig. 1. Proteins that bound to the beads were analysed by immunoblotting with monoclonal antibody against Itch
or polyclonal antibodies against NEDD4. Note that SNX9(SH3) does not pulldown NEDD4, a close functional homologue of Itch. The known
interaction of Melan-A with Itch and NEDD4 was used as a positive control, and the lack of interaction of GST–Itch–PWW with either protein
as a negative control. (B) Extracts were prepared from HEK293 cells co-transfected with VSV-tagged SNX9, myc-tagged Itch and myc-tagged
DPRD-Itch (lacking the proline-rich domain) in the indicated combinations. Extracts were immunoprecipitated using antibody against the myc
tag, resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibody against the VSV tag or the myc tag as indicated. Cell extracts
were analysed in parallel. (C) Extracts were prepared from HEK293 cells co-transfected with VSV-tagged SNX9, myc-tagged Itch and myc-
tagged DPRD-Itch (lacking the PRD) in the indicated combinations. Extracts were immunoprecipitated using antibody against the VSV tag,
resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibodies against the myc tag, ubiquitin or the VSV tag as indicated. Cell

extracts were analysed in parallel. (D) Extracts from HEK293 cells transfected with myc-tagged Itch or myc-tagged DPRD-Itch were analysed
by immunoblotting with antibodies against SNX9, actin or the myc tag as indicated.
C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9
FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2807
with VSV-tagged SNX9 into HEK293 cells. SNX9 was
immunoprecipitated and analysed by Western blotting
using an antibody to ubiquitin. Ubiquitylated forms of
VSV-tagged SNX9 were easily detected upon co-trans-
fection of SNX9 with full-length Itch, but not with the
deletion variant lacking the PRD (Fig. 4C). In agree-
ment with this finding, over-expression of full-length
Itch in HEK293 cells decreased the level of endogenous
SNX9, but over-expressing Itch protein lacking the
PRD had no impact on SNX9 levels (Fig. 4D). The
level of endogenous SNX9 in cells over-expressing
full-length Itch was about one-third of the amount in
non-transfected control cells.
Note that, in the analysis of cellular extracts in
Fig. 4B,C, co-expression of Itch and SNX9 did not
decrease the level of SNX9 compared to expression of
SNX9 alone, in contrast to results with endogenous
SNX9 shown in Figs 2B and 3D. This apparent
discrepancy was due to variability in co-transfection
efficiency (not shown).
Together, our results show that binding of SNX9 to
Itch is mediated by the SH3 domain of SNX9 and the
PRD of Itch. Furthermore, interaction with the PRD
of Itch is essential for ubiquitylation of SNX9 and its
intracellular regulation.
Phosphorylated SNX9 binds Itch

It has been reported that SNX9 can be phosphorylated
at tyrosine residues by ACK2 (activated Cdc42-associ-
ated tyrosine kinase 2) during membrane recruitment
[9,14]. Furthermore, in Drosophila, ACK was found to
phosphorylate Tyr56 within the SH3 domain of SNX9,
abrogating as a result the binding to proline-rich
sequences [10].
To examine whether tyrosine phosphorylation of the
human SNX9 protein had the same impact on interac-
tions with PRD-containing proteins as in Drosophila,we
investigated whether Itch interacts with tyrosine-phos-
phorylated SNX9. HEK293 cells were co-transfected
with plasmids encoding Itch and SNX9. Itch was immu-
noprecipitated from lysates, and SNX9 protein bound
to Itch was eluted and re-immunoprecipitated to analyse
its tyrosine phosphorylation status (Fig. 5). Phosphory-
lated SNX9 protein was detected, demonstrating that
tyrosine phosphorylated SNX9 still bound to Itch. As a
control, tyrosine phosphorylation of SNX9 immunopre-
cipitated directly from cell extracts was confirmed.
Itch interacts with SNX18
SNX18 (UniProtKB accession number Q96RF0) is the
closest relative of SNX9, with a very similar domain
structure, including an N-terminal SH3 domain. Little
is known about the function of SNX18. It has recently
been reported to induce membrane tubulation in AP-1-
positive endosomal trafficking [15]. In addition, like
SNX9, the SH3 domain of SNX18 can bind to dynam-
in-2 [15]. Given the similarity between SNX9 and
SNX18, we analysed the potential interaction of

SNX18 with the Ub ligase Itch by co-transfection of
myc-tagged SNX18 and HA-tagged Itch into HEK293
cells (Fig. 6). After immunoprecipitation of myc-tagged
SNX18, bound proteins were eluted and subjected to
Western blotting. The membrane was probed using an
antibody against Itch. SNX18 was found to interact
with over-expressed as well as endogenous Itch pro-
tein, suggesting that it may be regulated in a manner
similar to SNX9.
Discussion
In this study, we found that the sorting nexin SNX9 is
regulated by the E3 Ub ligase Itch. We showed that
the interaction is mediated by the SH3 domain of
SNX9 and the PRD of Itch, leading to ubiquitylation
Fig. 5. Tyrosine-phosphorylated SNX9 binds to Itch. Extracts were
prepared from HEK293 cells transfected with VSV-tagged SNX9,
myc-tagged Itch or both, using lysis buffer supplemented with phos-
phatase inhibitors. Extracts were immunoprecipitated using antibod-
ies against the VSV tag or the myc tag. Proteins eluted from beads
coupled to antibody against the myc tag were re-immunoprecipitated
using antibody against the VSV tag. Eluted proteins were resolved by
SDS ⁄ PAGE and subjected to immunoblotting with antibodies against
phospho-tyrosine (4G10), the VSV tag or the myc tag as indicated.
A fraction (75%) of eluted material from immunoprecipitated anti-
myc + anti-VSV was analysed using 4G10 antibody, and 10% of
eluted material was used for detection with antibody against the VSV
tag. From material immunoprecipitated using antibody against the
VSV tag, a fraction (50%) of eluted material was analysed with 4G10
antibody, and 10% of eluted material was used for detection with
antibody against the VSV tag. Cell extracts were analysed in parallel.

The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al.
2808 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS
and degradation of SNX9. Previous studies revealed
that the Itch substrate endophilin A1 also associates
via its SH3 domain with the PRD of the ligase [12].
Thus our study represents the second characterization
of an interaction between a substrate and a ubiquitin
ligase of the NEDD4 family that is mediated by a
sequence distinct from the conventional WW domains.
Importantly, while the SH3 ⁄ PRD interaction was
found to facilitate endophilin ubiquitylation in the case
of endophilin A1 [12], the interaction mediated by the
PRD of Itch appears to be essential for ubiquitylation
and degradation of SNX9. The ability of the PRD to
function as a substrate recognition domain in Itch
indicates that the pool of Itch substrates is not limited
to PY motif-containing proteins.
We showed that the interaction of SNX9 with Itch
is specific, as SNX9 does not bind to the PRD of the
related Ub ligase NEDD4. NEDD4 and Itch can inter-
act with the same PY motif-containing substrate pro-
tein, such as Melan-A [3], but the two ligases diverge
in the sequence of their proline-rich segments. Two
types of proline-rich binding motifs for SH3 domains
have been described, so-called class I and class II sites,
with the consensus sequences Arg-X-X-Pro-X-X-Pro
and Pro-X-X-Pro-X-Arg (X being any amino acid),
respectively [16,17]. It has been shown that the SH3
domain of SNX9 binds most strongly to the class I
ligand motif Arg-X-Ala ⁄ Pro-Pro-X-X-Pro [15,18].

Class I binding motifs are present in the PRDs of both
Itch and NEDD4. However, the amino acid context of
their binding sites is different, and may affect binding
to SH3 domains. Itch has a single discrete class I site
with the sequence Arg-Pro-Pro-Pro-Pro-Tyr-Pro, con-
forming to the preferred consensus SH3 ligand motif
of SNX9 previously reported [18]. In contrast, the
PRD of NEDD4 contains two class I sites that overlap
and may thus form a structure that cannot interact
with the SH3 domain of SNX9. In support of this
hypothesis, other SH3 domain-binding partners of
SNX9 such as dynamin-2 also present discrete class I
sites within their PRDs that mediate binding to SNX9
[15]. In addition to endophilin A1, another SH3
domain-containing protein, bPIX, has been found
to interact with the PRD of Itch [12,13]. The complex
of bPIX and Itch serves as a scaffold in G protein-
coupled receptor signalling [13]. Binding of these two
proteins to the PRD of NEDD4 has not been investi-
gated, and it is therefore not known whether the inter-
action can be mediated by either class I motif present
in Itch and NEDD4. It is likely that there are many as
yet unidentified proteins regulated by the NEDD4
family ligases Itch or NEDD4 through interactions
with their PRDs.
We found that SNX18, a close relative of SNX9 with
a similar domain structure, also interacts with Itch.
Little is known about the function of SNX18 [15,19]
and its regulation. SNX9 and SNX18 show more than
40% identity in the amino acid sequence of their SH3

domains, which share a preference for binding to
class I ligand motifs and dynamin-2 as a major inter-
acting protein [15]. It is therefore conceivable that
SNX18 is regulated through ubiquitylation by Itch in a
manner similar to SNX9. SNX33 is the third and final
member of the SNX family identified to date that
possesses an SH3 domain. It is closely related to SNX9
and SNX18, and has also been found to associate
with dynamin [20]. It will be interesting to determine
whether SNX18 and SNX33 are also substrates of Itch.
The SH3 domain of SNX9 is essential for the func-
tion of SNX9 in clathrin-mediated endocytosis. This
domain was shown to mediate the interaction of
SNX9 with dynamin-2 and N-WASP, a homologue of
the Wiskott–Aldrich syndrome protein (WASP)
[21–24]. Given that the Ub ligase Itch binds to the
SH3 domain of SNX9, one could speculate that Itch
competes for binding to the SH3 domain with other
interaction partners. Dynamin-2 and N-WASP are key
components of the actin polymerization machinery
[10,25,26]. Interaction of SNX9 with dynamin-2
recruits dynamin-2 to the clathrin-coated pit and stim-
ulates the GTPase activity required for vesicle scission
[21,23,27,28]. Interaction of SNX9 with N-WASP stim-
ulates actin remodelling during endocytosis by enhanc-
ing N-WASP activity [22,24,29,30]. It is possible that
functionally active SNX9 forming a complex with
dynamin-2 or N-WASP at membranes cannot be
targeted for degradation as the binding site for Itch is
not accessible. It may be speculated that, once the end-

ocytic process is completed, Itch competes for binding
to the SH3 domain of membrane-bound SNX9,
thereby destabilizing the complex of SNX9 with dyn-
amin-2 and ⁄ or N-WASP. Itch binding could enable
Fig. 6. SNX9 family member SNX18 also interacts with Itch. Extracts
were prepared from HEK293 cells transfected with myc-tagged
SNX18, HA-tagged Itch or both. Extracts were immunoprecipitated
using antibody against the myc tag, resolved by SDS ⁄ PAGE, and sub-
jected to immunoblotting with antibodies against Itch or the myc tag
as indicated. Cell extracts were analysed in parallel.
C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9
FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2809
the removal of SNX9 from the membrane, followed by
SNX9 ubiquitylation and degradation.
Alternatively, Itch may only bind to the SH3 domain
of cytosolic SNX9. In the cytosol, SNX9 forms a com-
plex with aldolase and dynamin-2 [27]. Thus, in this
scenario, Itch would have to compete with dynamin-2
for binding to the SH3 domain of SNX9. Ubiquityla-
tion of cytosolic SNX9 by Itch could serve as a mecha-
nism to regulate the pool of SNX9 protein in the
cell. Our results indicate that SNX9 is a very stable
protein with a half-life of approximately 24 h, at least
when over-expressed. Although increased Itch levels
increased the turnover of SNX9, the half-life of the
latter was still relatively long (approximately 15 h).
Given that endocytosis is a fast process, the hypothesis
of Itch-mediated removal of membrane-bound SNX9
upon endocytic vesicle release seems unlikely. The long
half-life of SNX9 instead supports the hypothesis that

Itch may regulate the steady-state levels of SNX9.
We found that tyrosine-phosphorylated SNX9 can
bind to Itch. At odds with our findings, tyrosine phos-
phorylation of the SH3 domain of Drosophila SNX9 by
ACK was found to reduce interactions with PRD-con-
taining proteins [10], hinting at differences between
human and Drosophila SNX9. It has also been reported
that tyrosine-phosphorylated human SNX9 can bind to
the PRD of ACK1 [11], again indicating different roles
of tyrosine phosphorylation in the human and Droso-
phila SNX9 proteins. Of note, in the human SNX9
protein, the presence of a tyrosine phosphorylation site
within the SH3 domain has not yet been formally demon-
strated. One could thus speculate that phosphorylation
of human SNX9 involves a tyrosine residue that is not
located within the SH3 domain, and may therefore not
affect the binding of SNX9 to PRD-containing proteins.
Future studies should address these issues in detail.
Phosphorylation of the ligase Itch might also affect
interaction with and ubiquitylation of SNX9. The
PRD of Itch was shown to be phosphorylated by the
serine ⁄ threonine kinase JNK (c-Jun N-terminal kinase)
upon treatment with epidermal growth factor (EGF)
[31–33]. This phosphorylation leads to an increase in the
ligase activity of Itch, resulting in increased ubiquityla-
tion and degradation of substrates such as endophi-
lin A1 and Casitas B-lineage lymphoma gene (Cbl)
[31]. Both endophilin and Cbl are involved in EGF
receptor down-regulation, and their increased degrada-
tion after EGF treatment has been proposed to serve as

a negative feedback mechanism to terminate internaliza-
tion of the EGF receptor [31]. Given that SNX9 is also
involved in down-regulation of the EGF receptor after
EGF treatment [9], one could speculate that, like endo-
philin and Cbl, the level of SNX9 ubiquitylation
increases upon EGF-induced phosphorylation of the
PRD of Itch.
In conclusion, we found that SNX9 is a new sub-
strate of the E3 Ub ligase Itch that binds to the PRD,
an unconventional substrate recognition domain in
Itch. Association of the SH3 domain of SNX9 with
the PRD of Itch mediates ubiquitylation and degra-
dation of SNX9. Binding of Itch to members of the
sorting nexin family with ensuing degradation is an
additional level at which E3 Ub ligases can regulate
endocytic processes. In the future, more SH3 domain-
containing substrates of the Ub ligases Itch and
NEDD4 are likely to be identified.
Experimental procedures
Antibodies
The rabbit antiserum against SNX9 was a generous gift from
S. Schmid (Scripps Research Institute, La Jolla, CA).
Another polyclonal antibody against SNX9 was produced in
rabbits by immunization with a full-length GST–SNX9
fusion protein (Eurogentec, Seraing, Belgium). The NEDD4
rabbit antiserum was provided by O. Staub (Department of
Pharmacology and Toxicology, University of Lausanne,
Switzerland). Monoclonal antibody against the myc tag
(clone 9E10) was a gift from R.D. Iggo (Institut Bergonie
´

,
Bordeaux, France). Commercial antibodies included:
monoclonal antibody against Itch (BD Biosciences ⁄
Pharmingen, San Diego, CA, USA), monoclonal antibody
against the HA tag (clone 16B12) (Covance, Princeton, NJ,
USA), polyclonal antibody against actin (Sigma-Aldrich,
St Louis, MO, USA), monoclonal antibody against the VSV
tag (clone P5D4) (Sigma-Aldrich), polyclonal antibody
against CD2AP (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), monoclonal antibody against ubiquitin (clone FK2)
(Biomol, Plymouth Meeting, PA, USA) and monoclonal anti-
body against phospho-tyrosine (clone 4G10) (Upstate
Biotechnology, Charlottesville, VA, USA). Secondary anti-
bodies were horseradish peroxidase-conjugated anti-mouse
IgG and anti-rabbit IgG (Amersham Biosciences, Pisca-
taway, NJ, USA) and horseradish peroxidase-conjugated
streptavidin (Dako, Glostrup, Denmark).
Plasmids and lentivirus production
The plasmids pGEX ⁄ Melan-A and pEGFP ⁄ Itch-HA have
been described previously [3]. The plasmid encoding Ub
HA
was a gift from S. Rothenberger (Institute of Microbiology,
University Hospital Lausanne, Switzerland). The pGEX ⁄
Itch-PWW vector was constructed by inserting a blunted
XbaI–BglII fragment from the pEGFP ⁄ Itch-HA vector [3]
into the SmaI site of the pGEX-4T-3 vector (Amersham
Biosciences). The full-length SNX9 coding sequence was
The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al.
2810 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS
cloned by RT-PCR from mRNA extracted from SK-MEL-

23 melanoma cells. The amplified fragment was digested
with EcoRI and XhoI and inserted into the corresponding
sites of the pCR-3 ⁄ Met-VSV vector (gift from P. Schneider,
Department of Biochemistry, University of Lausanne, Swit-
zerland) or the pGEX-4T-1 vector (Amersham Biosciences)
to generate the pCR3 ⁄ VSV-SNX9 and the pGEX ⁄ SNX9
plasmids, encoding SNX9 with an N-terminal VSV tag or
fused to GST, respectively. pGEX ⁄ SNX9(SH3) was con-
structed by PCR amplification of the SH3 domain of SNX9
from the same cDNA as above. The PCR product was
inserted into pGEX-4T-1 vector. The plasmids encoding
myc-tagged Itch and myc-tagged DPRD-Itch were a gener-
ous gift from T.P. Sakmar (Laboratory of Molecular Bio-
logy and Biochemistry, Rockefeller University, New York,
NY, USA). The plasmid encoding myc-tagged SNX18 was a
kind gift from S.R. Carlsson (Department of Medical Bio-
chemistry and Biophysics, Umea
˚
University, Sweden). The
plasmid pSuper-Itch, encoding Itch siRNA, was produced
by annealing two complementary DNA oligonucleotides tar-
geting nucleotides 88-108 of the ORF encoding human Itch,
as described previously [34], and ligating the fragment into
the pSuper vector (a gift from R. Agami, Nederlands Kan-
ker Instituut, Amsterdam, The Netherlands). Recombinant
lv ⁄ siItch was constructed by excising this siItch sequence
together with the H1-RNA promoter from the pSuper vec-
tor, and inserting the fragment into the pAB286 lentivector
(gift from R.D. Iggo, Institut Bergonie
´

, Bordeaux, France).
The same strategy was used to produce the control lentivec-
tor recombinant lv ⁄ siLamin to silence lamins A and C. The
plasmid pSuper ⁄ siLamin was a gift from R.D. Iggo (Institut
Bergonie
´
, Bordeaux, France) and has been described previ-
ously [35]. All constructs were verified by sequencing. Lenti-
virus production was performed as described elsewhere [3].
Cells and transfections
Human embryonic kidney HEK293 cells were maintained
in Dulbecco’s modified Eagle’s medium with 10% fetal
bovine serum and antibiotics. Pigmented Melan-A
+
SK-
MEL-23 human melanoma cells were maintained in RPMI-
1640 medium, supplemented with 10% fetal bovine serum
and antibiotics. Transfections were performed using Fugene
(Roche Diagnostics, Indianapolis, IN, USA) or Lipofecta-
mine (Invitrogen, Carlsbad, CA, USA), according to the
manufacturers’ instructions. Transfected cells were main-
tained for 48 h at 37 °C.
Silencing of Itch
Expression of Itch and lamins A and C (control) was
silenced in SK-MEL-23 melanoma cells by recombinant
lentivector transduction. Transduced cells were selected
using puromycin (2.5 lgÆmL
)1
) on day 2 and analysed
within 1–4 weeks of culture.

Ubiquitin assays
HEK293 cells were transfected with indicated plasmids.
Twenty-four hours after transfection, the cells were treated
for 2 h with 10 lm lactacystin (Biomol), a proteasome
inhibitor. Ubiquitylated proteins were immunoprecipitated
from cell lysates using monoclonal antibody against the
HA tag coupled to protein G–Sepharose. Proteins were
resolved by SDS ⁄ PAGE and immunoblotted using anti-
body against SNX9.
For the in vitro ubiquitylation assay, bacterially produced
GST–SNX9 or GST was adsorbed onto glutathione beads
and incubated for 0 or 24 h with cytosolic extracts from
3 · 10
6
HEK293 cells per reaction, supplemented with 2 lg
biotinylated ubiquitin (Biomol), 10 mm ATP, 5 mm Mg
acetate and 0.2 mm DTT. To avoid degradation, protea-
somes were removed from cytosol prior to the reaction with
GST–SNX9, as described previously [3]. Where indicated,
HEK293 cells were transfected with the plasmid encoding
Itch 48 h prior to lysis. After the ubiquitylation reaction,
10 mm EDTA was added and the immobilized material was
washed and eluted as described previously [3]. The superna-
tant was then diluted tenfold in cold lysis buffer and re-im-
munoprecipitated using antibody against SNX9. The
immunoprecipitated material was resuspended in SDS
sample buffer, resolved by SDS ⁄ PAGE and transferred
onto nitrocellulose. The ubiquitylated material was revealed
using horseradish peroxidase-conjugated streptavidin
(1 : 5000, Dako).

Preparation of GST fusion proteins and
pulldown assays
Bacteria cultures were grown to an attenuance at 600 nm
of 0.6–0.7. Protein expression was induced with 0.5 mm iso-
propyl thio-b-d-galactoside for 2 h at 37 °C, except for
GST–SNX9, which was induced for 4 h at 25 °C. Fusion
proteins were purified using the ProFound pulldown GST
protein:protein interaction kit (Pierce, Rockford, IL, USA),
according to the manufacturer’s instructions. Glutathione
beads (30 lL) were used for each purification and subse-
quent pulldown experiments. Pulldown experiments were
performed as described previously [3].
Immunoprecipitations and Western blots
Mammalian cells were lysed with Triton X-100 as described
previously [3], except for SNX9 immunoprecipitations,
where cells were lysed in 0.5% digitonin. For analysis of
tyrosine phosphorylated proteins, lysis buffer was supple-
mented with 50 mm sodium fluoride and 10 mm sodium
ortho-vanadate to inhibit phosphatases. The protein con-
tent was determined using a BCA protein assay kit (Pierce).
For immunoprecipitations, cleared cell extracts were incu-
bated either with agarose-conjugated antibodies against the
C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9
FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2811
HA tag (clone 3F10) (Roche) or antibodies against the
VSV tag (clone P5D4) (Sigma-Aldrich), Sepharose-conju-
gated antibodies against the myc tag (clone 9E10), or pro-
tein G beads (Pierce) plus polyclonal antibodies against
SNX9 or CD2AP (Santa Cruz Biotechnology). After 1–2 h
of incubation at 4 °C, immunoprecipitated material was

extensively washed, eluted in SDS sample buffer and boiled
at 95 °C for 5 min. Proteins were reduced, resolved on
SDS ⁄ PAGE and transferred onto nitrocellulose. Mem-
branes were subsequently incubated with blocking solution
(5% milk in NaCl ⁄ P
i
, except for 4G10 monoclonal anti-
body against where 1% gelatine in NaCl ⁄ P
i
⁄ Tween was
used) and primary antibody as indicated. Immunoreactivity
was detected using horseradish peroxidase-conjugated sec-
ondary antibodies and ECL (Amersham Biosciences)
according to the manufacturer’s instructions. Quantitative
analysis was performed using ImageJ quantification soft-
ware (National Institutes of Health, Bethesda, MD, USA).
Pulse–chase experiments
HEK293 cells were plated on 60 mm plates and transfected
with VSV-tagged SNX9 or a combination of VSV-tagged
SNX9 and myc-tagged Itch. Twenty-four hours after
transfection, cells were starved for 30 min in Dulbecco’s
modified Eagle’s medium lacking cysteine and methionine
(MP Biomedicals, Solon, OH, USA) supplemented with
10% dialysed fetal bovine serum, then incubated with Easy-
Tag Express
35
S Protein Labeling Mix (Perkin Elmer,
Waltham, MA), using 110 lCi per 2 mL per plate. After
30 min (pulse), radioactive medium was removed and cells
were incubated in Dulbecco’s modified Eagle’s medium sup-

plemented with 10% fetal bovine serum, 10 mm HEPES
and antibiotics for 6 or 24 h (chase). Cells were lysed for
20 min on ice in a buffer containing 80 mm Tris pH 7.6,
150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 1% sodium
deoxycholate and 0.05% SDS, supplemented with a cock-
tail of protease inhibitors. Aliquots of lysates were used
to measure the incorporated radioactivity using a liquid
scintillation counter. Lysates were subjected to immunopre-
cipitation as described above. Eluted material was resolved
by 10% SDS ⁄ PAGE. The gel was then fixed in 40% metha-
nol ⁄ 10% acetic acid for 15 min, dried, and exposed to an
imaging plate (Fuji Medical Systems, Stamford, CT, USA)
overnight. The plate was scanned using an FLA-3000 phos-
phorimager (Fuji Medical Systems) and BASReader 3.14
software (Raytest, Straubenhardt, Germany). aida image
analyzer software (Raytest) was used for quantitative analy-
sis. Autoradiography was performed by exposing the dried
gel to X-ray film (Kodak, Rochester, NY, USA).
Mass spectrometry
Gel bands were excised from SDS ⁄ PAGE and subjected to
in-gel proteolytic cleavage using sequencing-grade trypsin
(Promega, Madison, WI, USA) [36]. Proteolytic peptides
were analysed by LC-MS ⁄ MS on a SCIEX QSTAR Pulsar
hybrid quadrupole-time of flight instrument (Concord,
Ontario, Canada) equipped with a nanoelectrospray source
and interfaced to an Ultimate HPLC system (LC Packings,
Amsterdam, Netherlands). Peptides were separated on a
PepMap reverse-phase capillary C18 column (internal diam-
eter 75 lm, length 6.15 cm) at a flow rate of 200 nLÆmin
)1

using a 52 min gradient of acetonitrile (0–40%). The Ana-
lyst software (Applied Biosystems, Concord, Ontario,
Canada) was used for peak detection and to automatically
select peptides for collision-induced fragmentation. Non-
interpreted peptide tandem mass spectra were used for
direct interrogation of the uniprot (Swissprot + TrEMBL)
database using mascot 2.1 (rixscience.
com), limited to the subset of sequences from Homo
sapiens. The database used contained 64 386 sequences after
taxonomy filter. Generally, only proteins matched by at
least two peptides were accepted.
Acknowledgements
We thank Manfredo Quadroni (Department of Bio-
chemistry, University of Lausanne, Switzerland) and
the Protein Analysis Facility (Center for Integrative
Genomics, Faculty of Biology and Medicine, Univer-
sity of Lausanne, Switzerland) for mass spectrometry
analysis. We gratefully acknowledge the excellent tech-
nical assistance of Anne-Lise Peitrequin and Nicole
Le
´
vy. C.L. was supported by a post-doctoral fellowship
from the Wenner-Gren Foundation (Stockholm, Swe-
den). D.R. was supported in part by grants from the
Swiss National Funds (grant number 31003A-108283)
and the Faculty of Biology and Medicine of the Uni-
versity of Lausanne (grant number 22172). F.L. was
supported in part by grants from the Swiss National
Funds (grant number 310000-107686), the Cancer
Research Institute (New York, NY, USA), the Leena-

ards Foundation (grant number 2081) and the Faculty
of Biology and Medicine of the University of Lausanne
(grant number 22172).
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