Ubiquitination modifies proteins in a variety of ways, the
significance of which we only partially comprehend.
Ubiquitin can be attached: as an individual moiety to a
single or multiple lysine residues of substrate (mono- or
multiple monoubiquitination); as chains of ubiquitin
moieties that are interlinked through any one of the
seven lysine residues of ubiquitin (for example K48- or
K63-linked chains); or as branched chains, to name but a
few . The cell interprets each of these modifications as
a distinct signal. The first described role of ubiquitination
as mediating protein degradation through targeting to
the proteasome has now been complemented with
numerous other functions . For example, the signal
encoded by K63-linked chains can mediate functions as
diverse as receptor endocytosis [3,4], activation of
protein kinases in the NF-κB pathway and the initiation
of error-free DNA repair .
Signal transduction from transmembrane cell surface
receptors to nuclear transcription factors is regulated at
multiple levels by protein ubiquitination. The covalent
attachment of one, or often more, ubiquitin moieties has
emerged as the principal mechanism for termination of
signaling, by targeting the receptor for endocytosis and,
ultimately, degradation in the lysosome . This device
controls a vast array of mammalian signaling receptors,
such as receptor tyrosine kinases, G-protein coupled
receptors (GPCRs), growth hormone receptors, the
major histocompatibility complex I, NOTCH, various
channels and transporters, and cytokine and interferon
receptors . Receptors that are internalized after
activation are directed first into the endosomes of the
endocytic pathway, and then into multivesicular bodies
(MVBs), which undergo a process of maturation that
ends with fusion with the lysosome and delivery of the
contents for degradation. Ubiquitination of the receptor
provides the crucial signal for entering this pathway
Subsequent delivery of membrane receptors to the
lysosome requires accurate
ubiquitinated cargo by endosomal adaptors and sorting
proteins. For the EGF receptor, for example, these are
EPS15, epsin and ESCRTs (endosomal sorting complexes
required for transport), which contain one or more
ubiquitin-binding domains (UBDs) . EPS15 and epsins
act at the initial steps of internalization, serving to recruit
the enzymes required for ubiquitination of downstream
components of the endocytic pathway. ESCRTs act
sequentially at various points of the degradative route,
sorting the ubiquitinated cargo at the endosomal
membrane for inclusion into the intraluminal vesicle of
the MVB. ESCRT-0, composed of the two interacting
proteins HRS and STAM, is the first in this process.
Three additional complexes, ESCRT-I, ESCRTII and
ESCRT-III, are then involved in the generation of inward
vesicle budding, which is required for MVB maturation
(for reviews see [5,9]). The ubiquitin-binding ‘route
controllers’ that operate in this way to ferry the
internalized receptor inexorably towards a degradative
fate in lysosomes are also inducibly ubiquitinated [10,11]
The inducibility of the system illustrates the dynamic
nature of ubiquitin-based endocytic regulation. Indeed,
over the past 15 years, studies from different laboratories
recognition of the
Ubiquitin-dependent regulation of endocytosis plays
an important part in the control of signal transduction,
and a critical issue in the understanding of signal
transduction therefore relates to regulation of
ubiquitination in the endocytic pathway. We discuss
here what is known of the mechanisms by which
signaling controls the activity of the ubiquitin ligases
that specifically recognize the targets of ubiquitination
on the endocytic pathway, and suggest alternative
mechanisms that deserve experimental investigation.
Signaling-mediated control of ubiquitin ligases in
REVIEW Open Access
1IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139,
2Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di
Milano, Via di Rudinì 8, 20122 Milan, Italy.
© 2012 Polo; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Polo BMC Biology 2012, 10:25
have revealed a critical role for ubiquitination in receptor
down-regulation, a process that is essential for the spatial
and temporal resolution of receptor signaling .
The central component of this regulatory pathway is
the E3 ligase that attaches the ubiquitin molecules to the
receptor, or to the ubiquitin-binding endocytic adaptors
(Figure 1). A hierarchical set of three types of enzyme is
activating (E1), ubiquitin-conjugating (E2) and ubiquitin-
protein ligase (E3) enzymes. In mammals two E1-
activating enzyme transfer ubiquitin to roughly three
dozen E2s, which function together with several hundred
different E3 ubiquitin ligases to ubiquitinate thousands of
substrates. In the endocytic pathway distinct E3 enzymes
generally catalyze the ubiquitination of the cell-surface
receptors and the endosomal sorting proteins .
Therefore the endocytic sorting of a given target generally
involves more than one E3 ubiquitin protein ligase.
Here we focus on how signaling controls the activity of
the E3 ligases that ubiquitinate receptors and endocytic
adaptors. Although E3 ligases have largely been
considered to be constitutively active and regulated only
at the level of target binding, it has recently become
evident that they are subject to regulation by either E3 or
substrate phosphorylation, or by exploitation of adaptor
proteins that facilitate E3 activity. We discuss here the
various ways of regulating E3 ligases in the context of
for substrate ubiquitination: ubiquitin-
Ligand-induced E3 ligases for receptor
Protein phosphorylation, which is well known to function
in recruitment of the ubiquitination machinery to the
substrate , may also act directly to regulate the
activity of distinct ubiquitin ligases.
The best-characterized circuitry involves the E3 ligase
Cbl, which is responsible for the ubiquitination of several
receptor tyrosine kinases (RTKs) . The mammalian
Cbl protein family consists of the three homologs c-Cbl,
Cbl-b, and Cbl-3, all of which associate with a wide
variety of signaling proteins . Two highly conserved
amino-terminal domains contribute strongly to E3
regulatory function. First, the amino-terminal tyrosine
kinase binding (TKB) domain of Cbl recognizes
phosphotyrosine residues and allows Cbl to interact
directly with activated RTKs at the plasma membrane
(Figure 2). Second, the RING finger domain recruits
ubiquitin-loaded E2s, whose interaction with Cbl results
in the ubiquitination and subsequent degradation of the
associated RTK. In the case of the epidermal growth
factor receptor (EGFR) and the hepatocyte growth factor
receptor MET, the molecular mechanism of receptor
ubiquitination has been investigated in detail. In both
cases, Cbl binds directly to phosphotyrosine (pY)-sites on
the activated receptor through its TKB [14,15], as well as
indirectly through its constitutive partner GRB2, which is
recruited to receptors via other pY sites [7,16-18]. Both
direct and indirect interactions of Cbl with the EGFR or
MET are required for full ubiquitination of these
receptors (Figure 2). Once bound, the ligase is
phosphorylated and consequently activated . Two
Figure 1. Receptor internalization and the role of ubiquitin. The
example shown here is that of the EGF receptor. An activated EGFR
is ubiquitinated at the plasma membrane and recruits the endocytic
adaptor proteins EPS15 and EPSIN-1. These are ubiquitinated, in
turn, and direct the internalized receptor to the endosomal pathway
where it binds the sorting protein HRS. This is also ubiquitinated and
directs progression of the ubiquitinated receptors towards lysosomal
degradation through the ESCRT complexes. For simplicity, the EGF
receptor is depicted as monoubiquitinated: in reality, it is both
multimono- and polyubiquitinated. MVB, multivesicular body.
Endosomal sorting proteins
Polo BMC Biology 2012, 10:25
Page 2 of 9
structural studies have now shed light on the mechanism
of phosphorylation-induced activation of c-Cbl and
Cbl-b [20,21]. In the absence of substrate binding, the
TKB and RING domains form a compact structure that
masks the E2 binding site. Binding of the TKB to the
substrate induces a first rotation of the linker region,
allowing phosphorylation of tyrosine 371 (363 in Cbl-b).
This phosphorylation event induces a complete rotation
of the linker region that unmasks the RING E2 binding
surface and activates the ligase [20,21].
Another class of E3 ligases, the HECT NEDD4 family
, whose regulation has been extensively studied, also
regulates endocytosis and sorting of numerous signaling
receptors [3,5-7]. These enzymes present a conserved
modular organization with an amino-terminal C2
domain that is crucial for membrane localization,
between two and four WW domains capable of
recognizing substrates and adaptor proteins through PY
motifs, and a carboxy-terminal catalytic HECT domain.
In contrast to RING-based ligases in which the RING is
an allosteric activator of the E2, HECT-containing E3s
have intrinsic catalytic activity and directly ubiquitinate
their targets. In humans, there are nine members of this
family: NEDD4 (also known as NEDD4-1), NEDD4L
(also known as NEDD4-2), ITCH (also known as AIP4),
known as HECW1) and NEDL2 (also known as HECW2).
Rsp5 is the unique, essential member of the NEDD4
family in Saccharomyces cerevisiae. In normal conditions
most of them appear to be in an inactive state because of
an intramolecular inhibitory interaction between the
carboxy-terminal HECT and the amino-terminal C2
domain (in the case of SMURF2, NEDD4 and WWP2
) or the WW domains (in the case of ITCH ).
Activation of this class of enzyme can occur in various
ways that are briefly described below.
ITCH is the E3 ligase for the chemokine receptor
CXCR4 . The ubiquitin moiety on CXCR4 serves as a
signal on endosomes for entry into the degradative
pathway and long-term attenuation or downregulation of
signaling . ITCH can interact directly with CXCR4
through a non-canonical
interaction involving serine residues within the carboxy-
terminal tail of CXCR4. These serine residues are
phosphorylated upon agonist activation, and are critical
for mediating agonist-promoted binding of ITCH and the
subsequent ubiquitination and degradation of CXCR4
 (Figure 3a). Also in this case, the ligase appears to be
regulated by phosphorylation. ITCH phosphorylation is
activated by JNK1 , and is thought to lead to
WWP2, SMURF1, SMURF2, NEDL1 (also
Figure 2. EGFR ubiquitination by Cbl. Upon EGF-dependent receptor activation, the GRB2-Cbl complex binds to the receptor through
interactions of: i) the SH2 domain of GRB2 with pY1045 of EGFR, and ii) the TKB domain of Cbl (either c-Cbl or Cbl-b) with pY1068 or pY1086.
This substrate interaction may either stabilize or select for a partially open Cbl conformation (see bottom panel and main text). EGFR-bound Cbl
becomes phosphorylated on a critical tyrosine, leading to full rotation of the linker region. This, in turn, exposes the RING domain for ubiquitin-
charged E2 binding, resulting in the allosteric activation of the E2 by Cbl and ubiquitination of the EGFR. Note that, to simplify the picture, Cbl
bound to one receptor molecule is depicted to ubiquitinate the other molecule of the dimer. No available data suggest that this is indeed the case.
For simplicity, the EGF receptor is depicted as monoubiquitinated: in reality, it is both multimono- and polyubiquitinated.
Polo BMC Biology 2012, 10:25
Page 3 of 9
conformational changes that disrupt the inhibitory
intramolecular interactions between its WW and the
In the case of SMURF2, autoinhibition of the HECT
domain by the C2 domain helps in maintaining the
steady-state levels of this E3 ligase and can be relieved by
adaptor-mediated substrate targeting . SMURF1 and
SMURF2 bind to TGF-β family receptors through the
inhibitory Smad proteins, SMAD6 and SMAD7, to
induce their ubiquitin-dependent degradation. Wiesner
et al.  demonstrated that intramolecular interactions
between the C2 and HECT inhibit SMURF2 catalytic
activity interfering with ubiquitin thioester formation.
This in cis autoinhibition can be relieved by binding of
the amino-terminal domain (NTD) of the adaptor
protein SMAD7 to the E3 HECT domain (Figure 3b). The
SMAD7 NTD further enhances the catalytic activity of
the SMURF2 ligase by recruiting the E2 UbcH7 to the
HECT domain . By
autoinhibition, stimulating E2 binding, and recruiting
SMURF targets, SMAD7 functions at multiple levels to
control E3 activity and ensure specificity in SMURF-
Recently, a role for a UBD present on the N-lobe of the
HECT domain of NEDD4 and Rsp5 has been identified
[28,29]. The ability of the HECT domain to bind non-
covalently to the distal ubiquitin at the growing end of
the polyubiquitin chain on the substrate allows enzyme
Figure 3. Activation of E3 ubiquitin ligases through recruitment to activated receptors. (a) Ubiquitination of CXCR4 by ITCH. ITCH activity
is inhibited as a result of the intramolecular interaction between the WW domain and the carboxy-terminal catalytic HECT domain. Upon
agonist-mediated activation, CXCR4 becomes phosphorylated at Ser324 and Ser325 by an unknown kinase. This leads to the recruitment of
the ITCH, through its WW domain, and consequent release of the inhibitory intramolecular interaction, allowing ubiquitination of the receptor.
(b) Ubiquitination of the TGF-β receptor by the SMURF2-SMAD7 complex. SMURF2 activity is inhibited as a result of the intramolecular interaction
between the amino-terminal C2 and the carboxy-terminal catalytic HECT domain. The interaction with SMAD7 NTD displaces the C2 domain
of SMURF2 from the HECT domain and activates the ligase. The activated SMURF2-SMAD7 complex associates with activated TGF-β receptor
complexes at the membrane via the displaced C2 domain, causing receptor ubiquitination.
Polo BMC Biology 2012, 10:25
Page 4 of 9
processivity . It is tempting to attribute an inhibitory
role of the C2 binding for this critical feature of these
enzymes. Accessibility of the UBD may be restored in
response to upstream signaling events capable of inducing
phosphorylation and/or ubiquitination of critical sites in
the C2 or in the HECT domain, leading to full ligase
activation. While this hypothesis needs to be experimentally
verified, we notice that ubiquitination of NEDD4 is a
critical event for the coupled monoubiquitination of
EPS15 ( and below).
In some cases, such as for the epithelial Na+ channel
(ENaC), receptor:ligase interaction – and consequent
receptor ubiquitination – is the default pathway, with
phosphorylation negatively regulating ligase activity.
NEDD4-2 binds constitutively to ENaC PPxY-containing
motifs and catalyzes its ubiquitination, internalization
and lysosomal targeting. This prevents Na+ overload in
epithelial cells and is necessary for the maintenance of
salt and fluid balance in the body. To increase ENaC
abundance at the surface and enhance epithelial Na+
absorption, NEDD4-2 is phosphorylated by various
kinases, including protein kinase A (PKA), serum- and
glucocorticoid-inducible kinase (SGK), and IκB kinase
(IKK)β (Figure 4a). Phosphorylation induces binding of
14-3-3 proteins, which prevents NEDD4-2 from binding
to ENaC [31-33].
Regulation of receptor ubiquitination by adaptor
Finally, specific binding proteins can regulate the process
of ubiquitination by acting as adaptors to recruit E3
proteins to receptors that lack a direct binding motif for
the ligase (reviewed in ). The best evidence for this
mechanism so far comes from membrane transport
directed by the yeast HECT E3 ligase Rsp5, which is the
unique homolog of the mammalian NEDD4 family
proteins. While in humans there are nine members of the
NEDD4 family, yeast Rsp5 is sufficient on its own to
control most membrane traffic ubiquitination events at
the plasma membrane and at other biomembranes .
Cooperation with adaptors such as Bul1/Bul2, Bsd2/
Tre1/Tre2, or Ear1/Ssh4 enables Rsp5 to cope with this
large number of substrates , and the discovery of the
yeast family of ARR-related proteins (ARTs), which direct
Rsp5 activity to various plasma membrane receptors [36-
38], shows that the adaptor mechanism is even more
extensive than previously thought (Figure 4b). Does
receptor signaling regulate these HECT adaptor proteins?
Figure 4. Regulation of channels and transporters by ubiquitination. (a) ENaC ubiquitination by NEDD4-2. NEDD4-2 binds to PY motifs
on the epithelial Na+ channel ENaC and catalyzes its ubiquitination. This induces ENaC endocytosis and lysosomal targeting, resulting in fewer
channels at the cell surface. To increase Na+ transport, NEDD4-2 is phosphorylated by kinases, including PKA, SGK, and IKKβ, in turn activated by
various signaling pathways. Phosphorylation of NEDD4-2 induces binding of 14-3-3 dimers (not shown), which prevents NEDD4-2 from binding
to ENaC. As a result, endocytosis of ENaC is inhibited, and increased ENaC presence at the surface enhances epithelial Na+ absorption. (b) Rsp5
ubiquitinates permeases and transporters. In yeast, arrestin-related endocytic adaptors (ARTs) and the E3 ubiquitin ligase Rsp5 are recruited to the
plasma membrane in response to environmental stimuli that trigger the endocytosis of proteins such as permeases and transporters (for example,
the arginine transporter Can1). Through their PY motifs, ARTs bind to the WW domain of Rsp5 and mediate ubiquitination of cargo. ARTs are also
ubiquitinated by Rsp5, an event required for endocytosis.
Polo BMC Biology 2012, 10:25
Page 5 of 9
Two recent papers provide evidence that it does [39,40].
MacGurn et al.  demonstrated that signaling from
TORC1, which is a central regulator of cell growth in
response to amino acid availability, regulates Rsp5
targeting and endocytosis of nutrient transporters at the
plasma membrane. The effector mechanism that
regulates endocytosis involves a TORC1-Npr1 negative
kinase signaling cascade that tunes the phosphoinhibition
of the ubiquitin ligase adaptor Art1 . Leon and
colleagues  identified the α-arrestin Rod1/Art4
as a direct target of the glucose signaling pathway.
Glucose promotes Rod1/Art4 dephosphorylation and
its subsequent release from a phospho-dependent
interaction with 14-3-3 proteins. This allows Rsp5-
mediated Rod1 ubiquitination, a prerequisite for
transporter endocytosis . It is conceivable that other
signaling pathways may similarly regulate the activity of
other ART family proteins.
Does this mechanism also operate in mammals? And if
yes, how? These important gaps in our understanding
need to be filled. Of note, arrestin domain containing
protein 3 (ARRDC3) was recently shown to interact with
NEDD4 and to be an essential adaptor for β2-adrenergic
receptor (β2AR) ubiquitination .
Ligand-induced E3 ligases for adaptor
The adaptors we have just discussed function to recruit
E3 ligases to receptors. We now return to the endocytic
adaptors that are recruited to the ubiquitinated receptors
and direct their subsequent endocytosis. As with direct
receptor ubiquitination, the ubiquitination of endocytic
adaptors plays a critical role in endocytosis. The arrestin
(ARR) family of proteins is able to direct internalization
of GPCR cargo. Signaling from activated GPCRs is
terminated when GPCRs are phosphorylated by GPCR
kinases (GRKs), leading to the recruitment of ARR,
which binds to AP-2 and clathrin, structural components
of the vesicles formed at the plasma membrane causing
the whole complex to be internalized. Agonist-stimulated
ubiquitination of ARR mediated by the E3 ligase murine
double minute (MDM2), an important negative regulator
of p53, is critical for rapid receptor internalization .
MDM2-ARR binding is constitutive and does not persist
after receptor activation, suggesting that ubiquitin modi-
fication might cause a conformational change on ARR
required to promote internalization. GPCRs themselves can
also be ubiquitinated, most probably by NEDD4, an event
required for cargo degradation but not internalization .
Thus by analogy with the ‘phosphorylation code’ on the
receptor carboxy tail, ubiquitin modifications on both
adaptors and receptors result in a ‘ubiquitination code’
that fine-tunes signal strength, localization, and cellular
functions of GPCR (Figure 5a).
ARR is not the sole example of an endocytic adaptor
subjected to ubiquitin modification. Several components of
the downstream endocytic machinery are monoubiquitinated
upon RTK activation [10,11,44,45]. In most cases, these
adaptors are ubiquitin receptors that are ubiquitinated by
the E3 ligase NEDD4. The presence of a UBD is required
for monoubiquitination of the UBD-harboring adaptor,
in a process termed coupled monoubiquitination whose
molecular workings have been elucidated using the
endocytic proteins EPS15 and EPSIN-1 as models [30,46]
(Figure 5b). By contrast, the mechanism by which
NEDD4 recruitment is induced by the activated EGFR
remains to be clarified.
What is the role of
Monoubiquitination might permit the formation of
several tiers of ubiquitination-dependent interactions in
the endosome, by allowing binding of ubiquitinated cargo
(through UBDs) and recruiting another layer of ubiquitin
receptors through the monoubiquitin signal. The result
would be signal amplification and progression of
ubiquitinated cargoes along the endocytic pathway.
Monoubiquitination of ubiquitin receptors may also
result in an intra-molecular interaction between their
UBDs and monoubiquitinated residues, with resulting
dissociation from the ubiquitinated cargo [47,48]. These
two possibilities are not mutually exclusive and both
mechanisms may be involved in the regulation of
endocytic processes, possibly by acting at distinct steps
and/or regulating different endocytic adaptors.
Is the ubiquitination cascade like
the phosphorylation cascade?
It is important to realize that the power of ubiquitin stems
from its capacity to act as a protein-protein interaction
module that targets substrates to a plethora of downstream
effectors. In order to realize this network, complex molecular
machines generate and recognize signal diversity based
on ubiquitin-binding modules. The network is fine-tuned
by ubiquitinating (E3 ligases) and deubiquitinating
enzymes (DUBs) that balance the absolute levels of
protein ubiquitination, as well as the abundance and
localization of adaptors that contain docking sites for
specific ubiquitinated proteins (UBDs) .
One such network pivots around EGFR. EGF
stimulation promotes ubiquitination of EGFR and of
EGFR endocytic adapters, providing a striking example
of concerted regulation of signaling, ultimately regulating
the route of EGFR internalization [50,51]. Our own
approach to understanding the complex interplay
between EGFR-induced signaling circuitries has been the
recent elucidation of the EGF-induced ‘ubiproteome’
. This work has uncovered an extensive ubiquitin-
based signaling network that impinges on a wide array of
signaling circuitries and various aspects of cellular
Polo BMC Biology 2012, 10:25
Page 6 of 9
physiology including DNA repair, nuclear transport,
mRNA processing, metabolic pathways, and ribosome
biogenesis. Interestingly, many ubiquitin machinery
enzymes were detected in the EGF-induced ubiproteome.
Regardless of the initial triggering mechanism (which
necessarily involves the kinase activity of the EGFR), the
ubiquitin signal seems to be rapidly transmitted to, and
amplified through, the ubiquitin machinery. Just as in the
phosphorylation cascade, in which critical enzymes such
as kinases and phosphatases are often activated by
phosphorylation, ubiquitinating enzymes appear to be
regulated by ubiquitination. Moreover, a comparison of
the EGF-induced ubi- and pY-proteomes revealed a
significant overlap and identified many highly connected
‘hub’ proteins that are both phosphorylated and
ubiquitinated . These data suggest that two
complementary and interlinked enzymatic cascades drive
the flow of information from receptors to downstream
signaling molecules: kinases/phosphatases and E3
ligases/DUBs. In essence, these two post-translational-
modification-based networks can be conceptualized as
two overlapping, diffusely interconnected, matrices
Figure 5. Ubiquitination of adaptors. (a) Agonist induces rapid ubiquitination of GPCR-recruited ARR by MDM2, a process required for receptor
internalization. Once internalized, GPCRs can be dephosphorylated and rapidly recycled to the plasma membrane through a mechanism that
involves the sorting proteins EBP50 and NSF. (b) Activated EGFR is ubiquitinated at the plasma membrane by Cbl and recruits the UBD-containing
endocytic adaptors EPS15 and EPSIN-1 at the plasma membrane, and subsequently HRS at the endosomal membrane. These adaptors, in turn, are
ubiquitinated by NEDD4 through a process known as coupled monoubiquitination. This directs progression of the ubiquitinated receptors toward
lysosomal degradation through the ESCRT complexes. A similar mechanism can be envisioned for other RTKs.
Endosomal sorting proteins
To endocytosisTo endocytosis
Polo BMC Biology 2012, 10:25
Page 7 of 9
through which external signals are transduced and
interpreted by the cell. To understand how this is
achieved, a multidisciplinary approach is required. No
single ‘omics’ analysis can fully unravel the complexities
of the system. Complete understanding will be achieved
only by integrating information from high-resolution
molecular investigations, ‘omics’ approaches and ‘top-
down’ systems-based modeling.
Research in the Polo laboratory is supported by grants from the Associazione
Italiana per la Ricerca sul Cancro, the Italian University Research Program for
the Development of Research of National Interest and of Health (PRIN), the
Association of International Cancer Research, the CARIPLO Foundation, and
the EMBO Young Investigator Program.
Published: 15 March 2012
1. Komander D: The emerging complexity of protein ubiquitination. Biochem
Soc Trans 2009, 37:937-953.
2. Chen ZJ, Sun LJ: Nonproteolytic functions of ubiquitin in cell signaling. Mol
Cell 2009, 33:275-286.
3. Acconcia F, Sigismund S, Polo S: Ubiquitin in trafficking: the network at
work. Exp Cell Res 2009, 315:1610-1618.
4. Lauwers E, Erpapazoglou Z, Haguenauer-Tsapis R, Andre B: The ubiquitin
code of yeast permease trafficking. Trends Cell Biol 2010, 20:196-204.
5. Raiborg C, Stenmark H: The ESCRT machinery in endosomal sorting of
ubiquitylated membrane proteins. Nature 2009, 458:445-452.
6. Staub O, Rotin D: Role of ubiquitylation in cellular membrane transport.
Physiol Rev 2006, 86:669-707.
7. Miranda M, Sorkin A: Regulation of receptors and transporters by
ubiquitination: new insights into surprisingly similar mechanisms. Mol
Interv 2007, 7:157-167.
8. Dikic I, Wakatsuki S, Walters KJ: Ubiquitin-binding domains – from
structures to functions. Nat Rev Mol Cell Biol 2009, 10:659-671.
9. Hurley JH, Hanson PI: Membrane budding and scission by the ESCRT
machinery: it’s all in the neck. Nat Rev Mol Cell Biol 2010, 11:556-566.
10. Katz M, Shtiegman K, Tal-Or P, Yakir L, Mosesson Y, Harari D, Machluf Y, Asao H,
Jovin T, Sugamura K, Yarden Y: Ligand-independent degradation of
epidermal growth factor receptor involves receptor ubiquitylation and
Hgs, an adaptor whose ubiquitin-interacting motif targets ubiquitylation
by Nedd4. Traffic 2002, 3:740-751.
11. Polo S, Sigismund S, Faretta M, Guidi M, Capua MR, Bossi G, Chen H, De
Camilli P, Di Fiore PP: A single motif responsible for ubiquitin recognition
and monoubiquitination in endocytic proteins. Nature 2002, 416:451-455.
12. Gao M, Karin M: Regulating the regulators: control of protein
ubiquitination and ubiquitin-like modifications by extracellular stimuli.
Mol Cell 2005, 19:581-593.
13. Schmidt MH, Dikic I: The Cbl interactome and its functions. Nat Rev Mol Cell
Biol 2005, 6:907-918.
14. Levkowitz G, Waterman H, Ettenberg SA, Katz M, Tsygankov AY, Alroy I, Lavi S,
Iwai K, Reiss Y, Ciechanover A, Lipkowitz S, Yarden Y: Ubiquitin ligase activity
and tyrosine phosphorylation underlie suppression of growth factor
signaling by c-Cbl/Sli-1. Mol Cell 1999, 4:1029-1040.
15. Peschard P, Fournier TM, Lamorte L, Naujokas MA, Band H, Langdon WY, Park
M: Mutation of the c-Cbl TKB domain binding site on the Met receptor
tyrosine kinase converts it into a transforming protein. Mol Cell 2001,
16. Waterman H, Katz M, Rubin C, Shtiegman K, Lavi S, Elson A, Jovin T, Yarden Y:
A mutant EGF-receptor defective in ubiquitylation and endocytosis
unveils a role for Grb2 in negative signaling. EMBO J 2002, 21:303-313.
17. Jiang X, Huang F, Marusyk A, Sorkin A: Grb2 regulates internalization of EGF
receptors through clathrin-coated pits. Mol Biol Cell 2003, 14:858-870.
18. Huang F, Sorkin A: Growth factor receptor binding protein 2-mediated
recruitment of the RING domain of Cbl to the epidermal growth factor
receptor is essential and sufficient to support receptor endocytosis. Mol
Biol Cell 2005, 16:1268-1281.
19. Kassenbrock CK, Hunter S, Garl P, Johnson GL, Anderson SM: Inhibition of Src
family kinases blocks epidermal growth factor (EGF)-induced activation of
Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor. J
Biol Chem 2002, 277:24967-24975.
20. Kobashigawa Y, Tomitaka A, Kumeta H, Noda NN, Yamaguchi M, Inagaki F:
Autoinhibition and phosphorylation-induced activation mechanisms of
human cancer and autoimmune disease-related E3 protein Cbl-b. Proc Natl
Acad Sci USA 2011, 108:20579-20584.
21. Dou H, Buetow L, Hock A, Sibbet GJ, Vousden KH, Huang DT: Structural basis
for autoinhibition and phosphorylation-dependent activation of c-Cbl.
Nat Struct Mol Biol 2012, 19:184-192.
22. Rotin D, Kumar S: Physiological functions of the HECT family of ubiquitin
ligases. Nat Rev Mol Cell Biol 2009, 10:398-409.
23. Wiesner S, Ogunjimi AA, Wang HR, Rotin D, Sicheri F, Wrana JL, Forman-Kay
JD: Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2
domain. Cell 2007, 130:651-662.
24. Gallagher E, Gao M, Liu YC, Karin M: Activation of the E3 ubiquitin ligase Itch
through a phosphorylation-induced conformational change. Proc Natl
Acad Sci USA 2006, 103:1717-1722.
25. Marchese A, Raiborg C, Santini F, Keen JH, Stenmark H, Benovic JL: The E3
ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G
protein-coupled receptor CXCR4. Dev Cell 2003, 5:709-722.
26. Bhandari D, Robia SL, Marchese A: The E3 ubiquitin ligase atrophin
interacting protein 4 binds directly to the chemokine receptor CXCR4 via
a novel WW domain-mediated interaction. Mol Biol Cell 2009, 20:1324-1339.
27. Ogunjimi AA, Briant DJ, Pece-Barbara N, Le Roy C, Di Guglielmo GM, Kavsak P,
Rasmussen RK, Seet BT, Sicheri F, Wrana JL: Regulation of Smurf2 ubiquitin
ligase activity by anchoring the E2 to the HECT domain. Mol Cell 2005,
28. Maspero E, Mari S, Valentini E, Musacchio A, Fish A, Pasqualato S, Polo S:
Structure of the HECT:ubiquitin complex and its role in ubiquitin chain
elongation. EMBO Rep 2011, 12:342-349.
29. Kim HC, Steffen AM, Oldham ML, Chen J, Huibregtse JM: Structure and
function of a HECT domain ubiquitin-binding site. EMBO Rep 2011,
30. Woelk T, Oldrini B, Maspero E, Confalonieri S, Cavallaro E, Di Fiore PP, Polo S:
Molecular mechanisms of coupled monoubiquitination. Nat Cell Biol 2006,
31. Bhalla V, Daidie D, Li H, Pao AC, LaGrange LP, Wang J, Vandewalle A, Stockand
JD, Staub O, Pearce D: Serum- and glucocorticoid-regulated kinase 1
regulates ubiquitin ligase neural precursor cell-expressed,
developmentally down-regulated protein 4-2 by inducing interaction
with 14-3-3. Mol Endocrinol 2005, 19:3073-3084.
32. Ichimura T, Yamamura H, Sasamoto K, Tominaga Y, Taoka M, Kakiuchi K,
Shinkawa T, Takahashi N, Shimada S, Isobe T: 14-3-3 proteins modulate the
expression of epithelial Na+ channels by phosphorylation-dependent
interaction with Nedd4-2 ubiquitin ligase. J Biol Chem 2005,
33. Snyder PM: Down-regulating destruction: phosphorylation regulates the
E3 ubiquitin ligase Nedd4-2. Sci Signal 2009, 2:pe41.
34. Leon S, Haguenauer-Tsapis R: Ubiquitin ligase adaptors: regulators of
ubiquitylation and endocytosis of plasma membrane proteins. Exp Cell Res
35. Belgareh-Touze N, Leon S, Erpapazoglou Z, Stawiecka-Mirota M, Urban-
Grimal D, Haguenauer-Tsapis R: Versatile role of the yeast ubiquitin ligase
Rsp5p in intracellular trafficking. Biochem Soc Trans 2008, 36:791-796.
36. Lin CH, MacGurn JA, Chu T, Stefan CJ, Emr SD: Arrestin-related ubiquitin-
ligase adaptors regulate endocytosis and protein turnover at the cell
surface. Cell 2008, 135:714-725.
37. Nikko E, Sullivan JA, Pelham HR: Arrestin-like proteins mediate
ubiquitination and endocytosis of the yeast metal transporter Smf1. EMBO
Rep 2008, 9:1216-1221.
38. Polo S, Di Fiore PP: Finding the right partner: science or ART? Cell 2008,
39. Becuwe M, Vieira N, Lara D, Gomes-Rezende J, Soares-Cunha C, Casal M,
Haguenauer-Tsapis R, Vincent O, Paiva S, Leon S: A molecular switch on an
arrestin-like protein relays glucose signaling to transporter endocytosis. J
Cell Biol 2012, 196:247-259.
40. MacGurn JA, Hsu PC, Smolka MB, Emr SD: TORC1 regulates endocytosis via
Npr1-mediated phosphoinhibition of a ubiquitin ligase adaptor. Cell 2011,
Polo BMC Biology 2012, 10:25
Page 8 of 9
41. Nabhan JF, Pan H, Lu Q: Arrestin domain-containing protein 3 recruits the
NEDD4 E3 ligase to mediate ubiquitination of the beta2-adrenergic
receptor. EMBO Rep 2010, 11:605-611.
42. Shenoy SK, Modi AS, Shukla AK, Xiao K, Berthouze M, Ahn S, Wilkinson KD,
Miller WE, Lefkowitz RJ: Beta-arrestin-dependent signaling and trafficking
of 7-transmembrane receptors is reciprocally regulated by the
deubiquitinase USP33 and the E3 ligase Mdm2. Proc Natl Acad Sci USA 2009,
43. Shenoy SK, Xiao K, Venkataramanan V, Snyder PM, Freedman NJ, Weissman
AM: Nedd4 mediates agonist-dependent ubiquitination, lysosomal
targeting, and degradation of the beta2-adrenergic receptor. J Biol Chem
44. Haglund K, Shimokawa N, Szymkiewicz I, Dikic I: Cbl-directed
monoubiquitination of CIN85 is involved in regulation of ligand-induced
degradation of EGF receptors. Proc Natl Acad Sci USA 2002, 99:12191-12196.
45. Shih SC, Katzmann DJ, Schnell JD, Sutanto M, Emr SD, Hicke L: Epsins and
Vps27p/Hrs contain ubiquitin-binding domains that function in receptor
endocytosis. Nat Cell Biol 2002, 4:389-393.
46. Fallon L, Bélanger CM, Corera AT, Kontogiannea M, Regan-Klapisz E, Moreau F,
Voortman J, Haber M, Rouleau G, Thorarinsdottir T, Brice A, van Bergen En
Henegouwen PM, Fon EA: A regulated interaction with the UIM protein
Eps15 implicates parkin in EGF receptor trafficking and PI(3)K-Akt
signalling. Nat Cell Biol 2006, 8:834-842.
47. H Hoeller D, Crosetto N, Blagoev B, Raiborg C, Tikkanen R, Wagner S,
Kowanetz K, Breitling R, Mann M, Stenmark H, Dikic I: Regulation of
ubiquitin-binding proteins by monoubiquitination. Nat Cell Biol 2006,
48. Mattera R, Bonifacino JS: Ubiquitin binding and conjugation regulate the
recruitment of Rabex-5 to early endosomes. EMBO J 2008, 27:2484-2494.
49. Woelk T, Sigismund S, Penengo L, Polo S: The ubiquitination code: a
signalling problem. Cell Div 2007, 2:11.
50. Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP,
Polo S: Clathrin-independent endocytosis of ubiquitinated cargos. Proc
Natl Acad Sci USA 2005, 102:2760-2765.
51. Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP: Clathrin-
mediated internalization is essential for sustained EGFR signaling but
dispensable for degradation. Dev Cell 2008, 15:209-219.
52. Argenzio E, Bange T, Oldrini B, Bianchi F, Peesari R, Mari S, Di Fiore PP, Mann M,
Polo S: A proteomic snapshot of the EGF-induced ubiquitin network. Mol
Syst Biol 2011, 7:462.
Polo BMC Biology 2012, 10:25
Cite this article as: Polo S: Signaling-mediated control of ubiquitin ligases
in endocytosis. BMC Biology 2012, 10:25.
Page 9 of 9