Published OnlineFirst February 21, 2012; DOI: 10.1158/1535-7163.

MCT-11-0555

Molecular
Cancer
Therapeutics

Review

The Ubiquitin-Proteasome System Meets Angiogenesis

Nader Rahimi

Abstract
A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls
endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic
angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of
angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system
(UPS) plays a central role in fine-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR2, angiogenic signaling proteins (e.g., the PLCg1 and PI3 kinase/AKT pathways), and other non-VEGF
angiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins
control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that
link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in
angiogenesis-associated diseases. Mol Cancer Ther; 11(3); 53848. 2012 AACR.

Introduction
Angiogenesis, the growth of new blood vessels from
preexisting vessels, is an important physiological process
in the body that is required for normal wound-healing and
female reproduction. Pathologic angiogenesis (excessive
or insufficient) is now recognized as a common denominator underlying a number of deadly and debilitating
human diseases, including cancer, age-related macular
degeneration (AMD), diabetic retinopathy, and cardiovascular diseases (1, 2). Although in many ways these
diseases have distinct etiologies and mechanisms of
development, they all share abnormal angiogenesis. For
example, although cancer has nothing to do with AMD, it
shares one of its characteristics, angiogenesis, with AMD
(Fig. 1). Acquisition of angiogenesis by tumor cells is
considered the most critical step in tumor growth and
metastasis. To grow beyond 2 mm in diameter, a tumor
must acquire angiogenesis, which is often established by
hypoxia-induced expression of VEGF-A and other angiogenesis-inducing molecules (3). To support the growth of
the expanding tumor, an angiogenic switch is turned on,
causing normally quiescent endothelial cells to proliferate
and sprout (4). It is now clear that induction of VEGF-A
and other related angiogenesis inducers, and a reduction
in the expression of angiogenesis inhibitors, such as
thrombospondin (TSP-1), govern the tumor-induced
angiogenic switch (5, 6). Although it was initially thought
that angiogenesis plays a substantial role only when the

Author's Afliation: Departments of Pathology and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts
Corresponding Author: Nader Rahimi, Department of Pathology, Boston
University Medical Campus, 670 Albany St., Room 510, Boston, MA 02118.
Phone: 617-638-5011; Fax: 617-414-7914; E-mail: nrahimi@bu.edu
doi: 10.1158/1535-7163.MCT-11-0555
2012 American Association for Cancer Research.

538

tumor mass reaches a macroscopic size, it is becoming

increasingly apparent that angiogenesis is instigated in
the early stage of tumor development (5, 7), further supporting angiogenesis as a vital component of tumor
growth and metastasis.
VEGF-A also plays a central role in the development of
choroidal neovascularization, and indeed is responsible
for both neovascularization and vascular leakage in wet
AMD (8). The hallmarks of wet AMD are drusen formation [i.e., the focal deposition of debris between the retinal
pigment epithelium (RPE) and Bruchs membrane], choroidal neovascularization, RPE cell detachment, fibrovascular scarring, and vitreous hemorrhage. Aberrant blood
vessel growth and blood vessel leakage subsequently
result in the loss of central vision (9). Many cell types in
the eye, including RPE cells, pericytes, endothelial cells,
glial cells, M
uller cells, and ganglion cells, synthesize and
secrete VEGF. In addition to the vital importance of VEGF
in the pathology of AMD, elevated VEGF levels also
strongly correlate with retinal ischemia-associated neovascularization in diabetic retinopathy and retinopathy of
prematurity (8, 10).
Once the angiogenic switch is activated, different
sequential steps take place, including the activation of
various proteases from activated endothelial cells resulting in the degradation of the basement membrane surrounding the existing vessel, migration of the endothelial
cells into the interstitial space, endothelial cell proliferation, sprouting, lumen formation, generation of new basement membrane with the recruitment of pericytes, and
fusion of the newly formed vessels (11). In general, and in
most pathologic conditions, angiogenesis starts when
cells within a tissue respond to hypoxia (i.e., low oxygen)
or in certain circumstances when oncogenic gene products such as Ras and Myc induce expression of VEGF
along with other hypoxia-inducible genes (12). The VEGF
family of growth factors includes placental growth factor

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Ubiquitin-Proteasome System in Angiogenesis

Figure 1. Schematic presentation of tumor-induced angiogenesis and choroidal neovascularization as manifested in wet AMD. Expression of VEGF is
responsible for induction of angiogenesis, which is associated with tumor progression and worsening of wet AMD.

(PlGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E

(13). VEGF-A isoforms result from alternative splicing of
messenger RNA (mRNA), and VEGF-165 is considered to
be the most common VEGF-A isoform (14). Three different VEGF gene products (PlGF, VEGF-A, and VEGF-B)
have been identified as ligands for VEGFR-1. VEGF-A,
VEGF-D, and VEGF-C bind to and activate VEGFR-2 (13).
VEGF-C and VEGF-D are also known to recognize
VEGFR-3 (also called FLT4), a receptor tyrosine kinase
(RTK) that is expressed predominantly by lymphatic
endothelial and hematopoietic progenitor cells (15).
VEGF-E is a virally encoded VEGF-like protein that selectively binds to VEGFR-2 (16). VEGF family proteins
also interact with non-RTK cell surface receptors, including neuropilin-1 and neuropilin-2, which are characterized as coreceptors for VEGF family ligands (13, 17).
Among the VEGF receptors and coreceptors, activation
of VEGFR-2 by VEGF family ligands is considered the
most critical event in angiogenesis (18, 19). Beyond VEGF-

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mediated VEGFR-2 activation, recent studies have

shown that under certain circumstances, VEGFR-2 can
also be activated by non-VEGF family ligands, including heparan sulfate proteoglycans (20) and galectin-3, a
glycan-binding protein (21). Heparan sulfate proteoglycans have been proposed to potentiate VEGFR-2 activation through cis- and trans-binding with VEGFR-2
and VEGF complex (20), where galectin-3 is thought to
potentiate VEGFR-2 activation by prolonging its presence in the plasma membrane (21). Although VEGF
family proteins are prominent regulators of angiogenesis, several other growth factors and cytokines, including angiopoietin-1, Del-1, fibroblast growth factor,
hepatocyte growth factor, interleukin-8 (IL-8), and leptin, are known to stimulate angiogenesis (22), adding
further complexity to the regulation of angiogenesis.
The role of VEGFR-2 in angiogenesis is well established, and more-comprehensive reviews on the role
of VEGFR-2 in angiogenesis were recently published

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Rahimi

Figure 2. VEGF superfamily ligands and receptors. A schematic of VEGF ligands and their interactions with VEGF receptors is shown.

(13, 17, 19). Figure 2 summarizes VEGF superfamily

ligands and their interactions with VEGF receptors (Fig.
2). It is increasingly evident that angiogenic signaling is
established through an elegant and complex system of
VEGF and non-VEGF ligands, and VEGF receptors and
coreceptors, resulting in homo- and heterodimeric activation of VEGF receptors and in turn the complex
processes of angiogenesis.

Ubiquitin-Proteasome System
Ubiquitin is an evolutionarily conserved polypeptide
that consists of 76 amino acids and was named for
its ubiquitous expression in eukaryotes. Ubiquitin is
activated by a ubiquitin-activating enzyme, E1, in an
ATP-dependent manner and is transferred to a ubiquitin-conjugating enzyme, E2. Eventually, a ubiquitinprotein ligase, E3, specifically attaches the ubiquitin
molecule to a target protein through the e-amino group
of a lysine residue (Fig. 3). E3 ubiquitin ligases are a

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Mol Cancer Ther; 11(3) March 2012

large family of proteins (with almost 700 in the human

genome) that are known to be involved in regulating the
turnover and activity of many target proteins (23). E3
ligases are divided into 2 large groups: the homology to
the E6-associated protein carboxyl terminus (HECT)
domain-containing E3 ligases, and the Really Interesting New Gene (RING) domain-containing E3 ligases.
The RING-type E3 ligases include single subunit E3
ligases (such as Cbl family E3 ligases) and multisubunit
E3 ligases (such as Cullin-RING ubiquitin ligases). In
recent years, additional E3 ligases have been identified
that use different domains to recognize E2-conjugating
enzymes, such as plant homeodomain domain-containing E3 ligases and the U-box E3 ligases (23, 24).
Although conjugation of ubiquitin to target proteins
was initially recognized as a signal for protein degradation by the 26S proteasome, it is now recognized that
ubiquitination regulates a broad range of cellular functions, including protein processing, membrane trafficking, and transcriptional regulation (23, 25). Recent

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Ubiquitin-Proteasome System in Angiogenesis

Figure 3. Schematic of the UPS. Ubiquitin is activated by the ubiquitin-activating enzyme (E1) and then transferred to a ubiquitin-conjugating enzyme (E2). E2
transfers the activated ubiquitin moieties to the protein substrate that is bound specically to a particular ubiquitin ligase (E3). The transfer of ubiquitin
takes place either directly (in the case of RING nger ligases) or via an additional thiol-ester intermediate on the ligase (in the case of HECT domain
ligases). Repeated conjugation of ubiquitin moieties to each other generates a polyubiquitin chain that serves as the binding and degradation signal for the 26S
proteasome. The protein substrate is degraded, generating short peptides and free ubiquitin that can be further reused. Ub, ubiquitin.

studies showed that ubiquitination can also influence

cell signaling by targeting activation of proteins in a
proteolysis-independent manner (2628).
Multiple ubiquitin molecules can be attached to a target
protein by means of monoubiquitination (i.e., attachment of
a single ubiquitin to 1 or multiple lysine residues). Monoubiquitination is regarded as a signal for nonproteolytic
events such as endocytosis, histone regulation, DNA repair,
virus budding, and nuclear export (25). Alternatively, ubiquitin can be attached to a target protein in the form of
polyubiquitination, where multiple ubiquitin molecules
are attached to a single lysine residue. Ubiquitin contains
7 different lysine residues that potentially can be used for
ubiquitin-chain assembly. Lys48- and Lys29-linked polyubiquitination generally is associated with degradation of
target proteins by the 26S proteasome, whereas Lys63linked polyubiquitination is involved in DNA repair, signal
transduction, and endocytosis, but not degradation (23, 25).
Clearly, the ubiquitin machinery has evolved to play a
versatile role in protein functions ranging from protein
turnover to subcellular localization and kinase activation,
and consequently it is highly relevant to the pathobiology of
many human diseases.
The ubiquitin-proteasome system (UPS) consists of 2
major components: substrate-recruiting enzymes (E1, E2,
and E3) and substrate-degrading enzymes. E1 activates

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the polypeptide ubiquitin in an ATP-dependent manner,

enabling its transfer onto the ubiquitin carrier enzyme, E2.
Activated ubiquitin is then transferred by the ubiquitin
protein ligase, E3, to a substrate protein (29). The substrate-recruiting components of UPS then catalyze the
formation of an isopeptide bond between the C-terminal
glycine residue of ubiquitin and the e-amino group of a
substrate protein lysine residue. Continual addition of
ubiquitin moieties onto substrate (i.e., polyubiquitination)
facilitates recognition of the substrate by the proteolytic
machinery of the UPS, the 26S proteasome (29, 30). The 26S
proteasome complex is essentially composed of 1 20S and
2 19S units (Fig. 3). The 19S complex has 2 multisubunit
components, often described as the base and the lid. The
base contains 6 ATPases, which belong to the TRIPLE-A
family of ATPases, and 2 non-ATPase subunits, which
bind to the 20S catalytic core. The lid contains up to 10 nonATPase subunits (31, 32). Together, the base and lid
function in the recognition of ubiquitinated substrates
and their subsequent binding. The 20S complex is composed of 28 related subunits (14 different subunits) that
are arranged as 4 heptameric staggered rings. The 2 outer
rings contain the a subunits (a1a7). The 2 inner rings
contain 2 copies of the b subunits (b1b7). Within the 20S
proteasome, subunits b1, b2, and b5 exhibit postglutamyl
peptide hydrolyzing, trypsin-like, and chymotrypsin-like

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cleavage activity, respectively. More-comprehensive

reviews on the function and composition of the 26S
proteasome were recently published (29, 30, 32, 33).
Regulation of VEGF expression by von HippelLindau E3 ubiquitin ligase
The molecular mechanism by which hypoxia sets off
expression of VEGF has been extensively studied
(12, 34). In normoxic conditions (i.e., normal oxygen
levels), VEGF expression is generally inhibited by the
interaction of von Hippel-Lindau (pVHL) E3 ubiquitin
ligase with hypoxia-induced transcription factor 1a
(HIF-1a), leading to its ubiquitination and targeting
HIF-a for degradation by 26S-proteasome. Two of the
3 HIF-a isoforms, HIF-1a and HIF-2a, are closely related and can interact with hypoxia response elements to
induce VEGF expression (35, 36), whereas HIF-3a
appears to be involved in the negative regulation of
hypoxia-induced gene expression (37).
Oxygen-mediated posttranslational modification of
HIF-a through non-heme and iron-dependent oxygenases that uniquely hydroxylate specific HIF-a at proline
residues regulates its transcriptional activity. Hydroxylation of human HIF-1a at 2 proline residues (Pro402 and
Pro564) by prolyl hydroxylase domain (PHD) proteins
creates binding sites for the pVHL E3 ubiquitin ligase
complex that targets HIF-1a for proteasomal degradation
(38, 39). These proline hydroxylation sites contain a conserved LXXLAP (where X indicates any amino acid) motif
that is recognized by PHD proteins, leading to HIF-1a

hydroxylation by PHD (38, 40). Of interest, hydroxylation

of an asparagine residue (Asn803) in the C-terminal activation domain of HIF-1a by HIF asparagine hydroxylase,
termed factor inhibiting HIF (FIH), inhibits HIF-1a activity by blocking interaction of the HIF-1a C-terminal activation domain with the transcriptional coactivator, p300
(41). Under hypoxic conditions, however, VEGF is generally overproduced, which leads to pathologic angiogenesis. In response to low oxygen, pVHL E3 ubiquitin ligase
is S-nitrosylated (the covalent attachment of a nitrogen
monoxide group to the thiol side chain of cysteine), which
blocks HIF-a interaction with pVHL E3 ubiquitin ligase.
HIF-1a escapes from ubiquitin-mediated degradation as a
consequence of S-nitrosylation of pVHL (Fig. 4). Nitric
oxidemediated S-nitrosylation of HIF-1a at the cysteine
residue (C800) permits interaction of HIF-a with p300,
prompting its transcription activity and VEGF expression
(42).
Another important aspect and additional complexity of
HIF-a regulation is the function of heat shock protein 90
(Hsp90). Hsp90 is often upregulated under cellular stress
conditions, such as hypoxia (43), which appears to prevent HIF-1a degradation in a pVHL-independent manner
(4345). Consistent with the protective role of Hsp90 in
HIF-1a, agents that inhibit Hsp90 activity have also been
shown to promote ubiquitin-mediated degradation of
HIF-1a (44, 46). A recent study indicated that inhibition
of Hsp90 by hemin, a derivative of the protoporphyrin
compound, increases HIF-1a ubiquitination and hence
angiogenesis (47).

Figure 4. Role of pVHL in expression of VEGF and angiogenesis. A, in the presence of oxygen, proline residues in the oxygen-dependent degradation domain of
HIF are hydroxylated. This allows HIF-a to interact with pVHL. The interaction between HIF and pVHL causes degradation of HIF through ubiquitination.
B, in response to low oxygen (i.e., hypoxia), pVHL is S-nitrosylated, preventing HIF-a from interacting with pVHL, and the degradation of HIF-a is
disallowed. HIF-a stimulates expression of VEGF, which in turn stimulates angiogenesis, as manifested in cancer progression. S-nitrosylation is the covalent
attachment of a nitrogen monoxide group to the thiol side chain of cysteine.

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b-Transducin repeat-containing protein ubiquitin E3

ligase controls ubiquitination and degradation of
VEGFR-2
Activation of VEGFR-2 by VEGF family ligands mediates most of the known VEGF cellular responses
(13, 19, 48). Central to proper regulation of the angiogenic
activity of VEGFR-2 is the process by which VEGFR-2
triggers its own internalization and degradation, consequently terminating its angiogenic signaling. Upon stimulation with VEGF family proteins, VEGFR-2 is removed
from the cell membrane and undergoes clathrin-dependent endocytosis (49), which initiates its degradation (50)
and recycling (51). Of interest, VEGFR-2 internalization is
stabilized by cadherin-5 (52). Cadherin-5dependent stabilization of VEGFR-2 is established by reducing the
tyrosine phosphorylation of VEGFR-2, perhaps by recruiting tyrosine phosphatases to VEGFR-2 (49, 53). Ligandmediated degradation of VEGFR-2 requires tyrosine
kinase activity, and activation of the protein kinase C
(PKC) pathway accelerates its degradation (50). On the
other hand, activation of p38 mitogen-activated protein
kinase (MAPK) has been shown to stabilize VEGFR-2 (52),
suggesting that the stability and degradation of VEGFR-2
in endothelial cells are highly fine-tuned by the activities
of the PKC and p38 MAPK pathways. Initial studies
showed that the carboxyl terminal of VEGFR-2 plays a
pivotal role in VEGFR-2 stability and degradation. Progressive deletion of the carboxyl terminal of VEGFR-2 was
shown to inhibit ligand-dependent degradation of
VEGFR-2 (48, 50). A recent study identified the presence
of a PEST domain in the carboxyl domain of VEGFR-2,
which may account for the critical role of the carboxyl
domain in VEGFR-2 degradation (52). The PEST motif
[rich in proline (P), glutamic acid (E), serine (S), and
threonine (T)] is considered to be a signature of shortlived proteins that are degraded by the ubiquitin pathway
(54). It is thought that PEST sequences are unstructured
regions in certain protein sequences, which may serve as a
phosphodegron for the recruitment of F-boxcontaining
ubiquitin E3 ligases leading to ubiquitination and degradation (55, 56). Phosphorylation of Ser1188 and Ser1191 of
the PEST domain of VEGFR-2 recruits SCF-b-transducin
repeat-containing protein 1 (Trcp1) E3 ubiquitin ligase
to VEGFR-2, leading to SCF-bTrcp1dependent ubiquitination and degradation of VEGFR-2. Degradation of
VEGFR-2 is mainly attained through Lys48-linked polyubiquitination (52).
b-TrCPs (also called FWD1) belong to a larger family of
Fbw (F-box/WD40 repeat containing) proteins that are
generally characterized by the presence of a 4248 aminoacid F-box motif at the N-terminus and 7 WD40 repeats at
the C-terminus (57). b-TrCPs are highly conserved across
species, in particular within the F-box motif and WD40
repeats motif. Human b-TrCP1 and b-TrCP2 exist in
multiple isoforms due to alternatively spliced mRNA,
but all are conserved in F-box and all 7 WD40 repeats
(58, 59). The most notable differences in sequences
between b-TrCP1 and b-TrCP2 are found in their N-

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terminal regions, which are proximal to the F-box motif.

It is thought that the N-terminal sequences of b-TrCP1 and
b-TrCP2 allow these proteins to undergo homo- and
heterodimerization (60). It appears that both b-TrCP1 and
b-TrCP2 can promote ubiquitination of VEGFR-2, which
suggests that to some extent they may act in a redundant
fashion in VEGFR-2 ubiquitination (52). A redundant role
of mammalian b-TrCP1 and b-TrCP2 in ubiquitination
and degradation of other proteins, including IkB and
b-catenin, has also been suggested (61, 62).
Role of ubiquitination in phospholipase Cg1
activation and angiogenesis
Activation of phospholipase Cg1 (PLCg1) in endothelial
cells is considered to be one of the chief mediators of the
angiogenic signaling of VEGFR-2. It catalyzes the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol 4,5-bisphosphate (PIP2).
Phosphotyrosine 1173 on mouse VEGFR-2 (corresponding to Tyr1175 on human VEGFR-2) has been identified as the primary site responsible for the recruitment of
PLCg1 to VEGFR-2 (26, 63, 64). Substantial information
obtained from animal models links PLCg1 to angiogenesis. The initial evidence linking PLCg1 to endothelial cell
function and angiogenesis was provided by targeted
deletion of PLCg1, which resulted in early embryonic
lethality between embryonic days 9.5 and 10.5 due to
significantly impaired vasculogenesis and erythrogenesis
(65). Inactivation of PLCg1 in zebrafish was also shown to
be required for VEGF function and arterial development
(66). Additional evidence of the importance of PLCg1 in
angiogenic signaling of VEGFR-2 was obtained by pharmacological inhibition of PLCg1. U73122, a potent PLCg1
inhibitor, was shown to inhibit endothelial cell tube formation in vitro (64) and angiogenesis in vivo in a chorioallantoic membrane assay (67). Silencing the expression of
PLCg1 in primary endothelial cells by an siRNA strategy
also inhibits VEGF-mediated endothelial cell tube formation and proliferation (68), further underscoring the
importance of the PLCg1 pathway for angiogenic signaling of VEGF.
PLCg1 is a multidomain protein that consists of 2 SH2
domains and 1 SH3 domain between the catalytic
domains. The SH2 domains recognize phosphotyrosine
1173 on VEGFR-2 (64), whereas the SH3 domain recognizes proline-rich sequences (PXXP motifs). In addition to
its SH domains, PLCg1 also contains a C2 domain, EF
hand, and 2 putative PH domains. The presence of both Nand C-terminal SH2 domains is required for optimal
binding of PLCg1 to VEGFR-2 (64). PLCg1 also interacts
with c-Cbl through its proline-rich motif in a noninducible
manner (50). As a result of activation by VEGFR-2, c-Cbl
is recruited to VEGFR-2 and distinctly inhibits phosphorylation of Tyr783 on PLCg1 in an ubiquitination-dependent manner (26, 68). c-Cbl negatively regulates PLCg1
activation in a proteolysis-independent manner. Instead
of targeting it for degradation, c-Cbl distinctively
mediates ubiquitination of PLCg1 and suppresses its

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phosphorylation on Y783 (26). How PLCg1 can escape

from ubiquitin-mediated degradation but then enter into
a less enzymatic active state is a conundrum that warrants
further investigation.
Endothelial cells derived from c-Cbl knockout mice also
showed that loss of c-Cbl results in an increased phosphorylation of PLCg1 with no apparent effect on its halflife (68). Overexpression of c-Cbl in endothelial cells has
also been shown to inhibit tube formation and sprouting
of endothelial cells. Conversely, overexpression of c-Cbl
(70Z/3-Cbl), an E3 ligase-deficient variant form of c-Cbl,
or silencing its expression by siRNA elevated sprouting of
endothelial cells (26). Recent studies showed that VEGFand tumor-induced angiogenesis is highly elevated in
c-Cbl nullizygous mouse (67, 68). It appears that the role
of c-Cbl in angiogenesis is widespread, because laserinduced angiogenesis in c-Cbl knockout mice also results
in enhanced retinal neovascularization (67).
Role of ubiquitination in the PI3 kinase/AKT
pathway
The phosphoinositide 3-kinase (PI3K) signal transduction pathway is one of the main signaling routes
that VEGFR-2 uses to stimulate endothelial cell survival
and proliferation (6971). VEGFR-2 activates PI3K
through recruitment of p85 of PI3K involving Tyr799 and
Tyr1173 (67, 68). PI3K consists of an 85-kDa regulatory
subunit and a 110-kDa catalytic subunit. It is a lipid
kinase that converts the plasma membrane lipid PIP2 to
phosphatidylinositol-3,4,5-triphosphate (PIP3). Proteins
with pleckstrin-homology (PH) domains, such as protein
kinase B (PKB/AKT), phosphoinositide-dependent
kinase-1 (PDK-1), and PDK-2, bind to PIP3. AKT is activated by PIP3, PDK1, and PDK2, leading to phosphory-

lation of a host of other proteins that affect cell proliferation, cell cycle progression, and cell survival (73). The
Cbl-b ubiquitin E3 ligase is known to interact with the p85SH2 domain and catalyze p85 polyubiquitination (27,74).
Of interest, the Cbl-mediated ubiquitination does not lead
to degradation of p85 (27).
AKT, a serine/threonine protein kinase, is one of the
key PI3K substrates that play a central role in mediating
VEGFR-2dependent cellular events in endothelial (75,
76). It was recently shown that the carboxyl terminus of
Hsc-70-interacting protein (CHIP) interacts with AKT and
induces its ubiquitination (77). In addition, tetratricopeptide repeat domain 3 (TTC3) containing E3 ligase was
recently linked to AKT ubiquitination and degradation
(78, 79). Of interest, TTC3 interacts only with active AKT,
and not inactive AKT, in the nucleus (78, 79), indicating
that perhaps TTC3-mediated AKT ubiquitination is
important for controlling AKT signaling in the nucleus.
TTC3 itself is the target of AKT and is phosphorylated at
S378 by AKT, and this phosphorylation appears to be
necessary for TTC3 E3 ligase activity (78, 79). BRCA1 is
another E3 ligase that also interacts with activated AKT
and targets it for ubiquitination and degradation (80).
Recent studies also showed that AKT is ubiquitinated by
TRAF6 E3 ligase. TRAF6 directly interacts with and
induces AKT ubiquitination (28). TRAF6-mediated AKT
ubiquitination takes place through the K63-linked modification and does not trigger AKT degradation. K63 chain
polyubiquitination of AKT contributes to its membrane
localization, where it is phosphorylated (28). Figure 5
summarizes various ubiquitin E3 ligases that are involved
in fine-tuning the abundance and activation of key angiogenic proteins. Some ubiquitin E3 ligases, such as Cbl
family proteins, target multiple angiogenic proteins,

Figure 5. Regulation of core angiogenic proteins by ubiquitin E3

ligases. Expression of VEGF is regulated by activity of pVHL.
bTrcp1 ubiquitinates VEGFR-2 and subjects it to proteasomal
degradation. c-Cbl catalyzes ubiquitination of numerous
angiogenic signaling proteins, including VEGFR-1, Eph, Tie2, and
PLCg1. AKT abundance and activity are regulated by TRAF6,
CHIP, and TTC3. Itch regulates degradation of the Notch1
receptor.

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Ubiquitin-Proteasome System in Angiogenesis

whereas in other cases more than 1 ubiquitin E3 ligase is

involved in the ubiquitination of an angiogenic protein, as
illustrated for AKT (Fig. 5).

Dvl. Surprisingly, in neuronal cells, Dvl uniquely is ubiquitinated by the HECT-type E3 ligase NEDL1, and not by
KLHL12 (90), suggesting a cell-typespecific regulation of
Dvl by the UPS.

Role of Ubiquitination in Wnt Signaling

The ubiquitination system as a potential target for

antiangiogenesis and anticancer therapy
Given that the expression and degradation of core
proangiogenic proteins are regulated by the UPS, selectively targeting the different components of this pathway
may prove to be an effective strategy for antiangiogenesis
treatments. Our increasing understanding of the ubiquitination system and its role in angiogenesis is generating
great interest in the development of novel strategies to
block pathologic angiogenesis. The role of the ubiquitination pathway in human diseases in general, and in angiogenesis in particular, is still unclear because the molecular
mechanisms and gene products involved in the UPS are
not fully understood. Moreover, there has been no comprehensive analysis of the functional importance of the
UPS in angiogenesis-associated human diseases. The UPS
has many different components that potentially could be
targeted for inhibition or stimulation in the milieu of
angiogenesis. For example, the therapeutic value of the
proteasome inhibitor bortezomib (Velcade; Millennium
Inc.), the first UPS-targeting drug to be approved by the
U.S. Food and Drug Administration for treatment of
relapsed or refractory multiple myeloma (91), could be
explored in angiogenesis-associated diseases.
Recent studies have linked the potential therapeutic
value of inhibition of the proteasome pathway to angiogenesis. Indeed, treatment of endothelial cells in a cell
culture system with proteasome inhibitors was shown to
inhibit capillary tube formation of endothelial cells and
blood vessel formation in an embryonic chick chorioallantoic membrane assay (92, 93). Moreover, bortezomib
was shown to inhibit tumor angiogenesis in a murine
xenograft model (94). More recently, it was shown that
small molecules such as nutlins (Roche, Inc.) and RITA
can block p53 ubiquitination by inhibiting the activity of
MDM2 ubiquitin ligase, which is known to mediate p53
ubiquitination (9597). Loss of p53 activity is linked to
both tumor growth and angiogenesis, suggesting that, in
principle, the application of nutlins in cancer treatment
could target both tumor cells and angiogenesis. PR-171
(Proteolix, Inc.), a synthetic analog of epoxomicin, is
another proteasome inhibitor that was reported to irreversibly inhibit the chymotryptic site of the 26S proteasome, and initial studies suggested that it has more potent
anticancer activity than bortezomib (98). Moreover, recent
patent applications indicate that the ubiquitin activating
enzyme, E1, could also be targeted for possible therapeutic use (99, 100). More-comprehensive reviews on inhibition of the 26S proteasome and drug discoveries were
recently published (101, 102). Numerous ubiquitin E3
ligases are involved in the regulation of angiogenesis;
however, it remains to be determined whether a particular
ubiquitin E3 ligase can be exploited as a target for

The Wnt pathway is another key player in angiogenesis.

Binding of Wnt to its 7-span transmembrane receptor,
Frizzled (Fz), and its coreceptor, Lrp5/6, at the cell surface
initiates a signaling cascade that mediates angiogenesis
and other key developmental processes, including stem
cell maintenance, growth, and cell-fate specification, and
cell migration (81). Deregulation of the activation of Wnt/
b-catenin signaling has been linked to a range of human
diseases, including cancer (81, 82). During the resting state
of canonical Wnt signaling, several key Wnt-associated
signaling proteins, including b-catenin, are targeted via
ubiquitination for degradation. Initially, the adenomatous
polyposis coli protein forms a complex with glycogen
synthase kinase 3b (GSK-3b) and axin. This complex then
binds to b-catenin in the cytoplasm, which leads to phosphorylation of b-catenin by casein kinase 1 (CK1) and
GSK-3b. Phosphorylation leads to the creation of a phosphodegron motif on b-catenin that allows the ubiquitin E3
ligases (e.g., b-Trcp) and Jade-1 to recognize b-catenin. As
a result, b-catenin is targeted for ubiquitination, leading to
its 26S-proteasomemediated degradation (83, 84).
In addition, other ubiquitin E3 ligases (e.g., Siah1 and
Ozz) also target b-catenin for degradation in a cell-type
or context-specific manner (85, 86).
Removal of cytosolic b-catenin through the UPS prevents b-catenin from translocating into the nucleus, where
it acts as a transcription factor for genes associated with
various angiogenic events, such as proliferation of endothelial cells. In contrast, activation of the canonical Wnt
signaling pathway results in inhibition of b-catenin degradation, leading to increased cytosolic b-catenin, which
then translocates to the nucleus. In the nucleus, b-catenin
associates with at least one of a family of Tcf/Lef transcription factors and induces the expression of numerous
genes, such as cyclinD1 and c-myc (87), which are implicated in cellular proliferation.
In addition to Wnt pathwaymediated ubiquitination
of b-catenin, the stability of the b-catenin protein is regulated by ubiquitination of cadherins. For example, the
c-Cblrelated ubiquitin E3 ligase Hakai associates with
E-cadherin, promoting its degradation and resulting in
destruction of the cadherin-b-catenin complex and its
degradation (88).
Of interest, in addition to regulation of b-catenin protein
levels by ubiquitination, the levels and subcellular functions of Dvl are tightly regulated via multiple ubiquitindependent pathways.
Binding of the Kelch-like 12 (KLHL12) E3 ligase to Dvl is
regulated by Wnt stimulation. Subsequent ubiquitination
of Dvl leads to its proteasomal degradation (89), suggesting that KLHL12 acts as a Wnt-mediated negative regulator of the Wnt pathway by inducing the degradation of

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Rahimi

molecular therapeutic approaches in angiogenesis-associated diseases. Regardless of whether a particular ubiquitin E3 ligase can be targeted for therapeutic approaches
in angiogenesis-associated diseases, broad studies are
clearly required to increase our understanding of their
role in angiogenesis. In light of the current drug-discovery
activities involving the UPS, it is evident that by harnessing the UPS we may be able to design strategies for
different components of the UPS to selectively target
proteins for ubiquitination/degradation or inhibit protein
degradation. Hence, it is not unreasonable to expect more
drug-discovery efforts based on ubiquitin with the aim of
targeting proteins with pro- and antiangiogenesis activities in the near future.

however, these studies represent only the beginning of

our attempt to understand how this important pathway
functions in the regulation of angiogenesis. Characterizing the nature of this system in angiogenesis and the
complexity of the UPS will undoubtedly have many
therapeutic applications. Understanding the molecular
basis of the UPS and the target protein substrates in
endothelial cells may also provide a foundation for
learning to stimulate or inhibit angiogenesis. Finally, it
is reasonable to envision the UPS as a key component of
the angiogenic switch in cancer and other types of
pathologic angiogenesis.
Disclosure of Potential Conicts of Interest
No potential conflicts of interest were disclosed.

Conclusions and Perspectives

The emerging role of the UPS in regulating angiogenesis highlights the importance of investigating this
pathway in the milieu of angiogenesis. The reports
outlined in this review provide examples of regulation
of angiogenesis by various components of the UPS;

Grant Support
National Eye Institute, National Institutes of Health; Department of
Pathology, Boston University; Massachusetts Lions Foundation (to
Department of Ophthalmology, Boston University).
Received July 29, 2011; revised October 21, 2011; accepted November 11,
2011; published OnlineFirst February 21, 2012.

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Published OnlineFirst February 21, 2012; DOI: 10.1158/1535-7163.MCT-11-0555

The Ubiquitin-Proteasome System Meets Angiogenesis

Nader Rahimi
Mol Cancer Ther 2012;11:538-548. Published OnlineFirst February 21, 2012.

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