Regulation of angiogenesis is often viewed as a balance between pro-angiogenic and anti-angiogenic factors, and when the balance shifts in favour of angiogenesis stimulators, an angiogenic switch turns on the normally inactive endothelial cells to grow new blood vessels. Recent studies have shown that PLCγ1 (phospholipase Cγ1), a major signalling substrate of VEGFR-2 (vascular endothelial growth factor receptor 2), undergoes c-Cbl-mediated ubiquitination. c-Cbl suppresses tyrosine phosphorylation of PLCγ1 and with it VEGF (vascular endothelial growth factor)-induced endothelial cell proliferation and angiogenesis. Loss of c-Cbl in mice results in enhanced retinal neovascularization, VEGF- and tumour-induced angiogenesis. Notably, this observation suggests that c-Cbl-mediated ubiquitination pathway plays a central role in the ‘angiogenic switch’ employed by the VEGF system. The present article highlights the recent findings demonstrating a novel role for protein ubiquitination in angiogenesis and its potential in angiogenesis-based therapy.
A crucial aspect of many human diseases such as cancer, inflammatory diseases and AMD (age-related macular degeneration) is the formation of new blood vessels or angiogenesis. Regulation of angiogenesis is often viewed as a balance between pro-angiogenic and anti-angiogenic molecules, and when the balance shifts in favour of angiogenesis stimulators, an angiogenic switch turns on the normally quiescent endothelial cells to develop new blood vessels . The timing of endothelial cell activation must be under strict constraint, involving a network of signals that work together to dictate when angiogenesis could occur and come to an end. Although this regulation can be explained by the restricted expression of pro- and anti-angiogenesis molecules, it is less clear how endothelial cells integrate angiogenic signals such as VEGF (vascular endothelial growth factor) inputs quantitatively in order to balance the angiogenic switch, particularly in pathological conditions such as cancer and AMD. Activation of VEGFR-2 (VEGF receptor 2) by VEGF family ligands perhaps is the most critical event in VEGFR-2 signalling and angiogenesis, yet we know little regarding the dynamics of VEGFR-2 activation and its signalling partners. Our recent studies demonstrate that the protein ubiquitination pathway plays a key role in the angiogenic switch by restricting activation of PLCγ1 (phospholipase Cγ1), a key signalling substrate of VEGFR-2, ensuring that endothelial cells do not form new blood vessels.
Role of PLCγ1 in VEGFR-2 signalling and angiogenesis
Activation of VEGFR-2 in endothelial cells orchestrates a wide variety of biological responses that emanate from several key autophosphorylation sites within the cytoplasmic domain of VEGFR-2 [2,3]. Activation of PLCγ1 in endothelial cells is considered among the chief mediators of the angiogenic signalling of VEGFR-2. Phosphorylation of Tyr1173 on the mouse VEGFR-2 (corresponding to Tyr1175 on human VEGFR-2) is identified as a primary site responsible for the recruitment of PLCγ1 to VEGFR-2 [4,5]. Upon activation, PLCγ1 catalyses the hydrolysis of PtdIns(4,5)P2 into the second messenger molecules DAG (diacylglycerol) and Ins(1,4,5)P3. DAG binds to the C1 domain of classical PKC (protein kinase C) (α, βI, βII, γ) and novel PKC (δ, ε, η, θ) at the plasma membrane, resulting in their activation. Ins(1,4,5)P3 stimulates the opening of Ca2+ channels of the endoplasmic reticulum, leading to the release of Ca2+ ions into the cell (Figure 1). The interactions of both DAG and Ca2+ activate the kinase activity of PKCs . PLCγ1 is a multi-domain protein which contains two SH (Src homology) 2 domains and one SH3 (Src homology 3) domain between the catalytic domains. The SH2 domains recognize phosphotyrosine sequences, whereas the SH3 domain recognizes proline-rich sequences, particularly the PXXP motif . In addition to its SH domains, PLCγ1 also contains a C2 domain, an EF hand and two putative PH (pleckstrin homology) domains. The presence of both N- and C-terminal SH2 domains is required for optimal binding of PLCγ1 with VEGFR-2 .
Recruitment and activation of PLCγ1 by VEGFR-2
Substantial information has been obtained from animal models that link PLCγ1 to angiogenesis. The initial evidence linking PLCγ1 to endothelial cell function and angiogenesis was provided by targeted deletion of PLCγ1, which resulted in early embryonic lethality between E (embryonic day) 9.5 and E10.5 due to significantly impaired vasculogenesis and erythrogenesis . Furthermore, inactivation of PLCγ1 in zebrafish established that PLCγ1 is required for the function of VEGF and arterial development, indicating further that PLCγ1 activity is crucial for the downstream signalling of the VEGFR during embryogenesis . Consistent with these notions, another study also shows that mice homozygous for the mutant VEGFR-2Y1173F (a major PLCγ1-binding site on VEGFR-2) knockin allele dies between E8.5 and E9.5 without any organized blood vessels or yolk sac blood islands, and haemopoetic progenitors were severely decreased, similar to the case of VEGFR-2-null mice . It should be noted that multiple proteins, including PLCγ1, PI3K (phosphoinositide 3-kinase) and Shb all are known to be recruited to pTyr1173 of VEGFR-2, thus the observed phenotype for the VEGFR-2Y1173F knockin mouse could not be solely attributed to inactivation of the PLCγ1-binding site on VEGFR-2. Additional evidence for the importance of PLCγ1 in angiogenic signalling of VEGFR-2 comes from the use of U73122, a potent PLCγ1 inhibitor, which has been shown to inhibit VEGF-dependent endothelial cell tube formation in vitro  and angiogenesis in vivo in the CAM (chorioallantoic membrane) assay (D. Husain, E. Ahmed, E. Chou and N. Rahimi, unpublished work). Finally, silencing the expression of PLCγ1 in primary endothelial cells using an siRNA (small interfering RNA) strategy also inhibits VEGF-mediated endothelial cell tube formation and proliferation (D. Husain, E. Ahmed, E. Chou and N. Rahimi, unpublished work), underscoring further the importance of the PLCγ1 pathway for angiogenic signalling of VEGF. Finally, proteins that negatively regulate PLCγ1 activation such as c-Cbl also cause enhanced angiogenesis , supporting further a pivotal role of PLCγ1 activation in angiogenesis.
Ubiquitin is an evolutionarily conserved 76-amino-acid polypeptide and was named for its ubiquitous expression in eukaryotes. Ubiquitin is activated by a ubiquitin-activating enzyme (E1) in an ATP-dependent manner and it is transferred to a ubiquitin-conjugating enzyme (E2), and, eventually, a ubiquitin–protein ligase (E3) specifically attaches ubiquitin to a target protein through the ε-amino group of a lysine residue (Figure 2). E3 ubiquitin ligases are a large family of proteins (~700 in the human genome) that are known to be involved in the regulation of the turnover and activity of many target proteins . E3 ligases are divided into two large groups: the HECT (homologous with E6-associated protein C-terminus) domain-containing E3 ligases, and the RING (really interesting new gene) domain-containing E3 ligases. In the RING-type E3 ligases, there are single subunit E3 ligases (such as Cbl family E3 ligases) and multi-subunit E3 ligases (such as cullin–RING ubiquitin ligases). In recent years, additional E3 ligases have also been identified that use a different domain to recognize E2 conjugating enzymes such as the PHD (plant homeodomain) domain-containing E3 ligases and the U-box E3 ligases [12,13]. 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 [12,14]. It has been shown that ubiquitination can also have an impact on cell signalling by targeting activation of proteins in a proteolysis-independent manner. For example, Cbl-mediated ubiquitination of PLCγ1, a major substrate of VEGFR-2, suppresses its tyrosine phosphorylation and spares it from degradation .
Activation of c-Cbl and PLCγ1 by VEGFR-2 and c-Cbl-dependent ubiquitination of PLCγ1
Multiple ubiquitin molecules can be attached to a target protein by means of either mono-ubiquitination (i.e. attachment of a single ubiquitin to one or multiple lysine residues) or polyubiquitination. Mono-ubiquitination is regarded as a signal for non-proteolytic events such as endocytosis, histone regulation, DNA repair, virus budding and nuclear export . 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. There are seven different lysine residues in ubiquitin that can potentially be employed for ubiquitin chain assembly. Lys48- and Lys29-linked polyubiquitination is generally associated with degradation of target proteins by the 26S proteasome. Curiously, Lys63-linked polyubiquitination is involved in DNA repair, signal transduction and endocytosis, but not degradation by the 26S proteasome [12–14]. Clearly, the ubiquitin system could serve as a versatile protein modifier to control various key protein functions such as turnover, subcellular localization and activation.
c-Cbl mediates ubiquitination of PLCγ1 and inhibits its tyrosine phosphorylation
To translate quantitatively differences in extracellular VEGF ligands and VEGFR-2 activation and to ensure that PLCγ1 activity is appropriately turned off, mechanisms must exist to continuously inactivate PLCγ1. One mechanism by which this is achieved is by controlling tyrosine phosphorylation of PLCγ1. Activation of VEGFR-2 in endothelial cells stimulates phosphorylation of Tyr783 on PLCγ1, which is required for its activation [4,5]. One common and well-understood mechanism by which tyrosine phosphorylation of signalling substrates is controlled is the removal of a phosphate group by the action of PTPs (protein tyrosine phosphatases) . Interestingly, the role of individual PTPs involved in the dephosphorylation of PLCγ1 in endothelial cells or other cell types has not been fully investigated. However, a recent study has identified a distinct mechanism by which tyrosine phosphorylation of PLCγ1 is attained in endothelial cells . Upon stimulation by VEGF, VEGFR-2 recruits and activates a RING finger-containing ubiquitin E3 ligase, c-Cbl. pTyr1057, along with pTyr1052 on VEGFR-2 serve as binding sites to recognize the TKB (tyrosine kinase-binding) domain of c-Cbl (Figure 2). The TKB domain of c-Cbl directly recognizes phosphotyrosine residues in the context of a D(N/D)XpY(S/T)X(E/D) consensus sequence, with an aspartate residue at −3 and an asparagine residue at −2 relative to the phosphotyrosine residue being the most important determinants of specificity [11,16]. The primary amino acid sequence N-terminal to each of Tyr1052 and Tyr1057 on VEGFR-2 indicates that they also lie within a putative TKB domain-binding site. As a result of activation by VEGFR-2, c-Cbl inhibits phosphorylation of Tyr783 on PLCγ1 in a ubiquitin-dependent manner . c-Cbl exerts its effect on PLCγ1 by utilizing its C-terminus region, possibly interacting with the SH3 domain of PLCγ1 .
c-Cbl is involved in ubiquitination of several different RTKs (receptor tyrosine kinases), such as PDGFR (platelet-derived growth factor receptor), HGFR (hepatocyte growth factor receptor) and EGFR (epidermal growth factor receptor), acting as a negative regulator of RTKs by mediating the ubiquitination of its binding partner proteins, with the exception of VEGFR-2 which undergoes c-Cbl-independent ubiquitination and down-regulation . c-Cbl is a multi-domain protein, which contains a TKB domain, a RING finger domain, a proline-rich motif, a ubiquitin-associated domain and a leucine zipper domain. In general, c-Cbl-mediated ubiquitination, including RTKs such as PDGFR and EGFR, is considered to target them for degradation . However, ubiquitination of PLCγ1 by c-Cbl is not associated with its degradation. Graham et al.  showed that overexpression of c-Cbl in Jurket cells inhibits TCR (T-cell receptor)-mediated activation of PLCγ1 without altering its degradation. More recent studies have shown that, in endothelial cells, c-Cbl negatively regulates PLCγ1 activation by VEGFR-2 in a proteolysis-independent manner. Instead of targeting it for degradation, c-Cbl distinctively mediates ubiquitination of PLCγ1 and suppresses its phosphorylation on Tyr783 by VEGFR-2 . c-Cbl-dependent ubiquitination of PLCγ1 has no effect on its binding with VEGFR-2, suggesting that recognition of PLCγ1 by VEGFR-2 is not obstructed by ubiquitination . Further evidence for the role of c-Cbl is obtained from the analysis of endothelial cells derived from c-Cbl-knockout mice where it has been shown that loss of c-Cbl results in an increased phosphorylation of PLCγ1 with no apparent effect on its half-life. To functionally link c-Cbl to activation of PLCγ1, additional results from our laboratory showed that intracellular Ca2+ release is also elevated in endothelial cells deficient for c-Cbl (D. Husain, E. Ahmed, E. Chou and N. Rahimi, unpublished work).
Role of c-Cbl in angiogenesis
Although c-Cbl is not required for mouse embryonic development , the emerging new data now suggest that c-Cbl is involved in pathological angiogenesis. Initial evidence linking c-Cbl with angiogenesis came from overexpression of c-Cbl in PAE (porcine aortic endothelial) cells: overexpression of c-Cbl resulted in inhibition of tube formation and sprouting of PAE 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 PAE cells . To determine further whether c-Cbl is involved in pathological angiogenesis, multiple assays were employed on c-Cbl-nullizygous mice. First, in c-Cbl-nullizygous mice, VEGF-induced angiogenesis is highly elevated compared with wild-type mice. Secondly, tumour-induced angiogenesis is significantly higher in c-Cbl-nullizygous mice. Thirdly, laser-induced angiogenesis in c-Cbl-nullizygous mice results in enhanced retinal neovascularization. Finally, proliferation of endothelial cells derived from c-Cbl-nullizygous mice in response to VEGF is significantly higher than endothelial cells derived from wild-type mice. Early studies identified c-Cbl as a tumour-suppresser gene ; on the basis of our observations, we propose c-Cbl as an anti-angiogenic protein which, upon activation by VEGFR-2, inhibits the pro-angiogenic signalling of VEGFR-2 through ubiquitination of PLCγ1 (Figure 3). Further research is needed to establish whether c-Cbl could be used as a therapeutic target for the possible treatment of angiogenesis-associated diseases.
Proposed model for involvement of c-Cbl in angiogenesis
Molecular and Cellular Mechanisms of Angiogenesis: Biochemical Society Focused Meeting held at University of Chester, Chester, U.K., 15–17 July 2009. Organized and Edited by Ian Zachary (University College London, U.K.) and Sreenivasan Ponnambalam (Leeds, U.K.).
age-related macular degeneration
epidermal growth factor receptor
porcine aortic endothelial
platelet-derived growth factor receptor
protein kinase C
protein tyrosine phosphatase
really interesting new gene
receptor tyrosine kinase
small interfering RNA
vascular endothelial growth factor
Work is supported by grants from the National Institutes of Health and the Massachusetts Lions Foundation.