Ubiquitylation Of Epidermal Growth Factor Receptor Research Articles

Proteasomal deubiquitylase activity enhances cell surface recycling of the epidermal growth factor receptor in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) represents a top therapeutic target in the treatment of non-small cell lung cancer. EGFR expression is intricately modulated by receptor endocytosis, during which EGFR ubiquitylation and deubiquitylation play fundamental roles to govern receptor fate. This study aims to uncover novel aspects of the endocytic regulation of EGFR trafficking by deubiquitylases. The expression and ubiquitylation of EGFR in non-small cell lung cancer cells treated with deubiquitylase inhibitors were assessed by immunoblotting, immunoprecipitation and mass spectrometry analyses. The intracellular EGFR distribution was investigated using immunofluorescence and confocal microscopy assays, and colocalizations with endocytic compartments were examined using GFP-tagged Rab proteins as markers. The influence of the proteasomal deubiquitylase inhibitor b-AP15 on EGF- and HSP90 inhibitor-induced EGFR downregulation was evaluated by immunoblotting. The anticancer effects of b-AP15 were assessed by cell proliferation, colony formation and flow cytometry assays, as well as xenograft animal models. We found that b-AP15 caused a dramatically enhanced ubiquitylation of EGFR in lung cancer cells. Treatment with b-AP15 decreased cell surface EGFR levels and accumulated EGFR on recycling endosomes marked with Rab4A and Rab11A. b-AP15 effectively repressed EGF- and HSP90 inhibitor-induced EGFR degradation. Lung cancer cells exposed to b-AP15 showed markedly reduced cell propagation and significantly increased cell apoptosis. Furthermore, b-AP15 effectively inhibited tumor xenograft growth in nude mice. Proteasomal USP14 and UCHL5 act collectively to promote cell surface recovery of EGFR. Inhibition of proteasomal deubiquitylase activity induces increased EGFR ubiquitylation and retention on recycling endosomes. The USP14 and UCHL5 dual inhibitor b-AP15 elicits potent tumor-suppressive effects to deter cell proliferation and induce apoptotic cell death in lung cancer.

PTPIP51 inhibits non-small-cell lung cancer by promoting PTEN-mediated EGFR degradation

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts with two non-receptor tyrosine phosphatases and induces apoptosis. In the present study, we showed that PTPIP51 is downregulated in non-small cell lung cancer (NSCLC), and its elevated expression correlates with improved outcomes. PTPIP51 overexpression in NSCLC cells significantly inhibits downstream epidermal growth factor receptor (EGFR) signaling in PI3K/Akt, RAS/RAF/ERK, and JAK/STAT3 pathways. The efficacy of the EGFR inhibitor gefitinib improves in combination with PTPIP51 to accelerate apoptosis and inhibit NSCLC growth in vivo and in vitro. Here, we demonstrated that PTPIP51 interacts with phosphatase and tensin homolog (PTEN) to form a PTPIP51-PTEN-CK2 complex, which induces phosphorylation of the C-tail region of PTEN (p-PTEN Thr382 and Thr383). This subsequently induces ubiquitylation of EGFR and its degradation via lysosomes. Therefore, PTPIP51 acts as a tumor suppressor in NSCLC by inducing PTEN phosphorylation and by promoting EGFR degradation.

Knockout of c-Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation.

Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand‐dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c‐Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c‐Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up‐regulated. As compared to the control cells, EGF‐stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half‐life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2–3 h as compared to control cells. We propose antagonizing c‐Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling.

GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation.

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.

Development of c‐Cbl Antagonists to Promote Corneal Epithelial Wound Healing

Ligand mediated desensitization is a key negative regulator of cell surface receptors. In the human cornea, epidermal growth factor receptor (EGFR) desensitization, due to the relatively high levels of endogenous EGF in the tear fluid, limits the repair and restoration of the corneal epithelium. The E3 ubiquitin ligase, c‐Cbl is a critical modulator of EGFR desensitization, as it mediates the ubiquitylation of the activated receptor and targets it to the lysosome for degradation. Ultimately, this terminates receptor signaling. We have previously demonstrated that knock down of c‐Cbl results in an enhancement of EGFR‐mediated cell migration that is necessary for healing of corneal epithelial wounds. The goal of these experiments was to identify novel compounds that antagonize c‐Cbl activity and could be developed to enhance EGFR‐mediated corneal epithelial wound healing. Using the published crystal structures of the EGFR:c‐Cbl complex, we identified the point of interaction between the two proteins. We subsequently performed an in silico screen of ~25,000,000 compounds (from the ZINC15 library) to identify compounds that are predicted to antagonize c‐Cbl binding to and ubiquitylation of the EGFR. The top 50 candidates were screened using a biochemical assay for their ability to bind c‐Cbl using Differential Scanning Fluorimetry (Thermofluor™). From that screen, six compounds were identified as c‐Cbl binders and were tested in immortalized corneal epithelial cells to determine which compounds could inhibit c‐Cbl function. Two of the six compounds blocked EGF‐mediated EGFR ubiquitylation, albeit at relatively high concentrations (IC50 were ~50 μM and ~5 μM). Further, these compounds exhibited some cytotoxicity at 100 μM. These compounds represent two lead compounds that can be chemically optimized to enhance their affinity for c‐Cbl and become more potent inhibitors of c‐Cbl activity. The long‐term goal is to use these compounds as therapeutic agents to promote the restoration of damaged corneal epithelium, to minimize patient discomfort, reduce ocular infections, and minimize the likelihood of blindness.Support or Funding InformationResearch was supported by NIH/NEI grant EY027032.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EGF receptor signaling, phosphorylation, ubiquitylation and endocytosis in tumors in vivo.

Despite a well-established role for the epidermal growth factor receptor (EGFR) in tumorigenesis, EGFR activities and endocytosis in tumors in vivo have not been studied. We labeled endogenous EGFR with GFP by genome-editing of human oral squamous cell carcinoma cells, which were used to examine EGFR-GFP behavior in mouse tumor xenografts in vivo. Intravital multiphoton imaging, confocal imaging of cryosections and biochemical analysis revealed that localization and trafficking patterns, as well as levels of phosphorylation and ubiquitylation of EGFR in tumors in vivo closely resemble patterns and levels observed in the same cells treated with 20-200 pM EGF in vitro. Consistent with the prediction of low ligand concentrations in tumors, EGFR endocytosis was kinase-dependent and blocked by inhibitors of clathrin-mediated internalization; and EGFR activity was insensitive to Cbl overexpression. Collectively, our data suggest that a small pool of active EGFRs is sufficient to drive tumorigenesis by signaling primarily through the Ras-MAPK pathway.

Cell Surface Epidermal Growth Factor Receptors Increase Src and c-Cbl Activity and Receptor Ubiquitylation

There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple "moving parts," dissecting the spatial versus temporal contributions has been challenging. Blocking all endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation.

RASopathy-associated CBL germline mutations cause aberrant ubiquitylation and trafficking of EGFR.

Noonan syndrome, a congenital disorder comprising a characteristic face, short stature, heart defects, learning difficulties, and a predisposition to malignancies, is caused by heterozygous germline mutations in genes encoding components of RAS-MAPK signaling pathways. Mutations in the CBL tumor suppressor gene have been reported in patients with a Noonan syndrome-like phenotype. CBL encodes a multivalent adaptor protein with ubiquitin ligase activity, which promotes ubiquitylation and vesicle-mediated internalization and degradation of the epidermal growth factor (EGF) receptor (EGFR). We investigated the functional consequences of disease-associated CBL amino acid changes p.K382E, p.D390Y, and p.R420Q on ligand-induced EGFR trafficking. Expression of CBL(K382E), CBL(D390Y), or CBL(R420Q) in COS-7 cells resulted in increased levels of surface EGFR and reduced amounts of intracellular EGFR; both consequences indicate ineffective EGFR internalization. Accordingly, receptor-mediated uptake of EGF was decreased. Furthermore, the p.K382E, p.D390Y, and p.R420Q lesions impaired CBL-mediated EGFR ubiquitylation and degradation. Together, these data indicate that pathogenic CBL mutations severely affect vesicle-based EGFR trafficking. Since we detected enhanced ERK phosphorylation in cells expressing mutant CBL, we conclude that aberrant EGFR trafficking contributes to augmented RAS-MAPK signaling, the common trait of Noonan syndrome and related RASopathies. Thus, our data suggest that EGFR trafficking is a novel disease-relevant regulatory level in the RASopathy network.

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.

Rab5 Isoforms Differentially Regulate the Trafficking and Degradation of Epidermal Growth Factor Receptors

Ligand-mediated endocytosis is an intricate regulatory mechanism for epidermal growth factor receptor (EGFR) signal transduction. Coordinated trafficking of EGFR ensures its temporal and spatial communication with downstream signaling effectors. We focused our work on Rab5, a monomeric GTPase shown to participate in early stages of the endocytic pathway. Rab5 has three isoforms (A, B, and C) sharing more than 90% of sequence identity. We individually ablated endogenous isoforms in HeLa cells with short interfering RNAs and examined the loss-of-function phenotypes. We found that suppression of Rab5A or 5B hampered the degradation of EGFR, whereas Rab5C depletion had very little effect. The differential delay of EGFR degradation also corresponds with retarded progression of EGFR from early to late endosomes. We investigated the activators/effectors of Rab5A that can potentially separate its potency in EGFR degradation from other isoforms and found that Rin1, a Rab5 exchange factor, preferably associated with Rab5A. Moreover, Rab5A activation is sensitive to EGF stimulation, and suppression of Rin1 diminished this sensitivity. Based on our results together with previous work showing that Rin1 interacts with signal transducing adapter molecule to facilitate the degradation of EGFR (Kong, C., Su, X., Chen, P. I., and Stahl, P. D. (2007) J. Biol. Chem. 282, 15294-15301), we hypothesize that the selective association of Rab5A and Rin1 contributes to the dominance of Rab5A in EGFR trafficking, whereas the other isoforms may have major functions unrelated to the EGFR degradation pathway.

Conjugation to Nedd8 Instigates Ubiquitylation and Down-regulation of Activated Receptor Tyrosine Kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.

Cortactin Overexpression Inhibits Ligand-Induced Down-regulation of the Epidermal Growth Factor Receptor

Ligand-induced receptor down-regulation by endocytosis is a critical process regulating the intensity and duration of receptor tyrosine kinase signaling. Ubiquitylation of specific receptor tyrosine kinases, for example, the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, provides a sorting signal for lysosomal degradation and leads to termination of receptor signaling. Cortactin, which couples the endocytic machinery to dynamic actin networks, is encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin overexpression contributes to tumor progression is by enhancing tumor cell invasion and metastasis. However, in this study, we show that overexpression of cortactin in HeLa cells markedly inhibits ligand-induced down-regulation of the EGFR. This is independent of alterations in receptor autophosphorylation and correlates with impaired c-Cbl phosphorylation and association with the EGFR, reduced EGFR ubiquitylation, and sustained EGF-induced extracellular signal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell lines revealed that cortactin overexpression is associated with attenuated ligand-induced EGFR down-regulation. Importantly, RNAi-mediated reduction of cortactin expression in an 11q13-amplified HNSCC cell line accelerates EGFR degradation. This represents the first demonstration of modulation of growth factor receptor signaling by cortactin. Moreover, enhanced EGFR signaling due to cortactin overexpression may provide an alternative explanation for EMS1 gene amplification in human cancers.

Grb2 regulates internalization of EGF receptors through clathrin-coated pits.

The molecular mechanisms of clathrin-dependent internalization of epidermal growth factor receptor (EGFR) are not well understood and, in particular, the sequence motifs that mediate EGFR interactions with coated pits have not been mapped. We generated a panel of EGFR mutants and stably expressed these mutants in porcine aortic endothelial (PAE) cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2 completely abolished receptor internalization in PAE cells. Quantitative analysis of colocalization of EGF-rhodamine conjugate and coated pits labeled with yellow-fluorescent-protein-tagged beta2 subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants lacking Grb2 binding sites do not efficiently enter coated pits. The depletion of Grb2 from PAE as well as HeLa cells expressing endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial steps of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane.

Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors.

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

Human Sprouty Potentiates EGFR Signaling

Tight regulation of growth factor signaling through receptor tyrosine kinases (RTK) is critical to cell differentiation, growth, and survival. Sprouty (Spry) proteins are generally considered inhibitors of RTK signaling whose effects are mediated by interference with the mitogen-activated protein kinase (MAPK) cascade. Studies by Wong et al. and Egan et al. challenge this paradigm by demonstrating that human Sprouty2 (hSpry2) can potentiate epidermal growth factor (EGF) signaling by inhibiting ubiquitylation of the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl. Wong et al. used confocal microscopy to show that overexpressed hSpry2, but not truncated constructs or isoforms that don't bind c-Cbl, inhibited endocytosis of immunofluorescently labeled EGFR in COS-1 cells stimulated with EGF. Transfected c-Cbl accelerated EGF-stimulated receptor down-regulation, whereas hSpry2 enhanced cell surface retention of EGFRs as demonstrated by radiolabeled EGF to EGFR binding. Using Western analysis on immunoprecipitates of intact and mutant proteins from transfected COS-1 cells, as well as an in vitro ubiquitylation assay, Wong et al. demonstrated that hSpry2 inhibited c-Cbl-mediated ubiquitylation of EGFR. Analysis of immunoprecipitates of cells transfected with hSpry2 and UbcH7 (human E2 ubiquitin ligase) indicated that hSpry competed with UbcH7 for binding to the c-Cbl RING finger domain. Decreased EGFR ubiquitylation in response to hSpry correlated with sustained EGF-induced ERK phosphorylation, indicating potentiation of the MAPK pathway. In PC12 cells, transfected hSpry2 induced morphological differentiation--normally associated with nerve growth factor (NGF) or fibroblast growth factor (FGF) stimulation--presumably secondary to sustained EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, hSpry2 inhibited the response to FGF and NGF. These results indicate that, rather than being general inhibitors of RTK signaling, Sprys may inhibit certain specific pathways and potentiate others to fine tune the responses to RTK activation.E. S. M. Wong, M. Wong, C.W. Fong, J. Lin, P. Yusoff, B. C. Low, W. Y. Langdon, G. R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J. 21, 4796-4808 (2002). [Abstract] [Full Text] J. E. Egan, A. B. Hall, B. A. Yatsula, D. Bar-Sagi, The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins, Proc. Natl. Acad. Sci. U.S.A. 99, 6041-6046 (2002). [Abstract] [Full Text]

Human Sprouty Potentiates EGFR Signaling

Tight regulation of growth factor signaling through receptor tyrosine kinases (RTK) is critical to cell differentiation, growth, and survival. Sprouty (Spry) proteins are generally considered inhibitors of RTK signaling whose effects are mediated by interference with the mitogen-activated protein kinase (MAPK) cascade. Studies by Wong et al. and Egan et al. challenge this paradigm by demonstrating that human Sprouty2 (hSpry2) can potentiate epidermal growth factor (EGF) signaling by inhibiting ubiquitylation of the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl. Wong et al. used confocal microscopy to show that overexpressed hSpry2, but not truncated constructs or isoforms that don't bind c-Cbl, inhibited endocytosis of immunofluorescently labeled EGFR in COS-1 cells stimulated with EGF. Transfected c-Cbl accelerated EGF-stimulated receptor down-regulation, whereas hSpry2 enhanced cell surface retention of EGFRs as demonstrated by radiolabeled EGF to EGFR binding. Using Western analysis on immunoprecipitates of intact and mutant proteins from transfected COS-1 cells, as well as an in vitro ubiquitylation assay, Wong et al. demonstrated that hSpry2 inhibited c-Cbl-mediated ubiquitylation of EGFR. Analysis of immunoprecipitates of cells transfected with hSpry2 and UbcH7 (human E2 ubiquitin ligase) indicated that hSpry competed with UbcH7 for binding to the c-Cbl RING finger domain. Decreased EGFR ubiquitylation in response to hSpry correlated with sustained EGF-induced ERK phosphorylation, indicating potentiation of the MAPK pathway. In PC12 cells, transfected hSpry2 induced morphological differentiation--normally associated with nerve growth factor (NGF) or fibroblast growth factor (FGF) stimulation--presumably secondary to sustained EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, hSpry2 inhibited the response to FGF and NGF. These results indicate that, rather than being general inhibitors of RTK signaling, Sprys may inhibit certain specific pathways and potentiate others to fine tune the responses to RTK activation. E. S. M. Wong, M. Wong, C.W. Fong, J. Lin, P. Yusoff, B. C. Low, W. Y. Langdon, G. R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J. 21 , 4796-4808 (2002). [Abstract] [Full Text] J. E. Egan, A. B. Hall, B. A. Yatsula, D. Bar-Sagi, The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins, Proc. Natl. Acad. Sci. U.S.A. 99 , 6041-6046 (2002). [Abstract] [Full Text]

Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling.

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF- mediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DeltaN11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation.