Receptor Ubiquitylation Research Articles

Epigen, the Last Ligand of ErbB Receptors, Reveals Intricate Relationships between Affinity and Mitogenicity

Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands.

Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cbl-mediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation.

Kekkon proteins negatively regulate the epidermal growth factor receptor (EGFR) during oogenesis in Drosophila. Their structural relative in mammals, LRIG1, is a transmembrane protein whose inactivation in rodents promotes skin hyperplasia, suggesting involvement in EGFR regulation. We report upregulation of LRIG1 transcript and protein upon EGF stimulation, and physical association of the encoded protein with the four EGFR orthologs of mammals. Upregulation of LRIG1 is followed by enhanced ubiquitylation and degradation of EGFR. The underlying mechanism involves recruitment of c-Cbl, an E3 ubiquitin ligase that simultaneously ubiquitylates EGFR and LRIG1 and sorts them for degradation. We conclude that LRIG1 evolved in mammals as a feedback negative attenuator of signaling by receptor tyrosine kinases.

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases.

Growth factors and their transmembrane receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis, including multiple stages of cancer. The amplitude and kinetics of signaling by growth factor receptors are determined by an endocytic process, which sorts activated, autophosphorylated receptors to degradation in lysosomes. Recent studies uncovered the role of protein ubiquitylation in vesicular trafficking of growth factor receptors. Decoration of ligand-activated receptors by multiple monomeric ubiquitins distinguishes this degradative route from the proteasome-mediated pathway, which involves polymeric chains of ubiquitin. Although receptor ubiquitylation occurs at the cell surface, its major role is to sort internalized receptors to the lumen of the multivesicular body, en route to the lysosome. The ubiquitin ligases that control this late sorting event belong to the Cbl family of RING finger adaptors, which bind specific phosphotyrosine residues in the receptors upon activation by ligand. Another group of E3 ubiquitin ligases, the Nedd4 family, regulates the initial sorting event, which targets receptors to clathrin-coated regions of the plasma membrane. This step entails ubiquitin-dependent assembly of a clathrin-binding complex of adaptors such as epsins, which share ubiquitin-interacting motifs. The concerted action of both ubiquitin-binding adaptors of membrane coats and E3 ligases, as well as their regulation by protein phosphorylation and ubiquitylation, ensure robust endocytosis of growth factor receptors. Genetic defects and virus-mediated manipulations of the endocytic pathway divert receptors to a default recycling pathway, thereby enabling unrestrained signaling characteristic to transformed cells.

Endocytosis of the AT 1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region

Agonist-induced internalisation of the rat type 1A (AT 1A) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the α-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT 1A receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT 1A receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT 1A receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a β-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT 1A receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.

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.

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs, an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4.

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.

Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase.

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Here we show that Akt and Mdm2 form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, we demonstrate that the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation.

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling.

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.