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Differential Regulation of EGF Receptor Internalization and Degradation by Multiubiquitination within the Kinase Domain
Abstract
Ubiquitination of the EGF receptor (EGFR) is believed to play a critical role in regulating both its localization and its stability. To elucidate the role of EGFR ubiquitination, tandem mass spectrometry was used to identify six distinct lysine residues within the kinase domain of the EGFR, which can be conjugated to ubiquitin following growth factor stimulation. Substitution of these lysine residues with arginines resulted in a dramatic decrease in overall ubiquitination but preserved normal tyrosine phosphorylation of EGFR. Ubiquitination-deficient EGFR mutants displayed a severe defect in their turnover rates but were internalized at rates comparable to those of wild-type receptors. Finally, quantitative mass spectrometry demonstrated that more than 50% of all EGFR bound ubiquitin was in the form of polyubiquitin chains, primarily linked through Lys63. Taken together, these data provide direct evidence for the role of EGFR ubiquitination in receptor targeting to the lysosome and implicate Lys63-linked polyubiquitin chains in this sorting process.
... EGFR can be endocytosed via multiple different mechanisms, but whether an endocytosed receptor is subsequently degraded or recycled, is mainly governed by phosphorylation-dependent ubiquitination 79 . Upon ligand stimulation, activated EGFR phosphorylates up to 9 tyrosines on its intracellular C-terminal tail, which acts as binding sites for various downstream signaling molecules, including the E3 ligase CBL 80 , which can subsequently ubiquitinate EGFR. ...
... Furthermore, the CBL ubiquitination site (i.e. secondary degron) is on the EGFR protein kinase domain and thus distal to the site of CBL/GRB2 recruitment 79 . Ubiquitination, which is mostly K63-linked, subsequently recruits reader enzymes, which facilitate the direction of EGFR to nonclathrin-mediated endocytosis and lysosomal degradation 79 (Fig. 3). ...
... secondary degron) is on the EGFR protein kinase domain and thus distal to the site of CBL/GRB2 recruitment 79 . Ubiquitination, which is mostly K63-linked, subsequently recruits reader enzymes, which facilitate the direction of EGFR to nonclathrin-mediated endocytosis and lysosomal degradation 79 (Fig. 3). ...
Post-translational modifications (PTMs) can occur on specific amino acids localized within regulatory domains of target proteins, which control a protein’s stability. These regions, called degrons, are often controlled by PTMs, which act as signals to expedite protein degradation (PTM-activated degrons) or to forestall degradation and stabilize a protein (PTM-inactivated degrons). We summarize current knowledge of the regulation of protein stability by various PTMs. We aim to display the variety and breadth of known mechanisms of regulation as well as highlight common themes in PTM-regulated degrons to enhance potential for identifying novel drug targets where druggable targets are currently lacking. Here the authors summarize current knowledge of the regulation of protein stability by various post-translational modifications (PTMs) including methylation and phosphorylation. PTM-regulated degrons act as signals for protein degradation or stabilization.
... Thereby, the sorting and lysosomal degradation of activated EGFR are important mechanisms for terminating EGFR signaling. EGFR mutants found in tumor patients are not internalized or transported to the MVBs/lysosomes, resulting in enhanced and prolonged activation of EGFR and its downstream MAPK signaling that is essential for tumor cell proliferation and invasion (Huang et al., 2006;Goh et al., 2010). ...
... Accumulating evidence revealed that ubiquitination serves as a critical sorting signal for endocytic trafficking of EGFR (Clague et al., 2012). EGF engagement induces rapid ubiquitination of EGFR on lysine residues within its tyrosine kinase domain (TKD) (Stang et al., 2000;Huang et al., 2006). The endosomal complex required for transport (ESCRT) machinery then recognizes the ubiquitinated EGFR and sort the receptor into intraluminal vesicles of the MVBs for subsequent lysosomal degradation (Raiborg and Stenmark, 2009;Henne et al., 2011). ...
... It is well established that EGF stimulation induces EGFR ubiquitination, which is crucial for receptor sorting to the lysosome for degradation (Huang et al., 2006. ZNRF1 has been shown to mediate ubiquitination and degradation of AKT and CAV1 (Araki and Milbrandt, 2003;Wakatsuki et al., 2011;Lee et al., 2017), which prompted us to speculate that ZNRF1 might control EGFR trafficking by modulating EGFR ubiquitination. ...
Activation of the epidermal growth factor receptor (EGFR) is crucial for development, tissue homeostasis, and immunity. Dysregulation of EGFR signaling is associated with numerous diseases. EGFR ubiquitination and endosomal trafficking are key events that regulate the termination of EGFR signaling, but their underlying mechanisms remain obscure. Here, we reveal that ZNRF1, an E3 ubiquitin ligase, controls ligand-induced EGFR signaling via mediating receptor ubiquitination. Deletion of ZNRF1 inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. We further demonstrate that ZNRF1 and Casitas B-lineage lymphoma (CBL), another E3 ubiquitin ligase responsible for EGFR ubiquitination, mediate ubiquitination at distinct lysine residues on EGFR. Furthermore, loss of ZNRF1 results in increased susceptibility to herpes simplex virus 1 (HSV-1) infection due to enhanced EGFR-dependent viral entry. Our findings identify ZNRF1 as a novel regulator of EGFR signaling, which together with CBL controls ligand-induced EGFR ubiquitination and lysosomal trafficking.
... In some cell types, elimination of Cbl by siRNA or mouse knockout was sufficient for a robust inhibition of EGFR degradation (Duan et al., 2003;Crotchett and Ceresa, 2021). However, in most cell types, efficient elimination of both Cbl and Cbl-b was necessary to achieve an inhibitory effect on EGFR ubiquitination and endocytosis, suggesting that Cbl-b can be sufficient for EGFR ubiquitination and endocytosis (for example, Mitra et al., 2004;Huang et al., 2006;Pennock and Wang, 2008). While numerous studies examined effects of Cbl knockdown and mouse knockout on EGFR, effects of Cbl-b knockdown and knockout on endogenous EGFR were sparsely studied (Pennock and Wang, 2008), and whether Cbl-b regulates EGFR in a manner that is fully redundant to that of Cbl is unclear. ...
... Grb2 , c-Cbl and Cbl-b (Huang et al., 2006) siRNA duplexes were resuspended in 1 × siRNA universal buffer (Dharmacon, Lafayette, Colorado) to 20 μM before transfection. Nontargeting siRNA was from Qiagen. ...
Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant fashion by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking and signaling.
... binding promotes receptor dimerization, leading to the allosteric activation of EGFR kinase and to the transautophosphorylation of critical tyrosine residues in the cytoplasmic receptor tail, therefore triggering intracellular signaling cascades, such as the PI3K/AKT and RAS/ RAF/MEK/ERK pathways, which are critical for cell proliferation, survival, motility and differentiation [3,[5][6][7]. Once activated at the plasma membrane, EGFR is rapidly ubiquitinated by the E3 ligase c-Cbl and internalized to endosomes then eventually being degraded in lysosome [3,7,8]. The proteasomal pathway is also involved in EGFR degradation [9]. ...
... Immunofluorescence staining of EGFR and EEA1 revealed that GSDME knockdown promoted the colocalization of EGFR and EEA1, indicating that GSDME knockdown promoted the formation of early endosome-containing EGFR on the plasma membrane (Fig. 5D). c-Cbl is an E3 ubiquitin ligase that induces monoubiquitination and lysosome-mediated degradation of EGFR [8,37]. To test whether GSDME knockdown promotes EGFR ubiquitination and degradation by c-Cbl. ...
Background:
Lung cancer is the most lethal malignancy, with non-small cell lung cancer (NSCLC) being the most common type (~ 85%). Abnormal activation of epidermal growth factor receptor (EGFR) promotes the development of NSCLC. Chemoresistance to tyrosine kinase inhibitors, which is elicited by EGFR mutations, is a key challenge for NSCLC treatment. Therefore, more thorough understanding of EGFR expression and dynamics are needed.
Methods:
Human non-small cell lung cancer cells and HEK293FT cells were used to investigate the molecular mechanism of gasdermin E (GSDME) regulating EGFR stability by Western blot analysis, immunoprecipitation and immunofluorescence. GSDME and EGFR siRNAs or overexpression plasmids were used to characterize the functional role of GSDME and EGFR in vitro. EdU incorporation, CCK-8 and colony formation assays were used to determine the proliferation ability of non-small cell lung cancer cells.
Results:
GSDME depletion reduced the proliferation of non-small cell lung cancer cells in vitro. Importantly, both GSDME-full length (GSDME-FL) and GSDME-N fragment physically interacted with EGFR. GSDME interacted with cytoplasmic fragment of EGFR. GSDME knockdown inhibited EGFR dimerization and phosphorylation at tyrosine 1173 (EGFRY1173), which activated ERK1/2. GSDME knockdown also promoted phosphorylation of EGFR at tyrosine 1045 (EGFRY1045) and its degradation.
Conclusion:
These results indicate that GSDME-FL increases the stability of EGFR, while the GSDME N-terminal fragment induces EGFR degradation. The GSDME-EGFR interaction plays an important role in non-small cell lung cancer development, reveal a previously unrecognized link between GSDME and EGFR stability and offer new insight into cancer pathogenesis. Video abstract.
... Therefore, we speculated that there are other mechanisms that account for the promising efficacy of EGFR S645C. The activity of RTKs including EGFR is not only controlled by the extent of phosphorylation but also regulated by the duration of activated status (32). Firstly, we employed CHX treatment to detect the stability and quantify the half-life of EGFRwt and EGFRmut. ...
... Subsequently, a part of EGFR within early endosomes recycles back to the plasma membrane, and the residue is sorted for degradation (33,34). Ubiquitination of EGFR induced by Cbl (an E3 ubiquitin ligase with the RING domain) is an important means to mediate endocytosis, sorting, and degradation (32,35,36). Cbl recognizes the phosphorylated Y1045 site of EGFR and recruits ubiquitin to conjugate with EGFR, and the process is critically important in determining EGFR stability (37). ...
Background
The tightly controlled activity of EGFR is important for the homeostasis of self-renewal of human tissue. Mutations in the extracellular domain of EGFR are frequent and function as a novel mechanism for oncogenic EGFR activation in GBM, and impact the response of patients to small-molecule inhibitors.
Methods
We constructed glioblastoma cell lines stably expressing wild-type EGFR and the mutant of EGFR S645C. We detected cell growth in vitro and in vivo. We evaluated the anti-tumor activity and effectiveness of gefitinib and osimertinib in cells.
Results
In the present study, we identified an oncogenic substituted mutation of EGFR—S645C. The mutation can promote the proliferation and colony formation of glioblastoma in vitro and in vivo. Mechanistically, the EGFR S645C mutation potentially changes the formation of hydrogen bonds within dimerized EGFR and inhibits the degradation of EGFR to prolong downstream signaling. The mutation induces resistance to gefitinib but presents an opportunity for osimertinib treatment.
Conclusion
The study indicated a novel oncogenic mutation and advises on the precise treatment of individual patients with the EGFR S645C mutation.
... The canonical CME is initiated by phosphorylation of Y1068/Y1086 and Y1045 ( Fig. 12A) (F. Huang et al. 2006). Grb2 associates to phosphorylated Y1045 and one of the other phosphorylated tyrosyls. ...
... The early endosome gets sorted into recycling to the cell membrane or they become late endosome multivesicular bodies (MVB), which is sorted towards degradation upon fusion to the lysosome (F. Huang et al. 2006;Takei and Haucke 2001;Madshus and Stang 2009). ...
Transmembrane receptors are proteins that translate extracellular information into intracellular signals. These proteins are categorised based on the way they transduce signals through the plasma membrane. In many cases such transduction involves the formation of dimers, either ligand-induced or constitutive dimers. For example, while the enzyme-linked receptors, such as the Epidermal Growth Factor (EGF) receptor, may be ligand-induced dimers, the G protein-coupled receptors (GPCR) activated by the neurotransmitter glutamate are covalently linked constitutive dimers. Understanding how a signal can be transduced from the extracellular domain into the cell is important to develop novel drugs targeting such receptors.The EGF receptor is a member of the receptor tyrosine kinase (RTK) class and is involved in cell differentiation and proliferation. Inhibition of the EGF receptor by medication is used as a successful treatment for a variety of cancers, but side effects and resistance to medication remain common major obstacles. The full-length EGF receptor crystal structure is unknown, which leaves ambiguity to the precise activation process and the effect of inhibitors. Its activation mechanism has been described as either ligand-induced dimerization or conformational changes within pre-assembled dimers. This is followed by phosphorylation of the intracellular domain of the receptor and the generation of intracellular signalling cascades. By generating a fluorescent conformational biosensor, we showed that the activation of the EGF receptor by its endogenous agonists results from ligand-induced dimerization. We also found that some non-competitive tyrosine kinase (TK) inhibitors also induce dimer formation, without activating the receptor, through a direct contact of the TK domains. Strikingly, internalization induced by its endogenous ligand is not blocked by the non-competitive inhibitors, demonstrating it does not require TK domain activity and is likely mainly dependent on receptor dimer conformation. This is supported by our observation that TK inhibitors promoting dimer formation slowed down the internalization process. This finding shows that the activation/dimerization process of the EGF receptor can be changed by inhibitors and that internalization of the EGF receptor is regulated by monomeric/dimeric conformations rather than by phosphorylation of the receptor.In contrast to the EGF receptor, the metabotropic glutamate (mGlu) 4 receptor is a constitutive dimer that is important for the regulation of many synapses in the brain. Recently, an mGlu2-mGlu4 heterodimer was proven to exist in the brain, but its function remains unknown due to the lack of tools selectively controlling it. To further investigate mGlu4 homo- and heterodimers, an innovative nanobody that discriminates between these receptors was developed. The nanobody is selective for the human mGlu4 receptor, and stabilizes the active homodimer conformation, making it a full agonist. By combining molecular dynamics simulations and site-directed mutagenesis, the epitope and mechanism of action of the nanobody were identified, revealing a new way of activating mGlu receptors. Conversely, this nanobody is incapable of activating heterodimeric mGlu2-mGlu4, where it acts as positive allosteric modulator through asymmetric activation of the heterodimer. This nanobody is the first pharmacological tool that discriminates between homo- and heterodimers.Overall, our studies revealed how important is the conformation of dimeric receptors to control their signalling, as shown with the TK inhibitors controlling to conformation of the TK domain-induced dimers and the internalization process of the EGF receptor, or by showing that nanobodies can control specifically a receptor dimer made of specific subunits. These findings show the importance of allosteric and asymmetric regulation of dimeric transmembrane receptors.
... It has been shown that EGFR stability is tightly regulated upon EGF stimulation. E3 ubiquitin ligase c-Cbl can induce EGFR mono-ubiquitination, endocytosis, and lysosome-mediated degradation, resulting in attenuation of EGFR signaling upon EGF stimulation 7,8 . Although proteasome pathway is also involved in EGFR degradation 9 , the exact mechanisms underlying proteasome-dependent degradation of EGFR are largely unclear. ...
... FBXL2 binds to and promotes EGFR for polyubiquitin-and proteasome-mediated degradation in an EGF-independent manner. EGFR protein can be rapidly destabilized through c-Cbl-mediated lysosome pathway upon EGF stimulation 7,8 . Since EGFR protein levels are critically important in tumorigenesis, we reasoned that EGFR protein stability ought to be tightly controlled at multiple levels. ...
Abnormal activation of epidermal growth factor receptor (EGFR) drives non-small cell lung cancer (NSCLC) development. EGFR mutations-mediated resistance to tyrosine-kinase inhibitors (TKIs) is a major hurdle for NSCLC treatment. Here, we show that F-box protein FBXL2 targets EGFR and EGFR TKI-resistant mutants for proteasome-mediated degradation, resulting in suppression of EGFR-driven NSCLC growth. Reduced FBXL2 expression is associated with poor clinical outcomes of NSCLC patients. Furthermore, we show that glucose-regulated protein 94 (Grp94) protects EGFR from degradation via blockage of FBXL2 binding to EGFR. Moreover, we have identified nebivolol, a clinically used small molecule inhibitor, that can upregulate FBXL2 expression to inhibit EGFR-driven NSCLC growth. Nebivolol in combination with osimertinib or Grp94-inhibitor-1 exhibits strong inhibitory effects on osimertinib-resistant NSCLC. Together, this study demonstrates that the FBXL2-Grp94-EGFR axis plays a critical role in NSCLC development and suggests that targeting FBXL2-Grp94 to destabilize EGFR may represent a putative therapeutic strategy for TKI-resistant NSCLC. Aberrant EGFR activation is commonly found in non-small cell lung cancer (NSCLC). Here the authors show that E3 ubiquitin ligase FBXL2 targets EGFR and EGFR tyrosine kinase inhibitor (TKI)-resistant mutants for proteasome-mediated degradation to inhibit EGFR-driven NSCLC growth and TKI resistance.
... siRNA transfection and rescue experiments siRNAs (individual, ON-TARGET plus or siGENOME grade) were purchased from Dharmacon as follows: μ2: 59-AAGUGGAUGCCU UUCGGGUCA-39 (Huang et al., 2004); σ2 targeting 39UTR: 59-CCGUGUGUGUCCCGAGUAA-39 (J-011833-09-0005); CHC: D-004001-02 (Huang et al., 2004); Grb2: 59-CAUGUUUCCCCG CAAUUAUUUUU-39 ; Cbl-b: 59-GGACAGACG AAAUCUCACAUU-39 (Huang et al., 2006); and c-Cbl: 59-CCU CUCUUCCAAGCACUGAUU-39 (Huang et al., 2006). ...
... siRNA transfection and rescue experiments siRNAs (individual, ON-TARGET plus or siGENOME grade) were purchased from Dharmacon as follows: μ2: 59-AAGUGGAUGCCU UUCGGGUCA-39 (Huang et al., 2004); σ2 targeting 39UTR: 59-CCGUGUGUGUCCCGAGUAA-39 (J-011833-09-0005); CHC: D-004001-02 (Huang et al., 2004); Grb2: 59-CAUGUUUCCCCG CAAUUAUUUUU-39 ; Cbl-b: 59-GGACAGACG AAAUCUCACAUU-39 (Huang et al., 2006); and c-Cbl: 59-CCU CUCUUCCAAGCACUGAUU-39 (Huang et al., 2006). ...
Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.
... There was a 35% decrease in the rate of ligand:receptor endocytosis. Our data show the same trend as reported by Huang et al, 38 but we did not observe as robust of an effect from the c-Cbl knockout. This is likely due to the difference in cell line and level of receptor expression. ...
... In their studies, c-Cbl was transiently knocked down using siRNA in PAE cells expressing exogenous EGFRs. 38 The ...
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. Knockout of c‐Cbl slows EGFR endocytosis and enhances EGFR recycling
... OTUD4 regulates TβRI presence at the plasma membrane. Receptor ubiquitination has also been reported to promote their internalization and subsequent trafficking to the lysosomes, resulting in enhanced pathway activation or receptor recycling [28][29][30][31] . It is therefore possible that OTUD4 may be an important factor in the endocytosis and/or trafficking of TβR complex. ...
... Ubiquitin has long been implicated in regulating the endocytosis of plasma membrane receptors and though this is a disputed issue, K63-linked ubiquitin chains are believed to play a role in this process [29][30][31]40,41 . The fact that ectopic expression of OTUD4 leads to an increase in plasma membrane levels of this receptor subunit lends support to the notion that OTUD4-regulated ubiquitin chains affects endocytosis. ...
Systematic control of the transforming growth factor-β (TGFβ) pathway is essential to keep the amplitude and the intensity of downstream signalling at appropriate levels. Ubiquitination plays a crucial role in the general regulation of this pathway. Here we identify the deubiquitinating enzyme OTUD4 as a transcriptional target of the TGFβ pathway that functions through a positive feedback loop to enhance overall TGFβ activity. Interestingly we demonstrate that OTUD4 functions through both catalytically dependent and independent mechanisms to regulate TGFβ activity. Specifically, we find that OTUD4 enhances TGFβ signalling by promoting the membrane presence of TGFβ receptor I. Furthermore, we demonstrate that OTUD4 inactivates the TGFβ negative regulator SMURF2 suggesting that OTUD4 regulates multiple nodes of the TGFβ pathway to enhance TGFβ activity.
... Notably, exogenous EGF slightly upregulated EGFR phosphorylation also in Mel 599 P cells, suggesting that the EGFR constitutively expressed by these cells is functional. EGF stimulation triggered downregulation of EGFR, in line with literature data reporting degradation of activated EGFR (54)(55)(56)(57)(58)(59)(60)(61). Independent of the degree of constitutive EGFR phosphorylation, activation of the AKT pathway emerged as a common feature of VR cells, as demonstrated by the increased phosphorylation of AKT1 found in 3 out of 4 VR cell cultures as compared to their P isogenic cells. ...
Introduction
About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots.
Methods and results
By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells.
Conclusion
Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.
... Additionally, a notable decrease in total EGFR levels starting from Day 2, following the pattern of EGFR phosphorylation, suggests the possibility of ubiquitination and subsequent degradation of activated EGFRs, as reported previously. 28,29 While no significant difference was observed in the N-region phosphorylation at We previously reported the involvement of hepsin in the processing of pro-HGF and its influence on the activation of the downstream receptor, c-Met, in the liver. ...
Given the widespread use of partial hepatectomy for treating various liver pathologies, understanding the mechanisms of liver regeneration is vital for enhancing liver resection and transplantation therapies. Here, we demonstrate the critical role of the serine protease Hepsin in promoting hepatocyte hypertrophy and proliferation. Under steady-state conditions, liver-specific overexpression of Hepsin in adult wild-type mice triggers hepatocyte hypertrophy and subsequent proliferation, significantly increasing liver size. This effect is predominantly driven by the catalytic activity of Hepsin, engaging the EGFR-Raf-MEK-ERK signaling pathway. Significantly, administering Hepsin substantially enhances hepatocyte proliferation and facilitates liver regeneration following a 70% partial hepatectomy. Crucially, the proliferation induced by Hepsin is a transient event, without leading to long-term adverse effects such as liver fibrosis or hepatocellular carcinoma, as evidenced by extensive observation. These results offer substantial potential for future clinical applications and translational research endeavors in the field of liver regeneration post-hepatectomy.
... To further clarify whether JMJD5 affects EGFR stability through proteasome or lysosome pathway, we treated cells with proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ). Consistent with prior reports [13,35,36], in the absence of EGF induction, the degradation of EGFR protein proceeds primarily through the proteasome pathway, while following EGF stimulation, the EGFR protein was rapidly destabilized mainly through the lysosome pathway (Fig. S3C, D). The decrease of EGFR level caused by JMJD5 overexpression can be effectively reversed by MG132 but not by CQ, suggesting that proteasome-mediated degradation Fig. 1 JMJD5 is negatively related to EGFR expression and correlated with better survival of lung cancer. ...
Aberrant activation of epidermal growth factor receptor (EGFR) signaling is closely related to the development of non-small cell lung cancer (NSCLC). However, targeted EGFR therapeutics such as tyrosine kinase inhibitors (TKIs) face the challenge of EGFR mutation-mediated resistance. Here, we showed that the reduced JmjC domain-containing 5 (JMJD5) expression is negatively associated with EGFR stability and NSCLC progression. Mechanically, JMJD5 cooperated with E3 ligase HUWE1 to destabilize EGFR and EGFR TKI-resistant mutants for proteasomal degradation, thereby inhibiting NSCLC growth and promoting TKI sensitivity. Furthermore, we identified that JMJD5 can be transported into recipient cells via extracellular vesicles, thereby inhibiting the growth of NSCLC. Together, our findings demonstrate the tumor-suppressive role of JMJD5 in NSCLC and suggest a putative therapeutic strategy for EGFR-related NSCLC by targeting JMJD5 to destabilize EGFR.
... The extension of this colocalization under EGF to 5 minutes also implies the use of a "fast" endocytosis independent of clathrin. This would involve the ubiquitination of EGFR by the ubiquitin ligase Cbl, promoted by the adaptor protein Grb2 (27,28). As these elements are known from the literature, the most interesting result here was the significant increase in EGFR-Rab5 colocalization after stimulation with proNGF. ...
Head and Neck Squamous Cell Carcinoma (HNSCC) remains a cancer with a poor prognosis, with a 5-year survival rate of less than 50%. Although epidermal growth factor receptor (EGFR) is almost always overexpressed, targeted anti-EGFR therapies have modest efficacy and are mainly used in palliative care. Growth factors such as Nerve Growth Factor (NGF) and its precursor proNGF have been shown in our laboratory to play a role in tumor growth and aggressiveness. Interestingly, an interaction between Sortilin, a proNGF receptor, and EGFR has been observed. This interaction appears to interfere with the pro-oncogenic signaling of EGF and modulate the membrane expression of EGFR. The aim of this study was to characterize this interaction biologically, to assess its impact on clinical prognosis and to analyze its role in the cellular trafficking of EGFR. Using immunohistochemical staining on tumor sections from patients treated at our university center and PLA (Proximity Ligation Assay) labeling, we showed that Sortilin expression is significantly associated with reduced 5-year survival. However, when Sortilin was associated with EGFR, this association was not found. Using the Cal-27 and Cal-33 cancer cell lines, we observed that proNGF reduces the effects of EGF on cell growth by inducing the internalization of its receptor. These results therefore suggest a regulatory role for Sortilin in the degradation or renewal of EGFR on the membrane. It would be interesting in future work to show the intracellular fate of EGFR and the role of (pro)neurotrophins in these mechanisms.
... Prominent among these are the RING E3s of the Casitas B-lineage lymphoma (Cbl) (Liyasova et al., 2015) and membrane-associated RING-CH (MARCH) (Lin et al., 2019) families, as well as the HECT E3s neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) (Sicari et al., 2022) and Itchy homolog (ITCH) (Aki et al., 2015;Moser and Oliver, 2019). Ub marks conjugated to cell surface molecules usually take the form of multiple monoubiquitins or K63-linked polyubiquitin chains; however, other linkage types such as K48 and K11 have also been reported (Haglund et al., 2003;Huang et al., 2006). Ub ligase activities governing turnover of the cell surface proteome are in turn countered by DUBs and can themselves be regulated by Ub and Ubls (Nelson et al., 2016;Polo, 2012;Wang et al., 2020). ...
The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.
... A single ubiquitin is sufficient for recognition and sorting into ILVs, however, cargo often is polyubiquitinated (Haglund et al., 2003;Schuh and Audhya, 2014). The ubiquitination and sorting of EGFR is well characterized, and it was shown that stimulation of EGFR with ligand results in a 20-fold increase in ubiquitination of EGFR K63-linked polyubiquitin chains (Huang et al., 2006(Huang et al., , 2013. This demonstrated that activation of endocytic uptake and trafficking induces ubiquitination of target substrates. ...
The microtubule associated protein tau forms filamentous assemblies in the cytosol of neurons in several neurodegenerative diseases. In Alzheimer’s disease, tau pathology arises throughout the human brain in a spatio-temporal manner and closely correlates with disease severity. Assemblies of tau have been proposed to transit between cells of the brain in a ‘prion-like’ manner, resulting in templated aggregation of native tau in recipient neurons. Interactions between tau assemblies, surface receptor LRP1 and heparan sulphate proteoglycans promote the uptake of tau assemblies to membrane-bound vesicles. A subsequent escape from these vesicles is postulated for assemblies to enter the cytosol and contact cytosolic tau pools to induce seeded aggregation. However, the mechanistic details by which tau assemblies enter the cytosol are poorly defined. I have established a cell-based assay that permits the study of tau entry to the cytosol in real time and at physiological concentrations, in cell lines and neurons. Modulation of tau entry to the cytosol concomitantly modified levels of seeded aggregation, confirming the role of cytosolic entry as the rate-limiting, upstream step to seeding. Entry to a commonly used reporter cell line, HEK 293, occurred in a dynamin- and clathrin-dependent manner with late endosomal Rab7 GTPase involvement. In contrast, entry to primary and human neurons was via a clathrin- and dynamin-independent route that was sensitive to membrane cholesterol levels. Extraction of cholesterol from membranes rendered neurons extremely permissive to cytosolic entry and potentiated seeded aggregation at low concentrations of tau in 2D neurons and 3D organotypic slice cultures. Conversely, cholesterol supplementation or overexpression of the cholesterol transporter protein NPC1 reduced entry and almost completely blocked seeded aggregation. My findings establish entry to the cytosol as rate-limiting to seeded aggregation. They also demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting the entry of assemblies to the cytosol.
... Grb2 se lie à l'EGFR activé par son domaine SH2 (Batzer et al., 1994). La liaison EGFR / Grb2 conduit à deux effets opposés : l'un conduit à la régulation négative de l'EGFR par dégradation via le recrutement de l'ubiquitine ligase E3 (Cbl) qui induit une mono-ou polyubiquitinylation des résidus lysine de l'EGFR (Huang et al., 2006). L'autre effet de Grb2 conduit à l'activation de la cascade de signalisation Ras et Sos via son domaine SH3 (Chardin et al., 1993). ...
Le cancer bronchique reste la principale cause de décès par cancer dans le monde en raison de l’absence de diagnostic précoce et de sa difficulté de prise en charge. Cet échec des stratégies thérapeutiques est principalement dû à la présence de mutations et d’activations aberrantes de l’EGFR conduisant à une croissance tumorale exacerbée. Malgré une évolution et une diversification des thérapies ciblées visant à inactiver ces récepteurs, l’utilisation des inhibiteurs de tyrosine kinase ciblant l'EGFR montre toujours une efficacité initiale dont les bénéfices diminuent avec le temps. L’un des mécanismes de résistance permettant d’expliquer ce phénomène pourrait dépendre du switch entre la signalisation membranaire de l’EGFR et sa signalisation nucléaire. En effet, la relocalisation nucléaire de l’EGFR favoriserait l'expression de cibles génomiques responsable de l’échappement thérapeutique et de la récidive. Nos travaux antérieurs ont déjà permis d’identifier la fonction originale d’une protéine de transport, la sortiline dans l’inhibition de la signalisation membranaire de l’EGFR. Néanmoins, la découverte de la sortiline au niveau nucléaire met en évidence un nouveau rôle de la sortiline en tant que régulateur crucial des oncogènes contrôlés habituellement par l'EGFR nucléaire. Un large panel de gènes cibles de l’EGFR contrôlés par la sortiline a pu être caractérisé. Nos travaux nous ont conduit à analyser plus spécifiquement l’action de la sortiline sur l’expression de cMYC et CCND1. Cette dernière induirait en effet une diminution du recrutement de l’ARN pol II qui est cruciale pour leur transcription. Lors du traitement à l’osimertinib, la fixation de la sortiline sur la chromatine est augmentée ce qui conduit à une répression des gènes cibles tels que cMYC. Ceci suggère que la sortiline puisse être susceptible de réprimer l’expression de gènes cibles comme cMYC. Nos analyses transcriptomiques menées sur une cohorte de patients ont confirmé l’effet répresseur de la sortiline sur le gène cMYC. Ces résultats ouvrent des perspectives intéressantes quant au diagnostic du CBNPC, à la prédiction de récidive. A l’avenir la sortiline pourrait constituer un agent thérapeutique permettant la resensibilisation des tumeurs aux traitements.
... Phosphorylation of its carboxy-tail residues leads to downstream signaling events such as activation of ERK1/2 and PI3K/AKT/mTOR 10 . Conversely, receptor trafficking may be both dependent 11 and independent of the phosphorylation of carboxy terminal-tail residues 12,13 . Surprisingly, whether this process results from ligand-induced dimerization 14 or from conformational changes within pre-formed dimers 15 is still a matter of debate. ...
The structural basis of the activation of EGF receptors (EGFR) is still a matter of debate despite the importance of this target in cancer treatment. Whether agonists induce dimer formation or act on pre-formed dimers remain discussed. Here we provide direct evidence that EGFR activation results from EGF-induced dimer formation. This is well illustrated by i) a large increase in time resolved (TR)-FRET between snap-tagged EGFR subunits induced by agonists, ii) a similar effect of Erlotinib-related TK inhibitors despite the inactive state of the binding domain of the subunits, and iii) a similar TR-FRET efficacy in EGFR dimers stabilized by EGF or erlotinib with binding domains in active and inactive states, respectively. Surprisingly, TK inhibitors do not inhibit EGF-induced EGFR internalization despite their ability to fully block EGFR signaling. Only Erlotinib-related TK inhibitors promoting asymmetric dimers could slow down this process, while the lapatinib-related ones have almost no effect. These results reveal that the conformation of the intracellular TK dimer, rather than the known EGFR signaling is critical for EGFR internalization. These results also illustrate clear differences in the mode of action of TK inhibitors on the EGFR.
... Images were taken using a laser scanning confocal microscope. For EGF-EGFR complexes to be sorted for degradation, their ubiquitination, which follows phosphorylation, is required [175]. Ubiquitination is accomplished by EGFR's C-terminal interaction with the E3 ubiquitin-protein ligase Cbl (reviewed in [176]). ...
Simple Summary
Lung cancer is the leading cause of cancer-related deaths, with a low (<21%) 5-year survival rate. Lung cancer is often driven by the misfunction of molecules on the surface of cells of the epithelium, which orchestrate mechanisms by which these cells grow and proliferate. Beyond common non-specific treatments, such as chemotherapy or radiotherapy, among molecular-specific treatments, a number of small-molecule drugs that block cancer-driven molecular activity have been developed. These drugs initially have significant success in a subset of patients, but these patients systematically develop resistance within approximately one year of therapy. Substantial efforts towards understanding the mechanisms of resistance have focused on the genomics of cancer progression, the response of cells to the drugs, and the cellular changes that allow resistance to develop. Fluorescence microscopy of many flavours has significantly contributed to the last two areas, and is the subject of this review.
Abstract
Non-small cell lung cancer (NSCLC) is a complex disease often driven by activating mutations or amplification of the epidermal growth factor receptor (EGFR) gene, which expresses a transmembrane receptor tyrosine kinase. Targeted anti-EGFR treatments include small-molecule tyrosine kinase inhibitors (TKIs), among which gefitinib and erlotinib are the best studied, and their function more often imaged. TKIs block EGFR activation, inducing apoptosis in cancer cells addicted to EGFR signals. It is not understood why TKIs do not work in tumours driven by EGFR overexpression but do so in tumours bearing classical activating EGFR mutations, although the latter develop resistance in about one year. Fluorescence imaging played a crucial part in research efforts to understand pro-survival mechanisms, including the dysregulation of autophagy and endocytosis, by which cells overcome the intendedly lethal TKI-induced EGFR signalling block. At their core, pro-survival mechanisms are facilitated by TKI-induced changes in the function and conformation of EGFR and its interactors. This review brings together some of the main advances from fluorescence imaging in investigating TKI function and places them in the broader context of the TKI resistance field, highlighting some paradoxes and suggesting some areas where super-resolution and other emerging methods could make a further contribution.
... These rearrangements consist in conformational changes that will lead to the trans-autophosphorylation of tyrosines, triggering the signaling cascade (Lemmon et al., 2014). In the meantime, EGFR will also be ubiquitinated on several lysine residues (Huang et al., 2006) by ubiquitination proteins like E3 ligase Cb1 in complex with Grb2 (Waterman et al., 2002). These intracellular modifications will lead to the recruitment of the endocytic machinery that mediates the receptor endocytosis (Barbieri et al., 2016). ...
Neocortex development is highly regulated and mutations in genes involved in this process may lead to genetic diseases. Microlissencephaly is a congenital and poorly understood brain malformation characterized by the presence of both microcephaly (small brain) and lissencephaly (smooth brain). Our collaborators recently identified compound heterozygous mutations causing this pathology in the human WD repeat domain 81 (WDR81) gene. WDR81 is a poorly characterized protein involved in endosomal maturation through Pi3K regulation. The goal of my PhD project is to characterize the function of WDR81 in the developing brain, and identify how mutations in this gene may lead to pathological brain development.Using CRISPR/Cas9, I have generated a WDR81 knock-out mouse, which shows strong neuronal migration defects and microcephaly, mimicking the human phenotype. I demonstrated that the reduced brain size was not due to increased apoptosis or altered cell fate, but to reduced neural stem cell proliferation. I also observe this phenotype in patient-derived fibroblasts, altogether suggesting altered processing of proliferative signals. Fluorescent EGF uptake assay reveals a strong intracellular accumulation of EGF in early phases endosomes that fails to be processed and cleared giving as well defects in the activation of the receptor itself. My results indicate that mutant cells indeed poorly respond to EGF stimulation.I then investigated the exciting possibility that reduced brain growth (microcephaly) and increased brain growth (megalencephaly) may represent two sides of the same coin. Because my data point to opposite effects on similar pathways, I overexpressed megalencephaly-causing mutated factors in WDR81 KO brains, and tested whether they were able to rescue the microcephaly-causing proliferation defects. My result show that indeed a megalencephaly-causing mutation can overcome the effect of a microcephaly-causing mutation on the proliferation of radial glial progenitors. These two pathologies can therefore arise from a highly related cause: an imbalance in cell cycle regulation leading either to reduced brain growth or to brain overgrowth.
... Most of the internalized EGFR, however, is not recycled to the plasma membrane but rather routed to lysosomes for degradation. EGFR ubiquitynation is essential for lysosomal targeting (Huang et al. 2006). Of note, the different possible ligands of EGFR lead to different endocytic fates. ...
Cell migration is a fundamental process in the development and homeostasis of multicellular organisms. It is also central to many pathologies and it is especially important for metastatic dissemination. When migrating, cells use adhesion structures to push on their substrate in order to move forward. We recently showed that clathrin coated structures, primarily known as endocytic structures, can also serve as adhesion structures. In this manuscript, I show that some ligands internalized through clathrin mediated endocytosis can also bind to the extracellular matrix and orient cell migration using adhesive clathrin structures.I first showed that ligand-decorated collagen fibers are associated with more clathrin structures and more protrusions. I then showed that cells applied more forces to the ligand-decorated collagen fibers and this extra amount of forces requires the presence of clathrin structures. Finally, I showed that cells can migrate following collagen-bound ligands in 3D, this directed migration also requiring the presence of clathrin structures. Such migration mechanism could be used by cells to follow in vivo gradient of matrix-bound ligands and thus find their way when migrating inside the body.
... Using molecular modeling and single-molecule tracking studies it was shown that higher-order multimers are proposed [27][28][29]. Single-molecule tracking studies of EGFR in live cells shown that EGFR forms large clusters after activation [10,[30][31][32]. ...
Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) are two extensively studied membrane-bound receptor tyrosine kinase proteins that are frequently overexpressed in many cancers. As a result, these receptor families constitute attractive targets for imaging and therapeutic applications in the detection and treatment of cancer. This review explores the dynamic structure and structure-function relationships of these two growth factor receptors and their significance as it relates to theranostics of cancer, followed by some of the common inhibition modalities frequently employed to target EGFR and VEGFR, such as tyrosine kinase inhibitors (TKIs), antibodies, nanobodies, and peptides. A summary of the recent advances in molecular imaging techniques, including positron emission tomography (PET), single-photon emission computerized tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging (OI), and in particular, near-IR fluorescence imaging using tetrapyrrolic-based fluorophores, concludes this review.
... binds directly to p-Y1045 on the c-terminus of EGFR or via Grb2, leading to phosphorylation of Cbl on Y371, a critical requirement for Cbl E3 ligase activity [13]. Cbldependent ubiquitination of EGFR leads to clathrinmediated internalisation of the receptor and sorting into lysosomes where the receptor is degraded [10,14,15], leading to reduced EGFR signalling. In GB,~20% of classical tumours express a truncated form of EGFR that lacks exons 2-7 (EGFRvIII) [9,16] and is constitutively active. ...
Despite recent advances in our understanding of the disease, glioblastoma (GB) continues to have limited treatment options and carries a dismal prognosis for patients. Efforts to stratify this heterogeneous malignancy using molecular classifiers identified frequent alterations in targetable proteins belonging to several pathways including the receptor tyrosine kinase (RTK) and mitogen-activated protein kinase (MAPK) signalling pathways. However, these findings have failed to improve clinical outcomes for patients. In almost all cases, GB becomes refractory to standard-of-care therapy, and recent evidence suggests that disease recurrence may be associated with a subpopulation of cells known as glioma stem cells (GSCs). Therefore, there remains a significant unmet need for novel therapeutic strategies. E3 ubiquitin ligases are a family of >700 proteins that conjugate ubiquitin to target proteins, resulting in an array of cellular responses, including DNA repair, pro-survival signalling and protein degradation. Ubiquitin modifications on target proteins are diverse, ranging from mono-ubiquitination through to the formation of polyubiquitin chains and mixed chains. The specificity in substrate tagging and chain elongation is dictated by E3 ubiquitin ligases, which have essential regulatory roles in multiple aspects of brain cancer pathogenesis. In this review, we begin by briefly summarising the histological and molecular classification of GB. We comprehensively describe the roles of E3 ubiquitin ligases in RTK and MAPK, as well as other, commonly altered, oncogenic and tumour suppressive signalling pathways in GB. We also describe the role of E3 ligases in maintaining glioma stem cell populations and their function in promoting resistance to ionizing radiation (IR) and chemotherapy. Finally, we consider how our knowledge of E3 ligase biology may be used for future therapeutic interventions in GB, including the use of blood-brain barrier permeable proteolysis targeting chimeras (PROTACs).
... To examine the role of the observed EGFR relocalization to the endosomal network in achieving the productive infection, we employed an EGFR mutant. The EGFR re -5R mutant, having five point mutations of Lys (amino acids 692, 713, 843, 905, and 946) to Arg was reported to be less ubiquitinated to escape from the transport to the late endosome, with accumulation in the early endosome (18,19) (Fig. 4A and Fig. S6A). Expression of this EGFR re -5R mutant in EGFR-depleted cells supported much a lower level of HBV infection compared with the WT EGFR ( Fig. 4A (right panels and graph) and Fig. S6B). ...
Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.
... They ubiquitinate EGFR, provoking its internalization into early endosomes and sorting into multivesicular particles (MVPs). EGFR is then either degraded in lysosomes or recycled back to the cell membrane.43,44 Deubiquitinating enzymes also control EGFR turnover. ...
In non‐small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug‐tolerant cells ultimately leads to acquired resistance mechanisms. We performed an unbiased high‐throughput siRNA screen to identify proteins that abrogate the response of EGFR mutant NSCLC to EGFR targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase‐independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR‐specific co‐target that could improve the treatment efficacy of EGFR targeted therapies.
... Ubiquitination and phosphorylation show cooperativity at physiological levels of the receptor but become uncoupled when EGFR is overexpressed or the concentration of ligand exceeds normal levels [33]. Ubiquitination requires phosphorylation of EGFR at Y1045 and Y1068 (or Y1086) residues as these sites recruit the E3 ubiquitin ligase Cbl and adaptor protein Grb2 respectively to EGFR leading to the formation of K63-linked ubiquitin chains on EGFR [30,34]. Mass spectrometry analysis revealed that roughly 50% of all ubiquitin on EGFR exists in the form of mono-ubiquitin and~40% in the form of K63-linked poly-ubiquitin chains. ...
Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion.
... The balance between recycling and degradation depends on the EGFR ubiquitylation status, which invariably routes the activated receptor from the endosome to the lysosome, where it is degraded [6,7]. Ubiquitylation of the EGFR by the E3 ligase c-Cbl is dispensable for CME [8,9], while it ...
Deregulated epidermal growth factor receptor (EGFR) signaling is a key feature in different stages of oncogenesis. One important mechanism whereby cancer cells achieve increased and uncontrolled EGFR signaling is escaping down-modulation of the receptor. Ubiquitylation of the EGFR plays a decisive role in this process, as it regulates receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) may oppose the ubiquitylation process, antagonizing or even promoting receptor degradation. Here, we use qualitative and quantitative assays to measure EGFR internalization and degradation after Ubiquitin Specific Peptidase 25 (USP25) depletion. We show that, by acting at the early steps of EGFR internalization, USP25 restrains the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. This study establishes USP25 as a negative regulator of the EGFR down-modulation process and suggests that it is a promising target for pharmacological intervention to hamper oncogenic growth signals in tumors that depend on the EGFR.
... MVBs either fuse with lysosomes for the degradation of their membranous contents or with the plasma membrane for the secretion of exosomes. Conjugation of membrane proteins with ubiquitin can serve as a signal that directs these membrane proteins into the MVB pathway (Huang et al., 2006;Kamsteeg et al., 2006;Marchese et al., 2008;Stringer and Piper, 2011). In particular, monoubiquitination has been identified as an endocytosis signal that directs plasma membrane proteins to the endocytic pathway (Hicke, 2001). ...
Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes / lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono - ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral – but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non - ubiquitinatable mutant of Stx3 (Stx3 - 5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV / exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3 - 5R mutant acts as a dominant - negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3 - 5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.
... In addition, the presence of Lys11 linkages in heterotypic ubiquitin conjugates constitutes a strong proteasomal degradation signal (Meyer and Rape, 2014;Grice et al., 2015). In contrast, Lys63-linked chains are involved in the lysosomal degradation of membrane proteins (Huang et al., 2006;Mukhopadhyay and Riezman, 2007) and degradation of protein aggregates through autophagy, as autophagic receptors bind to Lys63-linked chains with higher preference (Kirkin et al., 2009). ...
Dysregulated proteostasis is one of the hallmarks of ageing. Damaged proteins may impair cellular function and their accumulation may lead to tissue dysfunction and disease. This is why protective mechanisms to safeguard the cell proteome have evolved. These mechanisms consist of cellular machineries involved in protein quality control, including regulators of protein translation, folding, trafficking and degradation. In eukaryotic cells, protein degradation occurs via two main pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Although distinct pathways, they are not isolated systems and have a complementary nature, as evidenced by recent studies. These findings raise the question of how autophagy and the proteasome crosstalk. In this review we address how the two degradation pathways impact each other, thereby adding a new layer of regulation to protein degradation. We also analyze the implications of the UPS and autophagy in ageing.
... This method requires educated guesses on the sites that may be modified by ubiquitin. Later on, MS was used to identify the modification sites on a specific protein after immunoprecipitation of the corresponding ubiquitylated protein through the identification of the ubiquitin remnant (GlyGly or diGly) on the modification sites after trypsin digestion [39,40]. In recent years, new reagents and high-throughput proteomic approaches have been developed to identify ubiquitylation sites at the proteome scale. ...
... K48linked Ub chains will point to proteasome-mediated degradation or ERAD, while K63 polyubiquitination is the signature of endocytosis or autophagy (Romero-Barrios and . Ub chain architecture can be determined through the use of chain-specific antibodies or sensors (Johnson and Vert, 2016;Romero-Barrios et al., 2020), the expression of linkage-specific deubiquitinases (Hospenthal et al., 2015), the co-expression of mutant forms of Ub unable to make a specific chain type (Yin et al., 2007;Mural et al., 2013), or by mass spectrometry if the protein of interest is purified in fully denaturing conditions to avoid the detection of Ub linkages from co-purified proteins (Huang et al., 2006). In the eventuality where the E3 ligase involved is known or suspected, in vitro ubiquitination assays will also rapidly provide evidence for ubiquitination of the target and involvement of this E3. ...
Organisms need to deal with the absolute requirement for metals, and their possible toxicity. This is achieved through an intricate network of signaling pathways integrated to ultimately fine tune iron uptake and metabolism. The mechanisms by which plants cope with iron limitation and the associated genomic responses are well characterized. On top of this transcriptional cascade is layered another level of regulation involving the post-translational modification and degradation. The ubiquitination and/or degradation of several transcription factors in the iron deficiency signaling pathways and metal transporters recently came to light. In this review we discuss about the mechanisms and on the possible roles of protein modification and turn over in the regulation of root iron deficiency responses. We also highlight the tight coupling between metal sensing by E3 ubiquitin ligases or bifunctional transporters and protein degradation.
... The number of SP-DiI fluorescent puncta (detected through the 561 nm channel), the sum fluorescence intensity of each individual puncta, and the sum of fluorescence intensity per single cell were calculated based on background-subtracted 3D images, using the segmentation-based method of SlideBook6 [53]. The segment mask was generated to select SP-DiI puncta using identical fluorescence intensity threshold parameters for all experimental variants. ...
Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for “endocytic escape” of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
Spontaneous dimerization of EGF receptors (EGFR) and dysregulation of EGFR signaling has been associated with the development of different cancers. Under normal physiological conditions and to maintain homeostatic cell growth, once EGFR signaling occurs, it needs to be attenuated. Activated EGFRs are rapidly internalized, sorted through early endosomes, and ultimately degraded in lysosomes by a process generally known as receptor down-regulation. Through alterations to EGFR trafficking, tumors develop resistance to current treatment strategies, thus highlighting the necessity for combination treatment strategies that target EGFR trafficking. This review covers EGFR structure, trafficking, and altered surface expression of EGFR receptors in cancer, with a focus on how therapy targeting EGFR trafficking may aid tyrosine kinase inhibitor treatment of cancer.
Bacterial infections are a major cause of human morbidity and mortality on a global scale. Many bacterial pathogens, such as Escherichia coli, can cause diseases intracellularly via cell entry and avoidance of the host immune system. Antibiotic resistance has caused such infections to be problematic, which has necessitated the development of new antimicrobials. Bacteriophages are a potent alternative due to their specificity and ease of genetic modification. We have engineered phage K1F, which is specific to E. coli K1 to express an epidermal growth factor (EGF) and green fluorescent protein (GFP) fusion on the minor capsid protein. Here, we demonstrate that EGF-labeled phage K1F can be internalized more readily in human cell lines to eradicate E. coli K1 infection intracellularly. Further, we establish that K1F-GFP-EGF enters human cells primarily through endocytosis following EGF receptor (EGFR) induction, subverting the phagocytic mode of entry and permitting its accretion in the cytosol to seek out its bacterial host.
Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.
Background:
Lactate is an important metabolite derived from glycolysis under physiological and pathological conditions. The Warburg effect reveals the vital role of lactate in cancer progression. Numerous studies have reported crucial roles for lactate in cancer progression and cell fate determination. Lactylation, a novel posttranslational modification (PTM), has provided a new opportunity to investigate metabolic epigenetic regulation, and studies of this process have been initiated in a wide range of cancer cells, cancer-associated immune cells, and embryonic stem cells.
Conclusion:
Lactylation is a novel and interesting mechanism of lactate metabolism linked to metabolic rewiring and epigenetic remodeling. It is a potential and hopeful target for cancer therapy. Here, we summarize the discovery of lactylation, the mechanisms of site modification, and progress in research on nonhistone lactylation. We focus on the potential roles of lactylation in cancer progression and cell fate determination and the possible therapeutic strategies for targeting lysine lactylation. Finally, we suggest some future research topics on lactylation to inspire some interesting ideas.
LZTR1 is the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase frequently mutated in sporadic and syndromic cancer. We combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations. Unbiased screens converged on EGFR and AXL receptor tyrosine kinases as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome. Pathogenic cancer-associated mutations of LZTR1 failed to promote EGFR and AXL degradation, resulting in dysregulated growth factor signaling. Conditional inactivation of Lztr1 and Cdkn2a in the mouse nervous system caused tumors in the peripheral nervous system including schwannoma-like tumors, thus recapitulating aspects of schwannomatosis, the prototype tumor predisposition syndrome sustained by LZTR1 germline mutations. Lztr1– and Cdkn2a-deleted tumors aberrantly accumulated EGFR and AXL and exhibited specific vulnerability to EGFR and AXL coinhibition. These findings explain tumorigenesis by LZTR1 inactivation and offer therapeutic opportunities to patients with LZTR1-mutant cancer.
Significance
EGFR and AXL are substrates of LZTR1-CUL3 ubiquitin ligase. The frequent somatic and germline mutations of LZTR1 in human cancer cause EGFR and AXL accumulation and deregulated signaling. LZTR1-mutant tumors show vulnerability to concurrent inhibition of EGFR and AXL, thus providing precision targeting to patients affected by LZTR1-mutant cancer.
Ubiquitin (Ub) ligases E3 are an important factor in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe containing the E2-binding site and a C-lobe containing the catalytic Cys residue that forms a thioester bond with Ub. These two lobes are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of this type of E3 ligases remain unclear. Here, we provide the first direct demonstration of structural dynamics of the E6AP HECT domain using high-speed atomic force microscopy. We also investigated structural dynamics of hinge loop flexibility restricted HECT domain, and we found that flexibility of the E6AP hinge loop has a great impact not only on its structural dynamics but also on the formation of free Ub chains mediated by E3-E3 interactions.
Protein-protein interaction networks (PPIs) govern the majority of biological processes, but how oncogenic mutations impact these interactions and their functions at a network scale is poorly understood. Mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) is a pre-requisition for EGFR tyrosine kinase inhibitor (TKI) treatment. Identification of interaction partners that bind to mutated EGFR can help understand the mechanism of action and pathways that mediate drug resistance. In this study, we characterized the dynamic interaction network of a pair of EGFR wildtype and mutant NSCLC cell lines. We performed immunoprecipitation of endogenous EGFR at various time points following EGF treatment and analyzed the associated proteins by quantitative mass spectrometry. Our results showed that the core signaling modules and key downstream pathways are maintained in the mutant cell line, but receptor internalization and intracellular trafficking in the mutant is delayed. Furthermore, we identified mutant EGFR-associated proteins that could affect EGFR functions in lung adenocarcinoma.
Significance
We analyzed the dynamic EGFR interaction network in NSCLC cell lines expressing wild-type and mutant EGFR. By comparing the similarities and differences in the EGFR proteome, we gained a better understanding of EGFR signal transduction network, and identified new factors for further functional characterizations and clinical significance assessment.
Type I Interferons (IFNs) induce the expression of >500 genes, which are collectively called ISGs (IFN-stimulated genes). One of the earliest ISGs induced by IFNs is ISG15 (Interferon-Stimulated Gene 15). Free ISG15 protein synthesized from the ISG15 gene is post‑translationally conjugated to cellular proteins and is also secreted by cells into the extracellular milieu. ISG15 comprises two ubiquitin-like domains (UBL1 and UBL2), each of which bears a striking similarity to ubiquitin, accounting for its earlier name ubiquitin cross-reactive protein (UCRP). Like ubiquitin, ISG15 harbors a characteristic β‑grasp fold in both UBL domains. UBL2 domain has a conserved C‑terminal Gly-Gly motif through which cellular proteins are appended via an enzymatic cascade similar to ubiquitylation called ISGylation. ISG15 protein is minimally expressed under physiological conditions. However, its IFN‑dependent expression is aberrantly elevated or compromised in various human diseases, including multiple types of cancer, neurodegenerative disorders (Ataxia Telangiectasia and Amyotrophic Lateral Sclerosis), inflammatory diseases (Mendelian Susceptibility to Mycobacterial Disease (MSMD), bacteriopathy and viropathy), and in the lumbar spinal cords of veterans exposed to Traumatic Brain Injury (TBI). ISG15 and ISGylation have both inhibitory and/or stimulatory roles in the etiology and pathogenesis of human diseases. Thus, ISG15 is considered a “double-edged sword” for human diseases in which its expression is elevated. Because of the roles of ISG15 and ISGylation in cancer cell proliferation, migration, and metastasis, conferring anti-cancer drug sensitivity to tumor cells, and its elevated expression in cancer, neurodegenerative disorders, and veterans exposed to TBI, both ISG15 and ISGylation are now considered diagnostic/prognostic biomarkers and therapeutic targets for these ailments. In the current review, we shall cover the exciting journey of ISG15, spanning three decades from the bench to the bedside.
Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies, aided by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity-biotinylation we show that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5- and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes, ensuring efficient receptor desensitization and lysosomal delivery.
Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.
Aging of the nervous system is typified by depressed metabolism, compromised proteostasis, and increased inflammation that results in cognitive impairment. Differential expression analysis is a popular technique for exploring the molecular underpinnings of neural aging, but technical drawbacks of the methodology often obscure larger expression patterns. Co-expression analysis offers a robust alternative that allows for identification of networks of genes and their putative central regulators. In an effort to expand upon previous work exploring neural aging in the marine model Aplysia californica , we used weighted gene correlation network analysis to identify co-expression networks in a targeted set of aging sensory neurons in these animals. We identified twelve modules, six of which were strongly positively or negatively associated with aging. Kyoto Encyclopedia of Genes analysis and investigation of central module transcripts identified signatures of metabolic impairment, increased reactive oxygen species, compromised proteostasis, disrupted signaling, and increased inflammation. Although modules with immune character were identified, there was no correlation between genes in Aplysia that increased in expression with aging and the orthologous genes in oyster displaying long-term increases in expression after a virus-like challenge. This suggests anti-viral response is not a driver of Aplysia sensory neuron aging.
Tumor metastasis is a leading cause of death in lung adenocarcinoma (LUAD) patients, but the molecular events that regulate metastasis have not been completely elucidated. STAMBP is a deubiquitinating enzyme of the Jab1/MPN metalloenzyme family that regulates the stability of substrates in cells by specifically removing ubiquitin molecules. We found that STAMBP expression was increased in the cytoplasm of tumor cells from LUAD patients. The STAMBP level was closely associated with tumor size, lymph node invasion and neoplasm disease stage. A high STAMBP level predicted poor overall survival and disease-free survival in LUAD patients. STAMBP overexpression promoted cell migration and invasion, whereas STAMBP knockdown attenuated these processes in LUAD cells after epidermal growth factor treatment. Mechanistically, increased STAMBP expression promoted the stabilization of Epidermal growth factor receptor (EGFR), whereas STAMBP knockdown induced the degradation of EGFR. STAMBP may deubiquitinate EGFR by localizing in early endosomes and increase EGFR membrane localization in LUAD cells. The overexpression of STAMBP triggered the activation of MAPK signaling after epidermal growth factor treatment. In contrast, this activation was attenuated in STAMBP knockdown cells. Small molecule inhibitors of EGFR and MAPK signaling pathway may block STAMBP-induced cell mobility and invasion as well as ERK activation in cells. Importantly, STAMBP knockdown suppressed LUAD tumor growth and metastasis by regulating the EGFR-mediated ERK activation in a xenograft mouse model. Our findings identified STAMBP as a novel potential target for LUAD therapy.
Digestive system cancers, including liver, gastric, colon, esophageal and pancreatic cancers, are the leading cause of cancers with high morbidity and mortality, and the question of their clinical treatment is still open. Previous studies have indicated that Ziyuglycoside II (ZYG II), the major bioactive ingredient extract from Sanguisorba officinalis L., significantly inhibits the growth of various cancer cells. However, the selective anti-tumor effects of ZYG II against digestive system cancers are not systemically investigated. In this study, we reported the anti-cancer effect of ZYG II on esophageal cancer cells (OE21), cholangiocarcinoma cells (HuCCT1), gastric cancer cells (BGC-823), liver cancer cells (HepG2), human colonic cancer cells (HCT116), and pancreatic cancer cells (PANC-1). We also found that ZYG II induced cell cycle arrest, oxidative stress and mitochondrial apoptosis. Network pharmacology analysis suggested that UBC, EGFR and IKBKG are predicted targets of ZYG II. EGFR signaling was suggested as the critical pathway underlying the anti-cancer effects of ZYG II and both docking simulation and western blot analysis demonstrated that ZYG II was a potential EGFR inhibitor. Furthermore, our results showed synergistic inhibitory effects of ZYG II and chemotherapy 5-FU on the growth of cancer cells. In summary, ZYG II are effective anti-tumor agents against digestive cancers. Further systemic evaluation of the anti-cancer activities in vitro and in vivo and characterization of underlying mechanism will promote the development of novel supplementary therapeutic strategies based on ZYG II for the treatment of digestive system cancers.
The tumor suppressor protein p53 participates in the control of key biological functions such as cell death, metabolic homeostasis and immune function, which are closely related to various diseases such as tumors, metabolic disorders, infection and neurodegeneration. The p53 gene is also mutated in approximately 50% of human cancer cells. Mutant p53 proteins escape from the ubiquitination-dependent degradation, gain oncogenic function and promote the carcinogenesis, malignant progression, metastasis and chemoresistance. Therefore, the stability of both wild type and mutant p53 needs to be precisely regulated to maintain normal functions and targeting the p53 stability is one of the therapeutic strategies against cancer. Here, we focus on compound-induced degradation of p53 by both the ubiquitination-dependent proteasome and autophagy-lysosome degradation pathways. We also review other posttranslational modifications which control the stability of p53 and the biological functions involved in these processes. This review provides the current theoretical basis for the regulation of p53 abundance and its possible applications in different diseases.
Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.
The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.
We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.
This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.
We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.
Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.
A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.
In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the alpha-factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH-induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin-dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.
Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.
Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis.
There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Here we report that mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin-protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination.
The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-A resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva(TM)). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.
STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs,
which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil
domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic
regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant
Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2
to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated
proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes
is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs
by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation
of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent
complex that sorts ubiquitinated proteins into the multivesicular body pathway.
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.
Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.
Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.
Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.
There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.
In the present study, we examined EGF-induced internalization, degradation and trafficking of the epidermal growth factor receptor (EGFR) mutated at serines 1046, 1047, 1057 and 1142 located in its cytoplasmic carboxy-terminal region. We found the serine-mutated EGFR to be inhibited in EGF-induced internalization and degradation in NIH3T3 cells. We therefore tested the hypothesis that these mutations affect ligand-induced c-Cbl association with the receptor, leading to inhibited receptor ubiquitination. EGF was unable to induce ubiquitination of the serine-mutated EGFR, yet EGF-induced phosphorylation of the c-Cbl-binding site at tyrosine 1045, and c-Cbl-EGFR association, was unaffected. To compare the relevance of these serine residues with tyrosine 1045 in their regulation of c-Cbl binding and receptor ubiquitination, we analysed an EGFR mutated at tyrosine 1045 (Y1045F). EGF-induced c-Cbl-EGFR binding was partially inhibited, and receptor ubiquitination was abrogated in cells expressing Y1045F-EGFR. In contrast, ligand-induced internalization and degradation of the Y1045F mutant was similar to that of wild-type EGFR. Together, our data indicate that the serine residues and tyrosine 1045 are essential for EGF-induced receptor ubiquitination, but only the serine residues are critical for EGFR internalization and degradation.
To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.
Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.
The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III. ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37. Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified. Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs. The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins. HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes. Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28. Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation. Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors.
Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis.
Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.
• internalization
• rafts
• caveolae
• ubiquitination
• ubiquitin-interacting motif
Mass-spectrometry-based proteomics has become an essential tool for the qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the ubiquitin system are well suited to proteomic studies. This review summarizes advances involving the identification of ubiquitinated proteins, the elucidation of ubiquitin-modification sites and the determination of polyubiquitin chain linkages, as well as offering a perspective on the application of emerging technologies for mechanistic and functional studies of protein ubiquitination.
Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.
We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.
Ubiquitin regulates protein transport between membrane compartments by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery. Monoubiquitin attached to integral plasma membrane proteins or to associated transport modifiers serves as a regulated signal for internalization into the endocytic pathway. Similarly, monoubiquitin attached to biosynthetic and endocytic membrane proteins is a signal for sorting of cargo into vesicles that bud into the late endosome lumen for delivery into the lysosome. Ubiquitination of trans-acting endocytic proteins is also required for transport, and key endocytic proteins are modified by monoubiquitin. Regulatory enzymes of the ubiquitination machinery, ubiquitin ligases, control the timing and specificity of plasma membrane protein downregulation in such diverse biological processes as cell fate specification and neurotransmission. Monoubiquitin signals appended by these ligases are recognized by endocytic proteins carrying ubiquitin-binding motifs, including UBA, UEV, UIM, and CUE domains. The UIM proteins epsins and Hrs are excellent candidates for adaptors that link ubiquitinated cargo to the clathrin-based sorting machinery at appropriate regions of the endosomal or plasma membranes. Other ubiquitin-binding proteins also play crucial roles in cargo transport, although in most cases the role of ubiquitin-binding is not defined. Ubiquitin-binding proteins such as epsins, Hrs, and Vps9 are monoubiquitinated, indicating the general nature of ubiquitin regulation in endocytosis and suggesting new models to explain how recognition of monoubiquitin signals may be regulated.
The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (∼350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.
Using a quantitative electron microscopic autoradiographic technique, we have localized the initial binding step of 125I-labeled epidermal growth factor (125I-EGF) to the plasma membrane of the human fibroblast. After initial binding, labeled EGF is internalized progressively by the cell in a time- and temperature-dependent fashion; when cell-associated radioactivity comes to steady state, approximately 1/3 of the autoradiographic grains are related to the plasma membrane and approximately 2/3 have been internalized. Under these conditions the internalized grains are almost exclusively related to lysosomal structures. When 125I-EGF associates with the cells for 2 hr at 4 degrees or 2 min at 37 degrees, 34% of grains localize to coated regions of the membrane. These coated regions make up less than 2% of the membrane surface. These data directly confirm kinetic studies and suggest that saturable binding of EGF is followed by adsorptive pinocytosis and cellular degradation of the ligand and possibly its cell surface receptor.
The distribution of cell surface receptors for transferrin–iron and epidermal growth factor (EGF) on the surface of cultured epitheloid (A431) cells has been identified by immunocytochemical electron microscopy. The patterns of movement displayed by these two receptor populations as they transfer to their sites of internalization on the cell surface are different. The movement of recycling transferrin receptors over the surface is ligand-independent whereas EGF receptors are more stable residents and remain monodisperse until they bind ligand.
Prior to uptake transferrin receptors cluster, predominantly within existing clathrin-coated pits while the aggregates formed by EGF ligand–receptor complexes induce new membrane invaginations. These results are discussed in relation to receptor populations concerned with constitutive, high capacity uptake processes and receptors involved in signal transduction.
Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.
Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.
TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.
The c-Cbl protooncogene can function as a negative regulator of receptor protein tyrosine kinases (RPTKs) by targeting activated receptors for polyubiquitination and downregulation. This function requires its tyrosine kinase binding (TKB) domain for targeting RPTKs and RING finger domain to recruit E2 ubiquitin-conjugating enzymes. It has therefore been proposed that oncogenic Cbl proteins act in a dominant-negative manner to block this c-Cbl activity. In testing this hypothesis, we found that although mutations spanning the RING finger abolish c-Cbl-directed polyubiquitination and downregulation of RPTKs, they do not induce transformation. In contrast, it is mutations within a highly conserved alpha-helical structure linking the SH2 and RING finger domains that render Cbl proteins oncogenic. Thus, Cbl transformation involves effects additional to polyubiquitination of RPTKs that are independent of the RING finger and its ability to recruit E2-conjugating enzymes.
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.
Binding of hormones, growth factors and other cell modulators to cell-surface receptors triggers a complex array of signal-transduction events. The activation of many receptors also accelerates their endocytosis. Endocytic transport is important in regulating signal transduction and in mediating the formation of specialized signalling complexes. Conversely, signal-transduction events modulate specific components of the endocytic machinery. Recent studies of protein tyrosine kinases and G-protein-coupled receptors have shed new light on the mechanisms and functional consequences of this bidirectional interplay between signalling and membrane-transport networks.
Binding of external factors to cell membrane receptors triggers intracellular signalling pathways that ultimately determine if the cell proliferates, differentiates or undergoes apoptosis. Activated receptors also initiate a cascade of events, called negative receptor signalling, that decreases the amplitude of positive signals and modulates the level of cell stimulation. Recent studies have revealed that negative signalling by receptor tyrosine kinases involves coordinated action of ubiquitin ligases (i.e. Cbl), adaptor proteins (i.e. Grb2 and CIN85), inhibitory molecules (i.e. Sprouty), cytoplasmic kinases (i.e. activated Cdc42-associated kinase) and phosphoinositol metabolites. These inhibitory signals are essential for normal cell functioning, and their deregulation often results in human diseases.
Cbl proteins have been implicated in the regulation of endocytic trafficking of epidermal growth factor receptor. However, the precise role of Cbl in epidermal growth factor receptor endocytosis is not defined. To directly visualize Cbl in cells and perform structure-function analysis of Cbl's role in epidermal growth factor receptor internalization, a yellow fluorescent protein-fusion of c-Cbl was constructed. Upon epidermal growth factor receptor activation, Cbl-yellow fluorescent protein moved with epidermal growth factor receptor to clathrin-coated pits and endosomes. Localization of Cbl-yellow fluorescent protein to these endocytic organelles was dependent on a proline-rich domain of c-Cbl that interacts with Grb2 as shown by fluorescence resonance energy transfer microscopy. In contrast, direct binding of Cbl to phosphotyrosine 1045 of the epidermal growth factor receptor was required for epidermal growth factor receptor polyubiquitination, but was not essential for Cbl-yellow fluorescent protein localization in epidermal growth factor receptor-containing compartments. These data suggest that the binding of Cbl to epidermal growth factor receptor through Grb2 is necessary and sufficient for Cbl function during clathrin-mediated endocytosis. Overexpression of c-Cbl mutants that are capable of Grb2 binding but defective in linker/RING finger domain function severely inhibited epidermal growth factor receptor internalization. The same dominant-negative mutants of Cbl did not block epidermal growth factor receptor recruitment into coated pits but retained receptors in coated pits, thus preventing receptor endocytosis and transport to endosomes. These data suggest that the linker and RING finger domain of Cbl may function during late steps of coated vesicle formation. We propose that the RING domain of Cbl facilitates endocytosis either by epidermal growth factor receptor monoubiquitylation or by ubiquitylation of proteins associated with the receptor.
Recent studies indicate that endocytic organelles can play a more active role in signal propagation and amplification than was recognised before. By deciphering the interplay between endocytosis and signalling, we will be able to gain a more sophisticated level of understanding of signal transduction mechanisms.
Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.
Signal transduction and endocytosis: close encounters of many kinds
- Sorkin
Sorkin, A., and Von Zastrow, M. (2002). Signal transduction and en-docytosis: close encounters of many kinds. Nat. Rev. Mol. Cell Biol. 3, 600–614.