The Cbl RING finger C-terminal flank controls epidermal growth factor receptor fate downstream of receptor ubiquitination

Evolutionarily conserved sequences of the E3/protein-ubiquitin ligase Cbl regulate epidermal growth factor receptor (EGF-R) signaling and degradation. These sequences encompass Cbl's tyrosine kinase-binding domain, linker region, RING finger (RF), and an uncharacterized flank C-terminal to the RF (residues 420-436). The latter domain, designated the RF tail, extends beyond Cbl's ubiquitin-conjugating enzyme (Ubc)-binding domain and has no known function. We report structure-function studies evaluating the impact of Cbl RF tail truncations on EGF-R fate in HEK 293 cells. All of the truncation mutants exhibit greatly reduced binding to activated EGF-R and lack proline-rich sequences that mediate direct Cbl association with SH3 proteins such as Grb2, yet a subset of mutants collectively enhances EGF-R ubiquitination, downregulation, and degradation. Significantly, EGF-R degradation correlates better with RF tail-dependent degradation of the Cbl substrate Sprouty2 than with EGF-R ubiquitination: expression of the RF tail truncation mutant Cbl 1-433 enhanced EGF-R ubiquitination while impeding Sprouty2 degradation, and Cbl 1-433 failed to enhance EGF-R downregulation or degradation. Our results suggest that EGF-R fate is controlled by a checkpoint downstream of receptor ubiquitination whose regulation by the Cbl RF tail may require Sprouty2 degradation.     

Epidermal growth factor induced apoptosis

Apoptosis, or programmed cell death, plays an important role in the pathogenesis of a number of human diseases, including cancers, autoimmune diseases, and neurodegenerative disorders. Recent evidence suggests that EGF induced signal transduction pathways which govern cell proliferation and cell cycle progression also mediate antiproliferative effects leading to increased apoptosis in cells that express high levels of epidermal growth factor receptors. Treatments designed to increase apoptosis have potential to change the natural progression of cancer and eventually lead to its successful control.     

Inhibition of autophosphorylation of epidermal growth factor receptor by a small peptide not employing an ATP-competitive mechanism

Previously we found that short peptides surrounding major autophosphorylation sites of EGFR (VPEY(1068)INQ, DY(1148)QQD, and ENAEY(1173)LR) suppress phosphorylation of purified EGFR to 30-50% at 4000 microM. In an attempt to improve potencies of the peptides, we modified the sequences by substituting various amino acids for tyrosine or by substituting Gln and Asn for Glu and Asp, respectively. Among the modified peptides, Asp/Asn- and Glu/Gln-substitution in DYQQD (NYQQN) and ENAEYLR (QNAQYLR), respectively, improved inhibitory potencies. The inhibitory potency of NYQQN was not affected by the concentration of ATP, while that of QNAQYLR was affected. Docking simulations showed different mechanisms of inhibition for the peptides: inhibition by binding to the ATP-binding site (QNAQYLR) and inhibition by binding to a region surrounded by alphaC, the activation loop, and the catalytic loop and interfering with the catalytic reaction (NYQQN). The inhibitory potency of NYQQN for insulin receptor drastically decreased, whereas QNAQYLR inhibited autophosphorylation of insulin receptor as well as EGFR. In conclusion, NYQQN is not an ATP-competitive inhibitor and the binding site of this peptide appears to be novel as a tyrosine kinase inhibitor. NYQQN could be a promising seed for the development of anti-cancer drugs having specificity for EGFR.     

Anti-epidermal growth factor receptor monoclonal antibodies affecting signal transduction

Monoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS-sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5--125 reacting with a site of the extracellular EGFR domain blocked EGF-binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5-D43, also defined by an SDS-sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine-specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5-D43. Since interaction of mab 5-D43 with EGFR induced most EGF-specific functions, although it did not bind to the EGF-specific site of EGFR, we have to assume that binding of mab 5-D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1-594, which is defined by an SDS-insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1-594 also abolished EGF-induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor receptor expression in human gliomas

Summary The expression of epidermal growth factor receptor (EGFR) was determined in cryosections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and two monoclonal antibodies (mAb) against EGFR. All gliomas were found to express EGFR when examined with B-EGF, whereas 33 expressed EGFR when examined with the two mAbs. The highly malignant gliomas (glioblastomas and anaplastic astrocytomas) had a more heterogeneous staining pattern and a larger proportion of tumour cells staining strongly with B-EGF than did the low-grade gliomas (astrocytomas, oligodendrogliomas, mixed gliomas, and ependymomas). This indicates that high-grade gliomas contain more tumour cells rich in EGFR than do the low-grade gliomas. Reactive astrocytes, ependymal cells, and many types of nerve cells (cerebral cortical pyramidal cells, pyramidal and granular hippocampal cells, Purkinje cells, cerebellar granular cells and neurons in the molecular layer of the cerebellum) expressed EGFR, whereas small neurons and normal glial cells were not found to express EGFR.     

表皮生长因子受体EGFR及其信号传导

表皮生长因子受体(epidermalgrowthfactorreceptor,EGFR)是ErbB家族成员之一,具有酪氨酸激酶活性,是一种重要的跨膜受体。EGFR被配体激活后启动胞内信号传导,经过细胞质中衔接蛋白、酶的级联反应,调节转录因子激活基因的转录,指导细胞迁移、黏附、增殖、分化、凋亡,且与肿瘤的形成和恶化密切相关。本文对EGFR的结构特性、几种重要的信号通路及各个信号通路之间的交联,以及信号的衰减等方面的研究进展进行了综述。     

Proteolytic domains of the epidermal growth factor receptor of human placenta

Microsomal membranes from human placenta, which bind 5–20 pmol of ¹²⁵I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with ¹²⁵I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.     

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using ¹²⁵I-labeled polyvinylpyrrolidone (¹²⁵I-PVP) as a fluid phase marker. We found that EGF initially induced a thereefold increase in the rate of ¹²⁵I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluie uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of ¹²⁵I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of ¹²⁵I-PVP uptake matched the kinetics of binding of ¹²⁵I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Syntaxin 9 is enriched in skin hair follicle epithelium and interacts with the epidermal growth factor receptor

We describe a novel syntaxin family member, syntaxin 9 (Syn 9), which does not possess a typical C-terminal hydrophobic tail anchor. Syn 9 has, however, a Q-SNARE domain and an overall homology to syntaxins (with the highest overall homology with mammalian syntaxin 11). Syn 9 is enriched in some epithelial cells, particularly that of the stomach lining and the skin. At the skin, it is found in the epidermal layers as well as structures associated with hair follicles. A biochemical interaction screen revealed that Syn 9 interacts specifically with the epidermal growth factor (EGF) receptor. Overexpression of Syn 9 perturbed EGF receptor endocytosis but does not appear to affect the internalization of the transferrin receptor. Syn 9 may therefore have a role in EGF receptor transport and signaling in certain epithelial cell types.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

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

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

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor receptor (EGFR) involvement in epithelial‐derived cancers and its current antibody‐based immunotherapies

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that is part of the family of tyrosine kinase receptors. The binding of EGFR to its cognate ligands leads to its autophosphorylation and subsequent activation of the signal transduction pathways involved in regulating cellular proliferation, differentiation, and survival. Accordingly, this receptor carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. Correspondingly, the loss of EGFR regulation results in many human diseases, with the most notable cancer. This receptor is overexpressed and/or mutated in multiple epithelial‐derived tumors, and associated with poor prognosis and survival in cancer patients. Here, we discuss in detail the role of EGFR in specific epithelial‐derived cancer pathologies; these include lung cancer, colorectal cancer, and squamous cell carcinomas. The development of multiple anticancer agents against EGFR diminished the progression and metastasis of tumors. Some of the most versatile therapeutic anti‐EGFR agents include the monoclonal antibodies (mAbs), demonstrating success in clinical settings when used in combination with cytotoxic treatments, such as chemotherapy and/or radiation. We thus discuss the development and application of two of the most notable therapeutic mAbs, cetuximab, and panitumumab, currently utilized in various EGFR‐related epithelial cancers.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。