Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling

Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution.

Structured summary of protein interactions

EEA1 and EGFRcolocalize by fluorescence microscopy (View interaction).     

The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells

CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, and adhesion. In addition, it has been known that CD9 can associate with other proteins. Here we demonstrated the physical and functional association of CD9 with epidermal growth factor receptor (EGFR) on MKN-28 cells. Double-immunofluorescent staining and immunoprecipitation demonstrated the complex formation of CD9-EGFR and CD9-beta(1) integrin, and that both complexes are colocalized on the cell surface especially at the cell-cell contact site. Anti-CD9 monoclonal antibody ALB6 induced a dotted or patch-like aggregation pattern of both CD9-EGFR and CD9-beta(1) integrin. The internalization of EGFR after EGF-stimulation was significantly enhanced by the treatment with ALB6. CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO-HER/CD9), which were transfected with pTJ/human EGFR/CD9. Furthermore expression of CD9 specifically attenuated EGFR signaling in CHO-HER/CD9 cells through the down regulation of surface expression of EGFR. These results suggest that CD9 might have an important role that attenuates EGFR signaling. Therefore, CD9 not only associates EGFR but also a new regulator, which may affect EGF-induced signaling in cancer cells.     

Interdependent epidermal growth factor receptor signalling and trafficking

Epidermal growth factor (EGF) receptor (EGFR) signalling regulates diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth and survival. EGFR signalling is critical during embryogenesis, in particular in epithelial development, and disruption of the EGFR gene results in epithelial immaturity and perinatal death. EGFR signalling also functions during wound healing responses through accelerating wound re-epithelialisation, inducing cell migration, proliferation and angiogenesis. Upregulation of EGFR signalling is often observed in carcinomas and has been shown to promote uncontrolled cell proliferation and metastasis. Therefore aberrant EGFR signalling is a common target for anticancer therapies. Various reports indicate that EGFR signalling primarily occurs at the plasma membrane and EGFR degradation following endocytosis greatly attenuates signalling. Other studies argue that EGFR internalisation is essential for complete activation of downstream signalling cascades and that endosomes can serve as signalling platforms. The aim of this review is to discuss current understanding of intersection between EGFR signalling and trafficking.     

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.     

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking

Background information. ARAP1 is an Arf (ADP‐ribosylation factor)‐directed GAP (GTPase‐activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1. Results. Here we report that ARAP1 associates with the CIN85 (Cbl‐interacting protein of 85 kDa). Arg⁸⁶ and Arg⁹⁰ of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl‐dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. Conclusion. ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1‐CIN85 complex drives exit of EGF—EGFR–Cbl complex from a pre‐early endosome into a pathway distinct from the early endosome/lysosome pathway.     

RME-8 regulates trafficking of the epidermal growth factor receptor

We recently identified receptor-mediated endocytosis 8 (RME-8), a DnaJ domain protein localized to endosomes. We now demonstrate that RME-8 depletion leads to decreased levels of epidermal growth factor receptor (EGFR) without influencing receptors that primarily recycle to the plasma membrane. Decreases in EGFR are detected at both surface and intracellular pools and result from increased rates of EGFR degradation. Interestingly, RME-8 depletion also decreases EGFR levels in breast cancer cell lines in which overexpression of the EGFR family member ErbB2 has been shown to protect EGFR from degradation. These data implicate RME-8 in sorting decisions influencing EGFR at the level of endosomes and point to RME-8 as a potential regulatory target in ErbB2-positive breast cancers.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Protein-tyrosine phosphatase 1B modulates early endosome fusion and trafficking of Met and epidermal growth factor receptors

The endoplasmic reticulum-localized non-receptor protein-tyrosine phosphatase 1B (PTP1B) is associated with oncogenic, metabolic, and cytokine-related signaling and functionally targets multiple receptor tyrosine kinases (RTKs) for dephosphorylation. Loss of PTP1B activity leads to enhanced ligand-dependent biological activity of the Met RTK among others. Here, we demonstrate that knockdown of PTP1B or expression of a PTP1B trapping aspartic acid-to-alanine substitution (D/A) mutant delayed ligand-induced degradation of the Met and EGF RTKs. Loss of PTP1B function abrogated trafficking of Met and EGF receptor to Rab5- and phosphatidylinositol 3-phosphate (Pl3P)-positive early endosomes and subsequent trafficking through the degradative pathway. Under these conditions, internalization of the Met and EGF receptors was unaltered, suggesting a block at the level of early endosome formation. We show that the N-ethylmaleimide-sensitive factor (NSF), an essential component of the vesicle fusion machinery, was hyperphosphorylated in PTP1B knockdown or PTP1B D/A-expressing cells and was a target for PTP1B. NSF knockdown phenocopied PTP1B knockdown, demonstrating a mechanism through which PTP1B regulates endocytic trafficking. Finally, we show that PTP1B dephosphorylated NSF and that this interaction was required for physiological RTK trafficking and appropriate attenuation of downstream signaling.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.     

Insulin-like growth factor I activates insulin receptor substrate 1 and Ras in human osteosarcoma cells.

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63, by acting through IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras cycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 cells were (1) IGF-I stimulated and lysates were immunoprecipitated with polyclonal IRS-1 antibody or (2) metabolically labeled with [32P]orthophosphoric acid and then cells were treated with IGF-I. Cell lysates were immunoprecipitated with p21Ras antibody (Y13-259) and bound nucleotides were analysed by thin-layer chromatography. We demonstrated tyrosine phosphorylation of IRS-1/2 immunoprecipitated from MG-63 cells stimulated with IGF-I. We also showed an increased level of GTP in p21Ras immunoprecipitates from IGF-I treated cells. Nuclear extracts prepared from 32P-labeled cells before and after addition of IGF-I were immunoprecipitated with c-Jun antibody. After electrophoresis and autoradiography, phosphorylation of the c-Jun band was seen to be IGF-I independent. Phosphoamino acid analysis of the c-Jun band showed that phosphoserine was the major species.     

TBC1D3, a hominoid-specific gene, delays IRS-1 degradation and promotes insulin signaling by modulating p70 S6 kinase activity

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.     

An integrated model of epidermal growth factor receptor trafficking and signal transduction

Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and approximately 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Our simulations predict that when EGFR is activated with TGF-alpha, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-alpha. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.     

Site-specific antibodies to the erbB oncogene product immunoprecipitate epidermal growth factor receptor

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.     

Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.