Late endosomal traffic of the epidermal growth factor receptor ensures spatial and temporal fidelity of mitogen-activated protein kinase signaling

Mitogen-activated protein kinase (MAPK) signaling is regulated by assembling distinct scaffold complexes at the plasma membrane and on endosomes. Thus, spatial resolution might be critical to determine signaling specificity. Therefore, we investigated whether epidermal growth factor receptor (EGFR) traffic through the endosomal system provides spatial information for MAPK signaling. To mislocalize late endosomes to the cell periphery we used the dynein subunit p50 dynamitin. The peripheral translocation of late endosomes resulted in a prolonged EGFR activation on late endosomes and a slow down in EGFR degradation. Continuous EGFR signaling from late endosomes caused sustained extracellular signal-regulated kinase and p38 signaling and resulted in hyperactivation of nuclear targets, such as Elk-1. In contrast, clustering late endosomes in the perinuclear region by expression of dominant active Rab7 delayed the entry of the EGFR into late endosomes, which caused a delay in EGFR degradation and a sustained MAPK signaling. Surprisingly, the activation of nuclear targets was reduced. Thus, we conclude that appropriate trafficking of the activated EGFR through endosomes controls the spatial and temporal regulation of MAPK signaling.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.     

Oncostatin M suppresses EGF-mediated protein tyrosine phosphorylation in breast cancer cells

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.     

Cytokine signaling through Stat3 activates integrins, promotes adhesion, and induces growth arrest in the myeloid cell line 32D

Hematopoietic cell development and function is dependent on cytokines and on intercellular interactions with the microenvironment. Although the intracellular signaling pathways stimulated by cytokine receptors are well described, little is known about the mechanisms through which these pathways modulate hematopoietic cell adhesion events in the microenvironment. Here we show that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules in the myeloid progenitor cell line 32D. We generated an erythropoietin receptor (EpoR) isoform (ER343/401-S3) that activates Stat3 rather than Stat5 by substituting the Stat3 binding/activation sequence motif from gp130 for the sequences surrounding tyrosines 343 and 401 in the receptor cytoplasmic region. Activation of Stat3 leads to homotypic cell aggregation, increased expression of intercellular adhesion molecule 1 (ICAM-1), CD18, and CD11b, and activation of signaling through CD18-containing integrins. Unlike the wild type EpoR, ER343/401-S3 is unable to support long term Epo-dependent proliferation in 32D cells. Instead, Epo-treated ER343/401-S3 cells undergo G(1) arrest and express elevated levels of the cyclin-dependent kinase inhibitor p27(Kip1). Sustained activation of Stat3 in these cells is required for their altered morphology and growth properties since constitutive SOCS3 expression abrogates homotypic cell aggregation, signaling through CD18-containing integrins, G(1) arrest, and accumulation of p27(Kip1). Collectively, our results demonstrate that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules, indicating that a role for Stat3 is to regulate intercellular contacts in myeloid cells.     

TACC3 Is Essential for EGF-Mediated EMT in Cervical Cancer

The third member of transforming acidic coiled-coil protein (TACC) family, TACC3, has been shown to be an important player in the regulation of centrosome/microtubule dynamics during mitosis and found to be deregulated in a variety of human malignancies. Our previous studies have suggested that TACC3 may be involved in cervical cancer progression and chemoresistance, and its overexpression can induce epithelial-mesenchymal transition (EMT) by activating the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinases (ERKs) signal transduction pathways. However, the upstream mechanisms of TACC3-mediated EMT and its functional/clinical importance in human cervical cancer remain elusive. Epidermal growth factor (EGF) has been shown to be a potent inducer of EMT in cervical cancer and associated with tumor invasion and metastasis. In this study, we found that TACC3 is overexpressed in cervical cancer and can be induced upon EGF stimulation. The induction of TACC3 by EGF is dependent on the tyrosine kinase activity of the EGF receptor (EGFR). Intriguingly, depletion of TACC3 abolishes EGF-mediated EMT, suggesting that TACC3 is required for EGF/EGFR-driven EMT process. Moreover, Snail, a key player in EGF-mediated EMT, is found to be correlated with the expression of TACC3 in cervical cancer. Collectively, our study highlights a novel function for TACC3 in EGF-mediated EMT process and suggests that targeting of TACC3 may be an attractive strategy to treat cervical cancers driven by EGF/EGFR signaling pathways.     

The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)-stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.     

Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.     

The hominoid-specific oncogene TBC1D3 activates Ras and modulates epidermal growth factor receptor signaling and trafficking

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.     

rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.     

EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. We recently reported that leucine-rich repeat kinase 1 (LRRK1) is involved in the trafficking of EGFR from early to late endosomes. In this study, we demonstrate that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper endosomal trafficking of EGFR. Phosphorylation of LRRK1 at Tyr-944 results in reduced LRRK1 kinase activity. Mutation of LRRK1 Tyr-944 (Y944F) abolishes EGF-stimulated tyrosine phosphorylation, resulting in hyperactivation of LRRK1 kinase activity and enhanced motility of EGF-containing endosomes toward the perinuclear region. The compartments in which EGFR accumulates are mixed endosomes and are defective in the proper formation of intraluminal vesicles of multivesicular bodies. These results suggest that feedback down-regulation of LRRK1 kinase activity by EGFR plays an important role in the appropriate endosomal trafficking of EGFR.     

Spatial regulation of EGFR signaling by Sprouty2

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. Here we show that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling [1], interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization.     

GAPex-5 mediates ubiquitination, trafficking, and degradation of epidermal growth factor receptor

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.     

Altered regulation of EGF receptor signaling following a partial hepatectomy

We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.