E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal growth factor receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human keratinocytes. Phosphatidylinositol 3-kinase (PI3K) and ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival factor. To test this hypothesis, the apoptotic response of UV B-irradiated primary keratinocytes infected with either control retrovirus, LXSN, or HPV16 2E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/2 MAPK pathways, respectively, suggesting that the PI3K and ERK1/2 MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/2 MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/2 activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were growth arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/2 MAPK signal pathways.     

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G₁/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis.     

Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration

Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.     

EGF Induces Cell Motility and Multi-Drug Resistance Gene Expression in Breast Cancer Cells

The epidermal growth factor receptor (EGFR) is important for normal development, differentiation, and cell proliferation. Deregulation of EGFR has been observed in breast cancer. EGFR and signal pathways activated by these receptors have been associated with an advanced tumor stage and a poor clinical prognosis in breast cancer, however, the precise mechanisms responsible for this process are still not known. Here we show that treatment of MCF-7 breast cancer cells with EGF activated Akt and ERK, induced morphological changes, and increased cell motility. In addition, the constitutive expression of Raf-1 and the use of a MEK inhibitor demonstrated the participation of the Raf/MEK/ERK pathway in these processes. Importantly we detected that EGF induced MRP-1, 3, 5 and 7 gene expression and an increase in MRP1 promoter activity. In conclusion, treatment of MCF-7 breast cancer cells with EGF, in the absence of other growth factors, resulted in activation of EGFR signal transduction pathways; which were related with cell motility and drug resistance.     

Urokinase receptor deficiency results in EGFR-mediated failure to transmit signals for cell survival and neurite formation in mouse neuroblastoma cells

Urokinase-type plasminogen activator uPA and its receptor (uPAR) are the central players in extracellular matrix proteolysis, which facilitates cancer invasion and metastasis. EGFR is one of the important components of uPAR interactome. uPAR/EGFR interaction controls signaling pathways that regulate cell survival, proliferation and migration. We have previously established that uPA binding to uPAR stimulates neurite elongation in neuroblastoma cells, while blocking uPA/uPAR interaction induces neurite branching and new neurite formation. Here we demonstrate that blocking the uPA binding to uPAR with anti-uPAR antibody decreases the level of pEGFR and its downstream pERK1/2, but does increase phosphorylation of Akt, p38 and c-Src Since long-term uPAR blocking results in a severe DNA damage, accompanied by PARP-1 proteolysis and Neuro2a cell death, we surmise that Akt, p38 and c-Src activation transmits a pro-apoptotic signal, rather than a survival.Serum deprivation resulting in enhanced neuritogenesis is accompanied by an upregulated uPAR mRNA expression, while EGFR mRNA remains unchanged. EGFR activation by EGF stimulates neurite growth only in uPAR-overexpressing cells but not in control or uPAR-deficient cells. In addition, AG1478-mediated inhibition of EGFR activity impedes neurite growth in control and uPAR-deficient cells, but not in uPAR-overexpressing cells. Altogether these data implicate uPAR as an important regulator of EGFR and ERK1/2 signaling, representing a novel mechanism which implicates urokinase system in neuroblastoma cell survival and differentiation.     

Wnt2b controls retinal cell differentiation at the ciliary marginal zone

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.     

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Wnt5a-dopamine D2 receptor interactions regulate dopamine neuron development via extracellular signal-regulated kinase (ERK) activation

The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R(-/-)) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R(-/-) mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R(-/-) mice. Co-immunoprecipitation and displacement of [(3)H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway.     

Phosphatidylinositol 3-kinase regulates early differentiation in human laryngeal keratinocytes

Summary Epidermal growth factor receptor (EGFR) signaling regulates a variety of cellular functions, including proliferation, gene expression, and differentiation. Infection of laryngeal epithelial cells by human papillomaviruses causes recurrent respiratory papillomas, benign tumors characterized by an altered pattern of differentiation. Papilloma cells overexpress the EGFR and have constitutively active extracellular signal-regulated kinase (ERK) and enhanced phosphatidylinositol 3-kinase (PI3K) activity, but overexpression of the lipid phosphatase PTEN (Phosphatase and Tensin Homolog) reduces activation of Akt by PI3K. We hypothesized that the altered differentiation of papillomas reflects these changes in signaling from the EGFR-ERK and PI3K-Akt pathways and that one or both of these pathways is required for the normal differentiation process in mucosal epithelium. Inhibiting either the enzymatic activity or the synthesis of PI3K in uninfected laryngeal cells blocked expression of keratin-13 (K13), a protein induced during normal differentiation. In contrast, inhibiting activation of ERK had minimal effect. Using ribonucleic acid interference to reduce protein levels of integrinlinked kinase 1 or phosphoinositide-dependent protein kinase 1, intermediates in the activation of Akt by PI3K, or reducing levels of Akt-1 itself did not inhibit K13 expression by normal laryngeal keratinocytes. We conclude that PI3K activation is an important regulator of expression of K13, a marker for the normal differntiation process in human mucosal keratinocytes, that this function does not require activation of Akt-1, and that the failure to express K13 in papilloma cells is not because of reduction in activated Akt.     

MUC1 contributes to BPDE-induced human bronchial epithelial cell transformation through facilitating EGFR activation

Although it is well known that epidermal growth factor receptor (EGFR) is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals), a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE), the active form of the cigarette smoke (CS) carcinogen benzo(a)pyrene (BaP)s. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Selenazoles (selenium compounds) facilitate survival of cultured rat pheochromocytoma PC12 cells after serum-deprivation and stimulate their neuronal differentiation via activation of Akt and mitogen-activated protein kinase, respectively

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.     

Frizzled-5 Receptor Is Involved in Neuronal Polarity and Morphogenesis of Hippocampal Neurons

The Wnt signaling pathway plays important roles during different stages of neuronal development, including neuronal polarization and dendritic and axonal outgrowth. However, little is known about the identity of the Frizzled receptors mediating these processes. In the present study, we investigated the role of Frizzled-5 (Fzd5) on neuronal development in cultured Sprague-Dawley rat hippocampal neurons. We found that Fzd5 is expressed early in cultured neurons on actin-rich structures localized at minor neurites and axonal growth cones. At 4 DIV, Fzd5 polarizes towards the axon, where its expression is detected mainly at the peripheral zone of axonal growth cones, with no obvious staining at dendrites; suggesting a role of Fzd5 in neuronal polarization. Overexpression of Fzd5 during the acquisition of neuronal polarity induces mislocalization of the receptor and a loss of polarized axonal markers. Fzd5 knock-down leads to loss of axonal proteins, suggesting an impaired neuronal polarity. In contrast, overexpression of Fzd5 in neurons that are already polarized did not alter polarity, but decreased the total length of axons and increased total dendrite length and arborization. Fzd5 activated JNK in HEK293 cells and the effects triggered by Fzd5 overexpression in neurons were partially prevented by inhibition of JNK, suggesting that a non-canonical Wnt signaling mechanism might be involved. Our results suggest that, Fzd5 has a role in the establishment of neuronal polarity, and in the morphogenesis of neuronal processes, in part through the activation of the non-canonical Wnt mechanism involving JNK.     

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal-regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de-phosphorylated phospho-ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper-phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over-expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock-down by Cdc25A siRNA resulted in a constitutive-like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR-devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho-ERK is likely another Cdc25A substrate, and Cpd 5-caused ERK phosphorylation is probably regulated by both EGFR-dependent and EGFR-independent pathways.     

Increased expression of epidermal growth factor receptor induces sequestration of extracellular signal-related kinases and selective attenuation of specific epidermal growth factor-mediated signal transduction pathways

Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.