Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.     

IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.

During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor receptor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor-induced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- 15% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility.To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamma and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.     

ERK-dependent threonine phosphorylation of EGF receptor modulates receptor downregulation and signaling

Epidermal growth factor (EGF) signaling is critical in normal and aberrant cellular behavior. Extracellular signal-regulated kinase (ERK) mediates important downstream aspects of EGF signaling. Additionally, EGFR undergoes MEK1-dependent ERK consensus site phosphorylation in response to EGF or cytokines such as growth hormone (GH) and prolactin (PRL). GH- or PRL-induced EGFR phosphorylation alters subsequent EGF-induced EGFR downregulation and signal characteristics in an ERK-dependent fashion. We now use reconstitution to study mutation of the sole EGFR ERK phosphorylation consensus residue, (669)T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A ((669)T changed to alanine) EGFRs at similar abundance. Treatment of cells with GH or EGF caused phosphorylation of WT, but not T669A EGFR, in an ERK activity-dependent fashion that was detected with an antibody that recognizes phosphorylation of ERK consensus sites, indicating that (669)T is required for this phosphorylation. Notably, EGF-induced downregulation of EGFR abundance was much more rapid in cells expressing EGFR T669A vs. WT EGFR. Further, pretreatment with the MEK1/ERK inhibitor PD98059 enhanced EGF-induced EGFR loss in cells expressing WT EGFR, but not EGFR T669A, suggesting that the ERK-dependent effects on EGFR downregulation required phosphorylation of (669)T. In signaling experiments, EGFR T669A displayed enhanced acute (15 min) EGFR tyrosine phosphorylation (reflecting EGFR kinase activity) compared to WT EGFR. Further, acute EGF-induced ubiquitination of WT EGFR was markedly enhanced by PD98059 pretreatment and was increased in EGFR T669A-expressing cells independent of PD98059. These signaling data suggest that ERK-mediated (669)T phosphorylation negatively modulates EGF-induced EGFR kinase activity. We furthered these investigations using a human fibrosarcoma cell line that endogenously expresses EGFR and ErbB-2 and also harbors an activating Ras mutation. In these cells, EGFR was constitutively detected with the ERK consensus site phosphorylation-specific antibody and EGF-induced EGFR downregulation was modest, but was substantially enhanced by pretreatment with MEK1/ERK inhibitor. Collectively, these data indicate that ERK activity, by phosphorylation of a threonine residue in the EGFR juxtamembrane cytoplasmic domain, modulates EGFR trafficking and signaling.     

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.     

Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3-F442A cells. Modulation of EGF-induced trafficking and signaling

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.     

Mechanisms for growth factor-induced pituitary tumor transforming gene-1 expression in pituitary folliculostellate TtT/GF cells

PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.     

Suppression of α-catenin and adherens junctions enhances epithelial cell proliferation and motility via TACE-mediated TGF-α autocrine/paracrine signaling

Sustained cell migration is essential for wound healing and cancer metastasis. The epidermal growth factor receptor (EGFR) signaling cascade is known to drive cell migration and proliferation. While the signal transduction downstream of EGFR has been extensively investigated, our knowledge of the initiation and maintenance of EGFR signaling during cell migration remains limited. The metalloprotease TACE (tumor necrosis factor alpha converting enzyme) is responsible for producing active EGFR family ligands in the via ligand shedding. Sustained TACE activity may perpetuate EGFR signaling and reduce a cell’s reliance on exogenous growth factors. Using a cultured keratinocyte model system, we show that depletion of α-catenin perturbs adherens junctions, enhances cell proliferation and motility, and decreases dependence on exogenous growth factors. We show that the underlying mechanism for these observed phenotypical changes depends on enhanced autocrine/paracrine release of the EGFR ligand transforming growth factor alpha in a TACE-dependent manner. We demonstrate that proliferating keratinocyte epithelial cell clusters display waves of oscillatory extracellular signal–regulated kinase (ERK) activity, which can be eliminated by TACE knockout, suggesting that these waves of oscillatory ERK activity depend on autocrine/paracrine signals produced by TACE. These results provide new insights into the regulatory role of adherens junctions in initiating and maintaining autocrine/paracrine signaling with relevance to wound healing and cellular transformation.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2

G protein-coupled receptor kinases （GRKs） mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors （GPCRs）. We investigate the role of GRK2 on epidermal growth factor （EGF） receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase （ERK/MAPK） activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold （P〈0.05） at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% （P〈0.01）. Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.     

Role of Ras in metal-induced EGF receptor signaling and NF-kappaB activation in human airway epithelial cells

We showed previously that epithelial growth factor (EGF) receptor (EGFR) signaling is triggered by metallic compounds associated with ambient air particles. Specifically, we demonstrated that As, Zn, and V activated the EGFR tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this study, we examined the role of Ras in EGFR signaling and the nuclear factor-kappaB (NF-kappaB) activation pathway and the possible interaction between these two signaling pathways in a human airway epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal significantly increased Ras activity, and this effect was inhibited by the EGFR tyrosine kinase activity inhibitor PD-153035. Adenoviral-mediated overexpression of a dominant-negative mutant form of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced IkappaBalpha serine phosphorylation, IkappaBalpha breakdown, and NF-kappaB DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced IkappaBalpha breakdown and NF-kappaB activation, while inhibition of MEK activity with PD-98059 failed to do so. In summary, exposure to As, Zn, and V initiated EGFR signaling and Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced Ras-dependent NF-kappaB nuclear translocation. EGFR signaling appears to cross talk with NF-kappaB signaling at the level of Ras, but additional signals appear necessary for NF-kappaB activation. Together, these data suggest that, in V-treated BEAS-2B cells, Ras-dependent signaling is essential, but not sufficient, for activation of NF-kappaB.     

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.     

Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to approximately 1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of (125)I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.     

Influence of kinetics of drug binding on EGFR signaling: a comparative study of three EGFR signaling pathway models

We used three models of the epidermal growth factor receptor (EGFR) signaling pathway mimicking three different cell lines to study the effects of kinetics of drug binding on influencing molecular signaling in the pathways. With no incubation of drugs before the external cue epidermal growth factor (EGF) was applied, we found that fast kinetics of binding to protein kinases was advantageous in suppressing the production of the Extracellular signal-regulated kinase (ERK) that triggers cell proliferation, with some exceptions. Incubation of a drug with a protein kinase target for an hour before a pathway was initiated with an external cue made kinetics less significant, so did high concentration of drugs. In addition, we found that applying a drug to a protein kinase mostly affected downstream signaling although upstream events were also affected in a few cases. In examining whether applying two drugs to two protein kinase targets in the pathways could produce synergistic effects, we found positive, negative, or no effects, depending on the protein kinases targeted and the pathway model considered.     

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.     

Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation

How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.     

Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.     

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells

Overexpressionof the epidermal growth factor receptors (EGFR) in polarized kidneyepithelial cells caused them to appear in high numbers at both thebasolateral and apical cell surfaces. We utilized these cells to lookfor differences in the regulation and signaling of apical vs.basolateral EGFR. Apical and basolateral EGFR were biologically activeand mediated EGF-induced cell proliferation to similar degrees.Receptor downregulation and endocytosis were less efficient at theapical surface, resulting in prolonged EGF-induced tyrosine kinaseactivity at the apical cell membrane. Tyrosine phosphorylation of EGFRsubstrates known to mediate cell proliferation, Src-homologous andcollagen protein (SHC), extracellularly regulated kinase 1 (ERK1), andERK2 could be induced similarly by activation of apical or basolateralEGFR. Focal adhesion kinase was tyrosine phosphorylated more bybasolateral than by apical EGFR; however, -catenin was tyrosinephosphorylated to a much greater degree following the activation ofmislocalized apical EGFR. Thus EGFR regulation and EGFR-mediatedphosphorylation of certain substrates differ at the apical andbasolateral cell membrane domains. This suggests that EGFRmislocalization could result in abnormal signal transduction andaberrant cell behavior.

     

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.