Identification of epidermal growth factor receptor- Grb2-associated binder-1-SHP-2 complex formation and its functional loss during neoplastic cell progression.

The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.     

Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10- to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.     

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen-activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)-induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen-activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time- and dose-dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP-GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5-altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho-extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5-induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth.     

A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2.

The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regions are responsible for this specificity. Here we describe a genetically engineered EGFR mutant containing a threonine for arginine substitution at position 662 in the EGFR juxtamembrane domain, corresponding to threonine 694 in gp185erbB-2. This mutant, designated EGFRThr662, displayed affinity for EGF binding and catalytic properties that were indistinguishable from those of the wild type EGFR. However, EGFRThr662 behaved much as gp185erbB-2 in a number of bioassays which readily distinguish between the mitogenic effects of EGFR and gp185erbB-2. Moreover, significant differences were detected in the pattern of intracellular proteins phosphorylated on tyrosine in vivo by EGFR and EGFRThr662 in response to EGF. Thus, small differences in the primary sequence of two closely related receptors have dramatic effects on their ability to couple with mitogenic pathways.     

Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor

Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.     

Single-molecule imaging of EGFR signalling on the surface of living cells

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.     

Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction     

Tissue inhibitor of metalloproteinases-2 (TIMP-2) suppresses TKR-growth factor signaling independent of metalloproteinase inhibition

The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. Here we demonstrate a direct role for TIMP-2 in regulating tyrosine kinase-type growth factor receptor activation. We show that TIMP-2 suppresses the mitogenic response to tyrosine kinase-type receptor growth factors in a fashion that is independent of MMP inhibition. The TIMP-2 suppression of mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and implicates cAMP as the second messenger in these effects. TIMP-2 neither altered the release of transforming growth factor alpha from the cell surface, nor epidermal growth factor (EGF) binding to the cognate receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (K(d) = 147 pm), that is not competed by the synthetic MMP inhibitor BB-94 and is independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation of EGFR and a concomitant reduction in Grb-2 association. TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation from immunoprecipitable EGFR complex and a selective increase in total SHP-1 activity. These studies represent a new functional paradigm for TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by short-circuiting EGFR activation.     

EGFR S1166 Phosphorylation Induced by a Combination of EGF and Gefitinib Has a Potentially Negative Impact on Lung Cancer Cell Growth

Phosphorylation of protein plays a key role in the regulation of cellular signal transduction and gene expression. In recent years, targeted mass spectrometry facilitates functional phosphoproteomics by allowing specific protein modifications of target proteins in complex samples to be characterized. In this study, we employed multiple reaction monitoring (MRM) to examine the influence of gefitinib (also known as Iressa) on the phosphorylation sites of EGFR protein before and after EGF treatment. By coupling MRM to MS/MS, 5 phosphotyrosine (Y1110, Y1172, Y1197, Y1069, and Y1092) and 1 S/T (T693) sites were identified on EGFR. Y1197 and T693 were constitutively phosphorylated. All phosphorylation sites were sensitive to gefitinib treatment except T693. Interestingly, gefitinib treatment induced phosphorylation of S1166 only in the presence of EGF. We further showed that lung cancer cells overexpressing phosphomimic S1166D EGFR mutant possessed significantly lower growth and proliferation property compared to wildtype EGFR-expressing cells. While the function and mode of regulation of S1166 remain unclear, our data supports the notion that S1166 represents a regulatory site that exerts a negative regulation on growth and proliferation of cancer cells. The data presented has implication in our understanding of dynamic drug (gefitinib)-target (EGFR) interaction and in improving the efficacy of target-directed therapeutics.     

Cytoskeletal association of epidermal growth factor receptor and associated signaling proteins is regulated by cell density in IEC-6 intestinal cells

Epidermal growth factor (EGF) mediates a variety of physiologic responses in rat intestine. EGF receptor (EGFR) responsiveness to EGF is mediated by the surface expression of high affinity EGFR, which is associated with the cytoskeleton (CSK). EGFR signal transduction appears to be mediated by the CSK association of EGFR and related signaling proteins. In the nontransformed intestinal cell line IEC-6, expression of EGFR, Src homology and collagen protein (SHC), phospholipase Cγ1 (PLCγ), and their tyrosine phosphorylation in response to EGF was assayed by immunoblot. The distribution of EGFR and tyrosine-phosphorylated EGFR was regulated by cell density. At confluence, EGFR and tyrosine-phosphorylated EGFR were predominantly associated with the Triton X-100-insoluble CSK at confluence, while predominantly Triton X-100-soluble at subconfluence. PLCγ was predominantly soluble at both states of confluence. Confluent but not subconfluent IEC-6 cells demonstrated a cascade of EGF-mediated events consisting of a transient CSK association of PLCγ with EGFR, a brief expression of tyrosine-phosphorylated PLCγ, a brief increase in PLCγ CSK association, and a prolonged soluble association of PLCγ with the EGFR. EGF led to an increase in the CSK association of SHC at both states of confluence and was greater at confluence. EGFR association with SHC was primarily soluble at subconfluence, while at confluence EGFR association was markedly increased and predominantly in the CSK. Thus, cell density regulates the CSK association of the EGFR and its ability to associate and activate signaling pathways in intestinal cells. J. Cell. Physiol. 172:126–136, 1997. © 1997 Wiley-Liss, Inc.     

Atypical receptor-mediated signal transduction events in the EGF-dependent growth-inhibited cell line, MDA-468.

It is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA-468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA-468 cells, both the EGFR and PLC-gamma are phosphorylated on tyrosine residues and can be co-immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF-treated MDA-468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology.     

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.     

Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response

Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.     

MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Single-chain antibody-mediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling.

ErbB-2 becomes rapidly phosphorylated and activated following treatment of many cell lines with epidermal growth factor (EGF) or Neu differentiation factor (NDF). However, these factors do not directly bind ErbB-2, and its activation is likely to be mediated via transmodulation by other members of the type I/EGF receptor (EGFR)-related family of receptor tyrosine kinases. The precise role of ErbB-2 in the transduction of the signals elicited by EGF and NDF is unclear. We have used a novel approach to study the role of ErbB-2 in signaling through this family of receptors. An ErbB-2-specific single-chain antibody, designed to prevent transit through the endoplasmic reticulum and cell surface localization of ErbB-2, has been expressed in T47D mammary carcinoma cells, which express all four known members of the EGFR family. We show that cell surface expression of ErbB-2 was selectively suppressed in these cells and that the activation of the mitogen-activated protein kinase pathway and p70/p85S6K, induction of c-fos expression, and stimulation of growth by NDF were dramatically impaired. Activation of mitogen-activated protein kinase and p70/p85S6K and induction of c-fos expression by EGF were also significantly reduced. We conclude that in T47D cells, ErbB-2 is a major NDF signal transducer and a potentiator of the EGF signal. Thus, our observations demonstrate that ErbB-2 plays a central role in the type I/EGFR-related family of receptors and that receptor transmodulation represents a crucial step in growth factor signaling.     

DNA microarray analysis of immediate response to EGF treatment in rat schwannoma cells

Epidermal growth factor (EGF) activates many intracellular effector molecules, which subsequently influence the expression levels of many genes involved in cell growth, apoptosis and signal transduction,etc. In this study, the early response of gene expressions due to EGF treatment was monitored using oligonucleotide DNA microarrays in rat schwannoma cell lines. An immunoblotting experiment showed the successful activation of EGF receptors and an effector protein, STATS, due to EGF treatment. The microarray study showed that 35 genes were significantly induced and 2 were repressed within 60 min after the treatment. The list of induced genes included early growth response 1, suppressor of cytokine signaling 3, c-fos, interferon regulatory factor 1 and early growth response 2,etc. According to the microarray data, six of these were induced by more than 10-fold, and showed at least two different induction patterns, indicating complicated regulatory mechanisms in the EGF signal transduction.