Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.     

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.     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.     

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.     

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb.E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2-/- cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.     

Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2(209-223) peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS-PAGE and LC/MS identified 14-3-3epsilon as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)(TG2+/+) cells but not from MEF(TG2-/-) cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.     

YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR

Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it sti mulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist. [BMB Reports 2014; 47(10): 581-586]     

Gefitinib-sensitive EGFR lacking residues 746-750 exhibits hypophosphorylation at tyrosine residue 1045, hypoubiquitination, and impaired endocytosis

Gefitinib-sensitive nonsmall cell lung cancers (NSCLC) are characterized by somatic mutations in the kinase domain of epidermal growth factor receptor (EGFR). The mutant EGFR forms are reported to mediate characteristic signal transduction pathways that are different from those mediated by the wild-type EGFR and are involved in transformation in vivo. We have examined signal transduction pathways initiated from a frequently identified gefitinib-sensitizing mutant EGFR lacking residues 746-750 by employing a mouse fibroblast cell line that is free of endogenous EGFR and transiently transfected COS-7 cells. Upon EGF stimulation, the deletion-mutant EGFR mediated prolonged downstream signals. The analysis of the phosphotyrosine patterns of the receptor revealed that the deletion-mutant EGFR lacked phosphorylation at tyrosine residue 1045, which is the major binding site of Cbl. The EGF-induced endocytosis of the deletion-mutant EGFR was impaired. The ubiquitination and downregulation of the deletion-mutant EGFR were also reduced. On the other hand, another mutant, EGFR, possessing a L858R substitution, exhibited phosphorylation at 1045 and its downstream signalings were not prolonged. These data suggest that the signal transduction pathways initiated from these mutant forms are different, and that impaired endocytosis might be responsible for the prolonged signals mediated by the deletion-mutant EGFR.     

Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.     

S-Nitrosylation of the epidermal growth factor receptor: A regulatory mechanism of receptor tyrosine kinase activity

Nitric oxide (NO) donors inhibit the epidermal growth factor (EGF)-dependent auto(trans)phosphorylation of the EGF receptor (EGFR) in several cell types in which NO exerts antiproliferative effects. We demonstrate in this report that NO inhibits, whereas NO synthase inhibition potentiates, the EGFR tyrosine kinase activity in NO-producing cells, indicating that physiological concentrations of NO were able to regulate the receptor activity. Depletion of intracellular glutathione enhanced the inhibitory effect of the NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) on EGFR tyrosine kinase activity, supporting the notion that such inhibition was a consequence of an S-nitrosylation reaction. Addition of DEA/NO to cell lysates resulted in the S-nitrosylation of a large number of proteins including the EGFR, as confirmed by the chemical detection of nitrosothiol groups in the immunoprecipitated receptor. We prepared a set of seven EGFR(C → S) substitution mutants and demonstrated in transfected cells that the tyrosine kinase activity of the EGFR(C166S) mutant was completely resistant to NO, whereas the EGFR(C305S) mutant was partially resistant. In the presence of EGF, DEA/NO significantly inhibited Akt phosphorylation in cells transfected with wild-type EGFR, but not in those transfected with C166S or C305S mutants. We conclude that the EGFR can be posttranslationally regulated by reversible S-nitrosylation of C166 and C305 in living cells.     

Negative regulation of MAPKK by phosphorylation of a conserved serine residue equivalent to Ser212 of MEK1

The MAPKKs MEK1 and MEK2 are activated by phosphorylation, but little is known about how these enzymes are inactivated. Here, we show that MEK1 is phosphorylated in vivo at Ser(212), a residue conserved among all MAPKK family members. Mutation of Ser(212) to alanine enhanced the basal activity of MEK1, whereas the phosphomimetic aspartate mutation completely suppressed the activation of both wild-type MEK1 and the constitutively activated MEK1(S218D/S222D) mutant. Phosphorylation of Ser(212) did not interfere with activating phosphorylation of MEK1 at Ser(218)/Ser(222) or with binding to ERK2 substrate. Importantly, mimicking phosphorylation of the equivalent Ser(212) residue of the yeast MAPKKs Pbs2p and Ste7p similarly abrogated their biological function. Our findings suggest that Ser(212) phosphorylation represents an evolutionarily conserved mechanism involved in the negative regulation of MAPKKs.     

Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.     

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.     

Transglutaminase down-regulates the dimerization of epidermal growth factor receptor in rat perivenous and periportal hepatocytes

Objective:  Recently, we found that transglutaminase 2 (TG2) might be involved in the difference in proliferative capacities between periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) through down-regulation of high-affinity epidermal growth factor receptor (EGFR). However, it is uncertain whether this high-affinity EGFR contributes to the hepatocyte growth signalling pathway. Here, we have investigated the influence of TG2 on EGF-induced EGFR dimerization and its phosphorylation, which are important steps in the hepatocyte proliferative/growth signalling pathway, in PPH and PVH.
Materials and methods:  PPH and PVH were isolated using the digitonin/collagenase perfusion technique. Amounts of TG2, EGFR dimerization and its phosphorylation were determined by Western blot analysis.
Results:  Pretreatment with monodansylcadaverine, an inhibitor of TG2, greatly increased EGF-induced EGFR dimerization and its phosphorylation in PVH compared with PPH. Conversely, treatment with retinoic acid, an inducer of TG2, significantly decreased EGF-induced EGFR dimerization and its phosphorylation with a significant increase in TG2 expression and its catalysed products, isopeptide bonds, in both subpopulations. It was found that EGFR served as a substrate for TG2.
Conclusion:  The present data showed good correlation with our previous data on EGF-induced DNA synthesis and EGFR-binding affinity to EGF. These results suggest that zonal difference in cell growth between PPH and PVH may be caused by down-regulation of EGFR dimerization and subsequent autophosphorylation through TG2-mediated cross-linking of EGFR.     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.     

Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding.

The extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) comprises four subdomains (I-IV) and mediates binding of several different polypeptide ligands, including EGF, transforming growth factor-alpha, and heparin-binding EGF. Previous studies have predominantly implicated subdomain III in ligand binding. To investigate a possible role for sequences in subdomain IV, we constructed several mutant EGFRs in which clusters of charged or aromatic amino acids were replaced with alanine. Analysis of stably transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of the wild-type receptor. Although tyrosine phosphorylation of most mutants was markedly induced by EGF, a cluster mutation (mt25) containing four alanine substitutions in the span of residues 521-527 failed to respond. EGF-induced tyrosine phosphorylation of an alternative mutant (DeltaEN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whereas the response to transforming growth factor-alpha was undetectable. These results suggest that mt25 might be defective with respect to either ligand binding or receptor dimerization. Quantitative analyses showed that binding of (125)I-EGF to mt25 and DeltaEN was reduced to near background levels, whereas binding of EGF to other cluster mutants was reduced 60-70% compared with wild-type levels. Among the mutants, only mt25 and DeltaEN failed to form homodimers or to transphosphorylate HER2/Neu in response to EGF treatment. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF family ligands. Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequently used for structure/function studies of the EGFR extracellular region.     

EGFR-independent activation of ERK1/2 mediates growth inhibition by a PTPase antagonizing K-vitamin analog

The K-vitamin analog Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone] is a potent cell growth inhibitor in vitro and in vivo, likely due to arylation of enzymes containing a catalytic cysteine. This results in inhibition of protein tyrosine phosphatase (PTPase) activity with resultant hyperphosphorylation of EGF receptors (EGFR) and ERK1/2 protein kinases, which are downstream to EGFR in the MAPK pathway. We used NR6 fibroblast cells, which lack endogenous EGFR and its variant cells transfected with different EGFR mutants to assess the contribution of the EGFR-mediated signaling pathway to Cpd 5-mediated ERK activation and cell growth inhibition. Cpd 5 treatment resulted in enhanced phosphorylation of EGFR at carboxyl-terminal tyrosines. This phosphorylation and activation of EGFR were found to be necessary neither for growth inhibition nor for the activation of the downstream kinases ERK1/2, since both occurred in EGFR-devoid mutant cells. U0126 and PD 098059, specific inhibitors of MEK1/2, the ERK1/2 kinases, antagonized both cell growth inhibition and ERK1/2 phosphorylation mediated by Cpd5. Cpd 5 was also found to inhibit ERK1/2 phosphatase(s) activity in lysates from all the cells tested, irrespective of their EGFR status. These results show that EGFR-independent ERK1/2 phosphorylation was involved in the mechanism of Cpd5 mediated growth inhibition. This is likely due to the observed antagonism of ERK phosphatase activity. A candidate PTPase was found to be Cdc25A, a recently identified ERK phosphatase.     

UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.