TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.     

A new EGFR inhibitor induces apoptosis in colon cancer cells

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.     

Hydrogel-based protein array for quantifying epidermal growth factor receptor activity in cell lysates

Epidermal growth factor receptor (EGFR) signaling plays an important role in a majority of solid tumors, and therapeutics targeted against EGFR has demonstrated promise in slowing growth of these tumors. However, many of these drugs either have failed to reproduce promising preclinical model results in clinical settings or have been successful in only a subgroup of cancer patients due partly to incomplete assessment of EGFR status in cancer. A patient-customized, predictive diagnostic for the effects of specific anti-EGFR therapies may improve outcomes. Here we report the development of a hydrogel-based protein array for quantitative and reproducible determination of the activity of EGFR directly from cellular extracts. In this study, we used glutathione S-transferase-fused Eps15 (GST–Eps15) fusion proteins immobilized within a polyacrylamide hydrogel as a substrate for quantifying EGFR kinase activity from the extracts of EGFR-expressing cell lines. Significant EGFR up-regulation was detected in a mixture containing 7% EGFR-overexpressing cell lysate diluted in lysate from a cell line expressing low levels of EGFR. In addition, the GST–Eps15 protein array was capable of detecting inhibition of EGFR activity when incubated with different tyrosine kinase inhibitors. These findings establish the potential of this protein–acrylamide copolymer hydrogel array not only to evaluate EGFR status in cancer cell lysates but also to screen for the most promising therapeutics for individual patients and monitor treatment progression.     

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.     

A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.     

Cloning and Expression of EGFR Tyrosine Kinase and Construction of the Screening Model for the Enzymatic Inhibitors

表皮生长因子受体(Epidermal growth factor receptor,EGFR)属受体酪氨酸激酶(Tyrosine kinase,TK)家族,其胞内的酪氨酸激酶在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。本研究从人脐静脉内皮细胞(HUVEC) 提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶催化域的编码基因。将其克隆至载体pET-30a,在E.coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化。通过对酶的活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,拟对微生物代谢产物进行筛选。     

Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.

Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.     

EGF receptor clustering is induced by a 0.4 mT power frequency magnetic field and blocked by the EGF receptor tyrosine kinase inhibitor PD153035

Atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy were used to investigate the effects of a 50 Hz 0.4 mT magnetic field (MF) on the clustering of purified epidermal growth factor receptors (EGFRs) and EGFRs in Chinese hamster lung (CHL) cell membrane. The results demonstrate that exposing purified EGFRs to the MF for 30 min induces receptor clustering. The peak height of apparent clusters increased from 1.42 +/- 0.18 (sham-exposed) to 3.08 +/- 0.38 nm (exposed) while the mean half-width increased from 21.7 +/- 2.2 to 33.0 +/- 4.0 nm. A similar effect was also observed by TEM. Treatment of purified EGFR with PD153035 (PD), an EGFR-specific tyrosine kinase (TK) inhibitor, inhibited the MF-induced EGFR clustering of the purified proteins, an effect also observed for the receptors in cell membrane in the absence of EGF. These results strongly suggest that the 50 Hz 0.4 mT MF interferes with the EGFR signaling pathway, most likely by interacting with the cytoplasmic TK domain.     

Epidermal growth factor induces oxidative neuronal injury in cortical culture

Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.     

Comparison of Elisa-Based Tyrosine Kinase Assays for Screening EGFR Inhibitors

Abstract

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu, Tyr)_1:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

Ligand-independent phosphorylation of Y869 (Y845) links mutant EGFR signaling to stat-mediated gene expression

Activating mutants of EGFR have been identified in a subset of non-small-cell lung cancers. To investigate mutant-driven signaling, we focused on Y869, a residue in the same activation loop where the L858R and L861Q mutations are located. We observed ligand-independent phosphorylation of Y869 in 32D cells EGFR(L858R) and EGFR(L861Q). The EGFR tyrosine kinase inhibitor (TKI) erlotinib inhibited Y869 P-EGFR in intact cells as well as in a cell-free kinase reaction. Expression of kinase domain of EGFR(L858R) and EGFR(L861Q) exhibited auto-phosphorylation of Y869; this was inhibited by EGFR TKIs but not by Src kinase inhibitor. P-Y859 of EGFR-mediated downstream component, STAT5, was also analyzed. Y694 P-STAT5 was eliminated by erlotinib treatment. Analysis of immune-complexes showed constitutive association of mutant EGFRs with STAT5 and Src which was unaffected by erlotinib or PP1. On the other hand, 32D-EGFR(WT) exhibited constitutive STAT5 phosphorylation and association of EGFR with JAK2. In these cells, a JAK2 inhibitor abrogated P-STAT5 whereas mutant EGFRs did not associate with JAK2. Expression of c-myc was regulated by EGFR/STAT5 signaling in cells expressing EGFR(L858R) and EGFR(L861Q). Our results suggest that ligand-independent and Src activity-independent phosphorylation of Y869 in mutant EGFR regulates STAT5 activation and c-myc expression.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G₁ arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G₁ arrest. This G₁ arrest was associated with up-regulation of p27^kip1, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G₁ arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.     

AZD9291 promotes autophagy and inhibits PI3K/Akt pathway in NSCLC cancer cells

AZD9291, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is highly selective against EGFR T790M-mutant non–small cell lung cancer (NSCLC). On investigating the growth inhibitory effects of AZD9291 on NSCLC and the underlying mechanism, we found that AZD9291 can trigger autophagy-mediated cell death in both A549 and H1975 cells by increasing the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3) and decreasing the expression of p62. In the presence of the autophagy inhibitor chloroquine, the AZD9291-induced increase in LC3 level was further augmented. AZD9291 decreased the levels of phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt. AZD9291-induced cell death was enhanced by Akt knockdown, and the levels of both EGFR and phosphorylated EGFR were decreased by AZD9291. AZD9291 was also found to significantly suppress the tumor growth in H1975 xenograft nude mice. Thus, AZD9291 was found to induce autophagy, decrease in EGFR levels, and show a strong inhibitory effect on NSCLC both in vitro and in vivo. Furthermore, the PI3K/Akt signaling pathway was found to play a critical role in AZD9291-induced cell death.     

Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post‐application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr¹⁰⁴⁵ and Tyr¹¹⁷³, we demonstrated that phosphorylation of Tyr¹¹⁷³ involved in stimulation of the MAPK (mitogen‐activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c‐Cbl and is necessary for targeting the receptor for lysosomal degradation. c‐Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination‐dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.     

Involvement of de novo synthesized palmitate and mitochondrial EGFR in EGF induced mitochondrial fusion of cancer cells

Increased expressions of fatty acid synthase (FASN) and epidermal growth factor receptor (EGFR) are common in cancer cells. De novo synthesis of palmitate by FASN is critical for the survival of cancer cells via mechanisms independent of its role as an energy substrate. Besides the plasma membrane and the nucleus, EGFR can also localize at the mitochondria; however, signals that can activate mitochondrial EGFR (mtEGFR) and the functions of mtEGFR of cancer cells remain unknown. The present study characterizes mtEGFR in the mitochondria of cancer cells (prostate and breast) and reveals that mtEGFR can promote mitochondrial fusion through increasing the protein levels of fusion proteins PHB2 and OPA1. Activation of plasma membranous EGFR (pmEGFR) stimulates the de novo synthesis of palmitate through activation of FASN and ATP-citrate lyase (ACLy). In vitro kinase assay with isolated mitochondria shows that palmitate can activate mtEGFR. Inhibition of FASN blocks the mtEGFR phosphorylation and palmitoylation induced by EGF. Mutational studies show that the cysteine 797 is important for mtEGFR activation and palmitoylation. Inhibition of FASN can block EGF induced mitochondrial fusion and increased the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. In conclusion, these results suggest that mtEGFR can be activated by pmEGFR through de novo synthesized palmitate to promote mitochondrial fusion and survival of cancer cells. This mechanism may serve as a novel target to improve EGFR-based cancer therapy.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.