Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.     

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.     

SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.     

Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.     

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.     

EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation

The epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase (RTK) superfamily and is involved in regulating cell proliferation, differentiation and motility. Growth factor binding induces receptor oligomerization at the plasma membrane, which leads to activation of the intrinsic RTK activity and trans-phosphorylation of tyrosine residues in the intracellular part of the receptor. These residues are docking sites for proteins containing Src homology domain 2 and phosphotyrosine-binding domains that relay the signal inside the cell. In response to EGF attached to beads, lateral propagation of EGFR phosphorylation occurs at the plasma membrane, representing an early amplification step in EGFR signalling. Here we have investigated an underlying reaction network that couples RTK activity to protein tyrosine phosphatase (PTP) inhibition by reactive oxygen species. Mathematical analysis of the chemical kinetic equations of the minimal reaction network detects general properties of this system that can be observed experimentally by imaging EGFR phosphorylation in cells. The existence of a bistable state in this reaction network explains a threshold response and how a high proportion of phosphorylated receptors can be maintained in plasma membrane regions that are not exposed to ligand.     

Prostate cancer: a model of integration of genomic and non-genomic effects of the androgen receptor in cell lines model

Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.     

Calcium ion-mediated regulation of the alpha-toxin pore of Staphylococcus aureus.

The water-soluble alpha-toxin monomers of Staphylococcus aureus become hexamers forming the transmembrane pore when exposed to the membranes. This pore is freely permeable to small hydrophilic molecules, e.g. carboxyfluorescein, and becomes less permeable in the presence of calcium ions. Calcium ion-mediated decrease of the carboxyfluorescein leakage could not be eliminated by EDTA added in the medium, but the carboxyfluorescein could be freed by EDTA added in the intraliposomal space. This result suggests that the alpha-toxin pore changes its conformation as the calcium ion is bound and that the binding site is exposed to the intraliposomal side of the membrane. The interaction between the alpha-toxin hexamer and 8-anilino-1-naphthalene-sulfonic acid (ANS) was monitored by determining the fluorescence in the presence and absence of calcium chloride. The mean distances between the tryptophan residues of the alpha-toxin hexamer and the bound ANS were calculated to be 1.90 and 1.80 nm in the absence and presence, respectively, of calcium ions. The results showed the calcium ion mediated conformational change of the membrane-embedded alpha-toxin hexamer.     

The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines

The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF–EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF–EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. Iressa is a trademark of the AstraZeneca group of companies.     

POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope

We have identified a concanavalin A-reactive glycoprotein of 150 kD that coenriches with isolated yeast nuclear pore complexes. Molecular cloning and sequencing of this protein revealed a single canonical transmembrane segment. Epitope tagging and localization by both immunofluorescence and immunoelectron microscopy confirmed that it is a pore membrane protein. The protein was termed POM152 (for pore membrane protein of 152 kD) on the basis of its location and cDNA-deduced molecular mass. POM152 is likely to be a type II membrane protein with its NH2-terminal region (175 residues) and its COOH-terminal region (1,142 residues) positioned on the pore side and cisternal side of the pore membrane, respectively. The proposed cisternally exposed domain contains eight repetitive motifs of approximately 24 residues. Surprisingly, POM152 deletion mutants were viable and their growth rate was indistinguishable from that of wild-type cells at temperatures between 17 and 37 degrees C. However, overproduction of POM152 inhibited cell growth. When expressed in mouse 3T3 cells, POM152 was found to be localized to the pore membrane, suggesting a conserved sorting pathway between yeast and mammals.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.     

Elevated prostaglandin EP2 receptor in endometrial adenocarcinoma cells promotes vascular endothelial growth factor expression via cyclic 3',5'-adenosine monophosphate-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 signaling pathways

Prostaglandin (PG) E(2) E-series prostanoid-2 (EP2) receptor is elevated in numerous carcinomas including the endometrium and has been implicated in mediating the effects of PGE(2) on vascular function. In this study, we investigated the intracellular signaling pathways that are activated by the EP2 receptor and their role in regulation of the expression of vascular endothelial growth factor in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with EP2 receptor cDNA in the sense or antisense directions. Treatment of Ishikawa cells with PGE(2) rapidly induced transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK1/2 via the EP2 receptor. Preincubation of cells with chemical inhibitors of protein kinase A, c-Src, and EGFR kinase abolished the EP2-induced activation of EGFR and ERK1/2. PGE(2) signaling via the EP2 receptor also promoted the mRNA expression and secretion of vascular endothelial growth factor protein in Ishikawa cells. This effect was inhibited by preincubation with chemical inhibitors of EGFR kinase, ERK1/2 signaling, and small inhibitory RNA molecules targeted against the EGFR. Therefore, we have demonstrated that elevated EP2 receptor expression may facilitate the PGE(2)-induced release of proangiogenic factors in reproductive tumor cells via intracellular cAMP-mediated transactivation of the EGFR and ERK1/2 pathways.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.     

Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization

Gangliosides are shed by tumor cells and can bind to normal cells in the tumor microenvironment and affect their function. Exposure of fibroblasts to exogenous gangliosides increases epidermal growth factor (EGF)-induced fibroblast proliferation and enhances EGF receptor (EGFR)-mediated activation of the mitogen-activated protein kinase signaling pathway (Li, R., Liu, Y., and Ladisch, S. (2001) J. Biol. Chem. 276, 42782-42792). Here we report that the EGFR itself is the target of this ganglioside effect: Preincubation of normal human dermal fibroblasts with G(D1a) ganglioside enhanced both EGF-induced EGFR autophosphorylation and receptor-tyrosine kinase activity. The enhancement was rapid (within 30 min), not due to alteration of time kinetics of the EGFR response to EGF, and reproduced in purified G(D1a)-enriched cell membranes isolated from ganglioside-preincubated fibroblasts. Evaluating the initial steps underlying activation, EGF binding, and EGFR dimerization, we found that G(D1a) enrichment of the cell membrane increased EGFR dimerization and the effective number of high affinity EGFR without increasing total receptor protein. Unexpectedly, G(D1a) enrichment also triggered increased EGFR dimerization in the absence of growth factor. This resulted in enhanced activation of the EGFR signal transduction cascade when EGF was added. We conclude that membrane ganglioside enrichment of normal fibroblasts (such as by tumor cell ganglioside shedding) facilitates receptor-receptor interactions (possibly by altering membrane topology), causing ligand-independent EGFR dimerization and, in turn, enhanced EGF signaling.