Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src@
© Chung et al; licensee BioMed Central Ltd. 2009
Received: 6 July 2009
Accepted: 30 November 2009
Published: 30 November 2009
Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear.
This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association.
The findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.
Epidermal growth factor receptor (EGFR) is a prototype of receptor tyrosine kinases (RTKs) which control critical cellular responses to extra-cellular growth factors during development and tissue homeostasis [1, 2]. Importantly, overexpression of EGFR and/or its ligands is frequently observed in human cancers, and recent studies have identified activating mutations in EGFR as direct determinants of oncogenic transformation in human cancers . For example, missense mutations or small in-frame deletions within the kinase domain, which render EGFR constitutively active, are observed in a subset of patients with non-small cell lung cancer (NSCLC) [4–6]. As mutational activation of EGFR imparts a higher sensitivity to inhibition by EGFR-selective tyrosine kinase inhibitors (TKIs), there is considerable interest in understanding biological mechanisms whereby mutant EGFRs mediate aberrant oncogenic signaling in cancer cells.
While the normal EGFR signaling cascade is initiated by ligand-dependent dimerization and subsequent trans-phosphorylation of tyrosine residues within the cytoplasmic tail of the receptor, constitutively active mutant EGFRs associated with human cancer are thought to engage downstream signaling pathways in a constitutive fashion. Indeed, biochemical analyses have demonstrated that NSCLC-associated EGFR mutants activate signaling through the Erk, Akt, Src and STAT pathways [4, 7, 8]. A notable finding from these studies has been that certain signaling pathways may be preferentially altered by mutationally-activated EGFRs. For example, phosphoinositide 3-kinase pathway activation by mutant EGFR was found to be highly sensitive to gefitinib, an EGFR tyrosine kinase inhibitor [4, 8]. Other studies have indicated a relatively selective activation of Src downstream of mutant EGFRs [7–9].
In the context of Src, use of Src inhibitors [9, 10] and mutation of Src-dependent phosphorylation sites within EGFR (Y845) [7, 11] have demonstrated a critical role for Src activity in linking mutant EGFRs to activation of several signaling pathways, to cell survival and to mutant EGFR-mediated oncogenic transformation. However, the reasons why certain signaling pathways, such as Src activity-dependent events, might be particularly activated by oncogenic EGFR mutants have not been addressed.
A crucial determinant of events downstream of RTKs such as EGFR is their endocytic traffic . Ligand-dependent internalization of EGFR with subsequent sorting of the internalized receptors for lysosomal degradation has emerged as a major mechanism for termination of signaling. While EGFR endocytosis is a pre-requisite for lysosomal targeting, the latter is not an invariant fate. It has become clear that endocytosed receptors undergo a sorting process whereby internalized receptors can either proceed to the lysosome through a series of vesicular fusion/maturation events or can be recycled back to the plasma membrane .
Recent studies have demonstrated that activation-dependent recruitment of the Cbl family of ubiquitin ligases is a major determinant of lysosomal targeting of EGFR [14, 15]. Cbl-dependent mono-ubiquitinylation of the cytoplasmic tail of EGFR serves as a signal for receptor sorting to the inner vesicles of the multi-vesicular bodies, a key step in lysosomal targeting of RTKs . Indeed, perturbation of Cbl protein expression or function alters the lysosomal degradation of EGFR and impacts the magnitude and/or duration of downstream signals [15, 17]. Additional mechanisms that function either in concert with Cbl-dependent ubiquitin modification, such as sprouty2, Sts-1/Sts-2 and cortactin [18–20], or independently (e.g. Sorting nexins)  further contribute to EGFR downregulation through lysosomal targeting.
In contrast to EGF-induced lysosomal targeting of EGFR, TGFα binding appears to promote the recycling of EGFR rather than its lysosomal degradation, correlating with a more potent signaling response [22–24]. Notably, TGFα stimulation is associated with a more transient EGFR-Cbl association and EGFR ubiquitinylation . EGFR heterodimerization with ErbB2, as is often observed in tumor cells, has also been shown to impair lysosomal degradation of EGFR apparently due to increased recycling and/or reduced internalization [25–27].
Given the importance of endocytic trafficking in dictating the lifespan of active EGFR and possibly the quality of downstream signaling events, it is of considerable interest to explore how oncogenic EGFRs traffic. In addition, the ability of mutant EGFRs to hyper-activate certain signaling pathways may be related to altered endocytic trafficking. Consistent with such a possibility, NSCLC-associated EGFR mutants appear to be impaired in their interaction with Cbl [28, 29]. More recent studies suggest that specific endocytic routes may dictate the type of biological responses to EGFR stimulation. For example, clathrin-dependent endocytosis appears to be critical for proliferative responses to EGF, whereas clathrin-independent endocytosis appears to primarily promote EGFR degradation . Furthermore, NSCLC-associated EGFR mutants have been shown to undergo EGF-independent internalization when expressed in a murine pro-B cell line . Intracellularly distributed EGFR was also observed in NSCLC cell lines . Here, we examined the subcellular localization of wild-type (wt) EGFR and oncogenic EGFR mutants in normal bronchial epithelial cells and NSCLC cell lines. Findings reported here demonstrate that mutant EGFRs undergo enhanced endocytic recycling and suggests a role of altered endocytic trafficking in mutant EGFR interaction with Src.
NSCLC-associated oncogenic EGFR mutants are constitutively endocytosed
Incubation of cells at 16°C allows for continued internalization from the cell surface but blocks further progression of endocytosed receptors and cargo along the endocytic pathway and into the endocytic recycling compartments, resulting in enhanced accumulation in sorting endosomes . Indeed, when NSCLC cell lines were incubated at 16°C, mutant EGFRs showed enhanced colocalization with labeled transferrin (Additional File 2). This result further suggests that mutant EGFRs transit along a transferrin-positive sorting compartment.
Treatment with monensin results in the accumulation of mutant EGFR, but not wtEGFR, in a perinuclear endocytic compartment
While our colocalization analyses demonstrated that constitutively endocytosed mutant EGFRs do transit to the lysosome, recent reports indicate that mutant EGFRs show reduced ligand-induced ubiquitinylation and degradation [28, 37] which could promote their entry into the endocytic recycling pathway; colocalization of mutant EGFRs with transferrin (Figure 2) is consistent with this idea. To further test this possibility, we examined the localization of mutant EGFRs after treating cells with monensin, an agent that has been shown to inhibit exit of internalized receptors and other endocytic cargo from sorting endosomes and the endocytic recycling compartment [34, 38, 39]. To demonstrate the ability of monensin to inhibit the cargo exit from the endocytic recycling compartment, we first assessed its effects on transferrin recycling in the NSCLC cell line H1666. As expected, labeled transferrin exit out of the perinuclear endocytic recycling compartment was essentially complete within the 60 min chase period; however, monensin treatment markedly delayed this process (Additional File 3A).
NSCLC-associated mutant EGFRs have been shown to attain sensitivity to Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) which targets the related receptor ErbB2 to degradation by enhancing its lysosomal targeting [37, 40, 41]. Notably, the presence of monensin prevented the lysosomal targeting of mutant EGFR and its degradation induced by 17-AAG (Additional File 4). 17-AAG treatment resulted in a decrease in mutant EGFR staining, indicating that mutant EGFR was targeted for degradation in the lysosomes. The 17-AAG-induced mutant EGFR downregulation was inhibited in monensin-treated cells and intracellular punctate staining of EGFR could still be observed. This is consistent with the concurrent effect of monensin to block traffic towards the lysosome .
To rule out the possibility that the perinuclear accumulation of mutant EGFRs may reflect an overall increase in the level of EGFR, we compared the EGFR expression levels in cells treated with DMSO versus monensin (Additional File 5A). Neither the overall EGFR levels nor the overall level of EGFR phosphorylation, as determined using anti-phosphotyrosine (PY) and anti-phospho-EGFR antibodies specific to pY845 and pY1173, showed a gross change upon monensin treatment (Additional File 5A).
Mutant EGFR colocalizes with markers of endocytic recycling compartment
Src association with mutant EGF receptors in the endocytic recycling compartment
Monensin treatment enhances the mutant EGFR-Src association
The outcome of RTK signaling involves a balance between various stimulatory and inhibitory mechanisms which in turn determine both the strength and duration of signals that are transmitted through networks of signaling cascades . In this respect, endosomal sorting plays a key role in the regulation of EGFR signaling .
NSCLC-associated kinase domain mutations in EGFR promote its constitutive activation, and a number of studies have focused on delineating the signaling pathways whose activation contributes to oncogenesis . The outcome of EGFR signaling is intimately linked to its endocytic traffic, which is normally triggered by ligand-induced dimerization  and phosphorylation-dependent as well as phosphorylation-independent recruitment of endocytic machinery components [51, 52]. The nature of endocytic trafficking of NSCLC-associated EGFR mutants and any relationship of altered traffic with oncogenic signaling remain poorly understood. Here, we have used NSCLC cell lines to demonstrate that oncogenic mutant EGFRs, but not wtEGFR, are constitutively endocytosed (Figure 1). Mutant EGFRs were found to localize in early and recycling endosomes based on colocalization with labeled transferrin (Figure 2) and GFP-tagged Rab4, Rab11, EHD1 and EHD3 proteins (Figure 4). Notably, blocking the exit of endocytosed receptors from endocytic recycling compartments with monensin led to a marked accumulation of mutant EGFR in a perinuclear endocytic compartment (Figure 3) and increased its colocalization with markers of sorting and endocytic recycling compartments (Figure 4B). Thus, these findings strongly suggest that mutant EGFRs transit through the endocytic recycling compartment.
Importantly, enhanced EGFR-Src as well as activated EGFR/phospho-Src colocalization was observed in endocytic vesicles of a mutant EGFR-expressing cell line (Figure 5). Furthermore, monensin treatment increased the colocalization of mutant EGFRs with Src in the perinuclear endosomal compartment (Figure 6) and enhanced the biochemical association between mutant EGFRs and Src (Figure 7). Given the emerging evidence for a critical role of the constitutive engagement of Src-mediated signaling pathways in mutant EGFR-dependent oncogenesis [7, 9, 10], our results suggest a potentially important role of altered endocytic trafficking in the oncogenic behavior of mutant EGFRs.
In view of the critical role of ligand-induced internalization and lysosomal targeting in limiting EGFR signaling, the constitutive activation of downstream signaling pathways by NSCLC-associated mutant EGFRs has generated interest into potential alterations of endocytic trafficking. For example, given the critical role for the Cbl-family of ubiquitin ligases in orchestrating EGFR ubiquitinylation and subsequent lysosomal sorting, it is notable that a recent analysis of NSCLC-associated mutant EGFRs showed reduced Cbl-dependent lysosomal downregulation [28, 29, 53]. However, another study in an NSCLC cell line reported that mutant EGFR traffics into lysosomes upon EGF stimulation . The present study extends beyond these observations by demonstrating that mutant EGFRs traffic through the endocytic recycling compartment. Our observations, that mutant EGFRs localize to the lysosomes (Figure 2) and block of their endocytic transit by low temperature incubation (Additional File 2) or monensin treatment led to reduced degradation (Additional File 4), are consistent with the idea of mutant EGFRs trafficking into lysosomes. However, our observations do not contradict the defective ubiquitin-dependent trafficking of mutant EGFRs reported by Shtiegman et al., and others [28, 29, 37], as our studies did not address this issue.
Whether the increased transit through the endocytic recycling compartment is an intrinsic property of mutant EGFRs or is a secondary consequence of their reportedly reduced interaction with Cbl and ubiquitin-mediated lysosomal sorting machinery are important questions that will need to be addressed through appropriate manipulations in NSCLC cells as well as the use of ectopic gene expression approaches. In this regard, it is noteworthy that conditions that prevent EGFR interaction with Cbl or its Cbl-dependent ubiquitinylation lead to a more prolonged stay of EGFR in early/recycling endosomal compartments [15, 55, 56]. Physiologically, ligands such as TGFα that promote EGFR recycling rather than lysosomal degradation appear to engage the Cbl and ubiquitin machinery more transiently [22, 57]. In addition to altered ubiquitinylation of mutant EGFRs, other defects in their signaling or protein-protein interactions could contribute to their propensity to enter the endocytic recycling compartment. For example, deubiquitinylating enzymes [58, 59] as well as other factors (e.g. secretory membrane carrier protein SCAMP3) can regulate EGFR recycling versus lysosomal degradation [13, 60]. Future studies to elucidate whether or not mutant EGFRs might aberrantly interact with such proteins will therefore be of considerable interest.
NSCLC-associated mutant EGFRs (both gefitinib-sensitive deletion mutants and gefitinib-resistant L858R/T790 M mutant) are constitutively active and constitutively endocytosed (Figure 1 and Additional File 1). Recent studies have demonstrated that NSCLC-associated kinase domain mutations promote constitutive dimerization of EGFR . As dimerization is critical to EGFR endocytosis and may promote internalization in a kinase-dependent  or kinase-independent [50, 62] manner, constitutive dimerization may play an important role in the transit of mutant EGFRs into the endocytic recycling compartment. In this context, our observations using kinase inhibitors indicate that the kinase activity of EGFR is not essential for the constitutive endocytic localization of mutant EGFR (Additional File 6A). The intracellular localization of mutant EGFR was also unaffected by Src inhibitor PP2, indicating that there may be another determinant of constitutive endosomal localization of mutant EGFRs.
Transit of the constitutively-active mutant EGFR through the endocytic recycling compartment is likely to be biologically relevant. Analyses of EGFR as well as other RTKs have demonstrated that endocytic recycling, in addition to returning the internalized receptors for additional rounds of ligand-binding and signaling, can directly participate in signaling events . For example, inhibition of EGFR internalization reduced the level of activation of Akt and MAPK downstream of the receptor [30, 63]. Notably, initiation of EGFR activation directly at the level of endosomes has been shown to be sufficient to activate Erk and Akt, as well as promote cell survival and proliferation [34, 64]. However, monensin treatment did not enhance Erk, Akt and STAT3 phosphorylation levels (Additional File 5B). The lack of monensin effect on downstream signaling is likely to reflect its ability to affect multiple endocytic compartments and/or its effects on other cellular processes [38, 42, 65]. Nevertheless, our observations of EGFR and Src colocalization and association are consistent with a role of signaling at the level of the endocytic recycling compartment in the biology of mutant EGFR.
Our analyses of mutant EGFR recycling in the context of Src were based on prior evidence that Src-dependent signaling is critical for EGFR-mediated oncogenesis; this has been established in vitro using Src inhibitors as well as mutational approaches , and Src is overexpressed or hyperactive in NSCLC as well as other cancers where EGFR mutations or overexpression have been implicated in oncogenesis [9, 10]. Importantly, Src has been shown to localize to endosomes , and recent studies have shown that Src specifically localizes on recycling endosomes [45, 66]. Thus, it appears plausible that mutant EGFRs, by virtue of their transit through the endocytic recycling compartment, may gain enhanced access to Src, providing a potential explanation for the higher level of constitutive Src-mutant EGFR association [7, 48]. Confocal image analyses indeed support this possibility, as Src and mutant EGFRs show a detectable colocalization (versus essentially little detectable colocalization of Src with wtEGFR) (Figure 5); moreover, this colocalization was further increased by inhibiting the exit of EGFR from the endocytic recycling compartment using monensin (Figure 6). Also, a predominant pool of activated EGFR colocalized with activated Src (Figure 5), and Src inhibitor slightly decreased the mutant EGFR-Src association (additional File 6), which suggest that Src activity might be important for colocalization and association with mutant EGFR. In a different study, a Src inhibitor did not inhibit mutant EGFR-Src association . The difference between the two studies may be due to different types of inhibitor and/or cell lines tested.
Rather interestingly, monensin treatment led to a higher level of biochemically detectable EGFR-Src complexes (Figure 7). This, together with higher constitutive Src-mutant EGFR association, suggests the likelihood that Src-mutant EGFR complexes are either formed or more stable in the endocytic recycling compartment. As Src-dependent signaling is critical for mutant EGFR-mediated oncogenic transformation , these findings suggest that altered trafficking of mutant EGFRs into the endocytic recycling compartment may contribute to their oncogenic behavior. Further studies to perturb the endocytic recycling of oncogenic EGFR mutants should help address the biological role of the altered endocytic trafficking identified here.
It has been reported that a gefitinib-resistant version of H1650 NSCLC cell line showed increased internalization of EGFR upon ligand stimulation when compared to the parental gefitinib-sensitive cell line . Notably, the wtEGFR in the gefitinib-resistant cell line did not undergo ligand-induced lysosomal sorting, even though the receptor was found in endocytic vesicles . In our analyses, we observed a comparable pattern of subcellular localization and endocytic trafficking of gefitinib-sensitive (deletion) and gefitinib-resistant (L858R/T790 M) EGFR mutants (Figures 1, 2, 3 and Additional Files 2 and 3). Similarly, both gefitinib-resistant H1975 and gefitinib-sensitive H1650 cell lines showed delayed internalization of labeled EGF in comparison to the wtEGFR-expressing cell line H358 . However, there were subtle differences among different cell lines harboring mutant EGFRs in the perinuclear accumulation of the mutant EGFR induced by monensin in the regular growth condition (Additional File 3B); the perinuclear accumulation of EGFR was dramatic in HCC827 and HCC4006, intermediate in H1650, and not readily apparent in H1975. Similarly, quantitative assessments of EGFR localization under steady-state conditions (Figure 2E) suggested differences between different NSCLC lines: the mutant EGFR is evenly divided between Tf-positive and LAMP1-positive vesicles in H1650, HCC827 and HCC4006 showed much more mutant EGFR in LAMP1-positive than in Tf-positive vesicles; and gefitinib-resistant mutant EGFR in H1975 colocalized more with Tf than with LAMP1. In addition, H1650 cell line displayed more sensitivity to EGF than other mutant EGFR-expressing cell lines (Figure 1 and Additional File 5A). Whether EGFR expression levels, the nature of EGFR mutations, and/or activities of EGFR regulatory factors such as Src, Cbl or PTEN, which has been shown to be absent in the H1650 cell line , might contribute to the differences in the localization of mutant EGFR and their endocytic trafficking remain open questions. While it is possible that altered endocytic trafficking of EGFR relates to gefitinib resistance, extensive future studies are needed to determine if this is the case.
In summary, the results presented here show that mutant EGFRs in NSCLC cell lines constitutively transit through the sorting and endocytic recycling compartments. Impairment of EGFR exit from the endocytic recycling compartment enhances the mutant EGFR colocalization with Src in the endocytic recycling compartments and increases the Src-mutant EGFR association. Given the critical role of Src-mediated signaling in mutant EGFR-mediated oncogenic transformation, our findings suggest a potentially important role for altered endocytic trafficking in the biology of NSCLC-associated EGFR mutants.
The EHD1-GFP and EHD3-GFP expression constructs in the pcDNA-DEST47 vector were described previously . The Rab4-GFP and Rab11-GFP expression constructs in the EGFPN1 vector  were provided by Dr. Victor Hsu (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). The lentiviral expression vectors pLenti6-V5-UbC GFP, wtEGFR-GFP, EGFR L858R-GFP, and EGFR Δ746-750-GFP were generated using the Gateway cloning technology (Invitrogen, Carlsbad, CA). EGFR was PCR amplified from pcDNA 3.1 EGFR using primers CACCATGCGACCCTCCGGGACGG and TGCTCCAATAAATTCACTGCTTTG, and the amplified fragment was inserted into pENTR/SD/D-TOPO vector using the pENTR/SD/D-TOPO cloning kit (Invitrogen). LR recombination reaction was performed to insert EGFR into the pDEST47 vector to generate an EGFR-GFP chimera. EGFR-GFP was PCR amplified using primers CACCATGCGACCCTCCGGGACGG and TTATTTGTAGAGCTCATCCATGCC, inserted into the pENTR vector, and finally into the pLenti6-V5-UbC vector. PLenti6-V5-UbC EGFR L858R-GFP, and EGFR Δ746-750-GFP were generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA) as previously described . All PCR reactions were performed using the QuikChange II XL Site-Directed Mutagenesis Kit following the manufacturer's instructions.
Human bronchial epithelial cell line immortalization and lentiviral transfection
Primary normal bronchial epithelial cells (HBEC) obtained from a bronchoscopic biopsy sample were provided by Dr. Ravi Salgia (University of Chicago). Cells were transduced with retroviral supernatants of human papilloma virus (HPV) E6 and E7 and selected for several weeks to generate an immortalized human bronchial epithelial cell (HBEC) cell line. Lentiviral supernatants generated as per Gateway cloning technology protocol were used to make the HBEC cell line stably expressing pLenti6-V5-UbC vector, GFP, wtEGFR-GFP, EGFR L858R-GFP, or EGFR Δ746-750-GFP.
Cell culture and transient transfection
Immortalized bronchial epithelial cell line HBE135 (ATCC, Manassas, VA) and HBEC were grown in the DFCI-1 medium described in Band et al. . NSCLC tumor cell lines H1666, H1650, HCC827, HCC4006 and H1975 (ATCC) were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (FBS, Hyclone Inc., Logan, UT), 20 mM HEPES (pH 7.35), 1 mM sodium pyruvate, 1 mM each of nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine and 55 μM 2-Mercaptoethanol (all supplements were from Invitrogen) at 37°C in 5% CO2. Cells were transiently transfected with the indicated plasmids using the FuGene6 Transfection Reagent (Roche, Indianapolis, IN) following the manufacturer's protocol.
Antibodies and other reagents
The following antibodies were obtained from commercial sources: rabbit polyclonal (pAb) anti-EGFR (1005), pAb anti-phospho-AKT (pAKT1/2/3) (Ser 473), pAb anti-phospho-Erk 1/2 (Thr 202/Tyr 204), pAb anti-Erk1 (K-23), and pAb anti-Src (SRC 2) were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal (mAb) anti-phospho-EGFR (activated form) was from BD Biosciences (San Jose, CA); pAb anti-phospho-Src (Tyr416), pAb anti-phospho-EGFR (Tyr1173), pAb anti-STAT3, Rabbit monoclonal anti-phospho-STAT3 (Tyr705), and pAb anti-phospho-EGFR (Tyr845) were from Cell Signaling Technology (Danvers, MA); mAb anti-β actin (Clone AC-15) was from Sigma-Aldrich (St Louis, MO); mAb anti-LAMP1 (H4A3) was from Developmental Studies Hybridoma Bank (Iowa City, IA); mAb anti-EGFR (clone 528; ATCC) was Protein G purified from hybridoma supernatants. Purified anti-phosphotyrosine mAb 4G10  was provided by Dr. Brian Druker (Oregon Health Science University, Portland, OR). Purified mouse EGF, human holo-Transferrin, and monensin were from Sigma-Aldrich. EGFR inhibitor, Erlotinib (Tarceva), was obtained from the Hospital Pharmacy. Src inhibitor PP2 was from Calbiochem (San Diego, CA). Hsp90 inhibitor 17-AAG was from Biomol International (Plymouth, PA, U.S.A.).
Preparation of cell lysates, SDS-PAGE and immunoblotting
Cells at 50-60% confluence were incubated in normal growth medium, growth factor-deprived D3 medium (HBE135)  or 0.1% FBS-containing medium (H1666, H1650, HCC827, HCC4006 and H1975) for 48 hr. For EGF stimulation, cells preincubated in growth factor-deficient medium were either left as such or EGF was added at 10 ng/ml 10 min before cell lysis. Cell lysates were prepared in cold Triton X-100-based lysis buffer , and SDS-PAGE and immunoblotting were performed as previously described .
Cells were grown, EGF stimulation performed, and cell lysates prepared as above with the exception that the lysis buffer contained 0.25% NP-40 (instead of 0.5% Triton X-100), 50 mM Tris (pH 8.0), and 100 mM sodium chloride. Cell lysate aliquots were incubated with anti-EGFR (528) antibody, and immune complexes were captured using Protein A-Sepharose beads (GE Healthcare, Piscataway, NJ). Subsequent SDS-PAGE and immunoblotting were performed as described above.
Cells were plated on glass coverslips (VWR, Batavia, IL) at 50-60% confluence and incubated in normal growth medium or growth factor-deficient medium for 48 hr. Cells were either left unstimulated or stimulated with EGF (10 ng/ml) for 30 min, washed in phosphate buffered saline (PBS, Cellgro, Manassas, VA), fixed in 3.7% formaldehyde (Sigma) in PBS for 20 min at RT, blocked in 2% FBS/PBS/0.02% sodium azide at 4°C for 24 hr, and permeabilized in immunostaining buffer with 0.05% Saponin (Sigma) and 0.2% BSA (Sigma) in PBS for 15 min. Cells were stained with primary antibodies diluted in immunostaining buffer for 1 hr and with Alexa 488- or Alexa 647-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Invitrogen) for 1 hr. Coverslips were mounted on microscope slides with VECTASHIELD® Hard Set™ Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Confocal fluorescence images were obtained with a LSM510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) under a 63× oil immersion lens. Colocalization coefficients for each channel were calculated using the LSM510 Image Examiner software. Colocalization parameters were either set automatically by the software or thresholds were set using the scattergrams. Colocalization coefficients from at least three images were obtained, and averages were either represented as percentages or normalized and plotted with standard deviation as error bars.
For analyses involving immunoblotting or immunoprecipitation, cells were starved in D3 or 0.1% FBS-containing media and preincubated in DMSO (0.1%) or 10 μM monensin for 4 hr. Cells were then continued as such or EGF (10 ng/ml) was added for 30 min followed by cell lysis. For immunofluorescence analyses, starved cells were preincubated in DMSO or monensin as above and loaded with 10 ug/ml of Alexa Fluor 546-conjugated transferrin (Invitrogen) for 45 min. Cells were then washed twice in PBS and either left unstimulated or stimulated with EGF (10 ng/ml) for 30 min. Cells were immunostained as described above.
Epidermal growth factor
Epidermal growth factor receptor
Lysosomal-associated membrane protein 1
Non small cell lung cancer
Receptor tyrosine kinase
Tyrosine kinase inhibitor
We thank Dr. Victor Hsu for the Rab4-GFP and Rab11-GFP constructs; Dr. Brian Druker for the 4G10 antibody; Dr. Mark Rainey for critical reading of the manuscript; members of the Band laboratories for helpful suggestions and discussions; and Janice Taylor and James Talaska of the Confocal Laser Scanning Microscope Core Facility at the University of Nebraska Medical Center (supported by the Nebraska Research Initiative and the Eppley Cancer Center) for their technical assistance. The H4A3 monoclonal antibody developed by J. Thomas August and E.K. Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by: the NIH grants CA105489, CA99900, CA87986, CA116552 and CA99163 to HB, and CA94143, CA96844 and CA81076 to VB; Department of Defense Breast Cancer Research Grants W81XVVH-08-1-0617 (HB), DAMD17-02-1-0508 (VB), and W81XWH-08-1-0612 (MG); the Jean Ruggles-Romoser Chair of Cancer Research (HB) and the Duckworth Family Chair of Breast Cancer Research (VB); and the Malkin Scholarship from Northwestern University (BMC). UNMC-Eppley Cancer Center is supported by an NCI Cancer Center Core Grant.
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