BACKGROUND
epidermal growth factor receptor  is a prototype of receptor tyrosine kinases  which control critical cellular responses to extra-cellular growth factors during development and tissue homeostasis  <cit> .
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  <cit> .
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   <cit> .
as mutational activation of egfr imparts a higher sensitivity to inhibition by egfr-selective tyrosine kinase inhibitors , 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  <cit> .
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  <cit> .
other studies have indicated a relatively selective activation of src downstream of mutant egfrs  <cit> .
in the context of src, use of src inhibitors  <cit>  and mutation of src-dependent phosphorylation sites within egfr   <cit>  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  <cit> .
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  <cit> .
recent studies have demonstrated that activation-dependent recruitment of the cbl family of ubiquitin ligases is a major determinant of lysosomal targeting of egfr  <cit> .
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  <cit> .
indeed, perturbation of cbl protein expression or function alters the lysosomal degradation of egfr and impacts the magnitude and/or duration of downstream signals  <cit> .
additional mechanisms that function either in concert with cbl-dependent ubiquitin modification, such as sprouty <dig>  sts-1/sts- <dig> and cortactin  <cit> , or independently   <cit>  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  <cit> .
notably, tgfα stimulation is associated with a more transient egfr-cbl association and egfr ubiquitinylation  <cit> .
egfr heterodimerization with erbb <dig>  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  <cit> .
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  <cit> .
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  <cit> .
furthermore, nsclc-associated egfr mutants have been shown to undergo egf-independent internalization when expressed in a murine pro-b cell line  <cit> .
intracellularly distributed egfr was also observed in nsclc cell lines  <cit> .
here, we examined the subcellular localization of wild-type  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.
RESULTS
nsclc-associated oncogenic egfr mutants are constitutively endocytosed
to examine the cell surface versus intracellular  localization of egfr, we carried out confocal immunofluorescence imaging analyses of a normal bronchial epithelial cell line hbe <dig> expressing wtegfr and nsclc cell lines expressing wtegfr  or mutant egfrs   <cit> .
as anticipated for unstimulated wtegfr  <cit> , egfr was essentially exclusively localized at the cell periphery in growth factor-deprived, unstimulated hbe <dig> and h <dig> cell lines, and egfr was only observed in punctate intracellular vesicles after egf stimulation of these cells .
in contrast, all of the cell lines expressing mutant egfrs showed predominantly punctate egfr staining with a much lower level of peripheral cell surface staining when examined without egf stimulation; the intracellular punctate staining increased further upon egf stimulation .
mutant egfrs ectopically expressed in hbec cell lines as egfr-gfp chimeras showed similar results, indicating that the constitutive intracellular localization of mutant egf receptors observed in figure  <dig> was not due to cell type specificity .
correlating with the immunofluorescence results, mutant egfrs displayed high levels of basal phosphorylation  whereas the phosphorylation of wtegfr was observed only upon egf stimulation , as expected from previous biochemical analyses  <cit> .
to determine the identity of intracellular vesicles in which the mutant egfrs reside, we loaded cells with fluorescently-labeled transferrin, an early and recycling endosomal marker, followed by immunostaining for egfr and lamp <dig>  the latter as a late endosomal/lysosomal marker .
labeled transferrin loading for  <dig> min allowed for an examination of both early and recycling endosomes  <cit> .
mutant egfrs in all of the cell lines examined  colocalized both with transferrin and lamp <dig>  albeit at different levels in the steady-state conditions .
this data indicates that under steady-state conditions the mutant egfrs are located in multiple endosomal compartments within the cell.
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  <cit> .
indeed, when nsclc cell lines were incubated at 16°c, mutant egfrs showed enhanced colocalization with labeled transferrin .
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  <cit>  which could promote their entry into the endocytic recycling pathway; colocalization of mutant egfrs with transferrin  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  <cit> .
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 h <dig>  as expected, labeled transferrin exit out of the perinuclear endocytic recycling compartment was essentially complete within the  <dig> min chase period; however, monensin treatment markedly delayed this process .
to assess the impact of recycling inhibition on mutant versus wild-type egfr, we carried out concurrent egf stimulation and labeled transferrin chase in hbe <dig> and nsclc cell lines with or without pre-incubation in monensin .
while the relatively low uptake of transferrin in the hbe <dig> cell line did not permit a clear assessment of transferrin accumulation upon monensin treatment, all of the nsclc cell lines, including the wtegfr-expressing cell line h <dig>  showed a marked increase in perinuclear labeled transferrin staining in the presence of monensin, indicating an effective inhibition of cargo exit from the endocytic recycling compartment.
importantly, monensin treatment induced the perinuclear accumulation of egfr in h <dig>  hcc <dig>  hcc <dig> and h <dig> cell lines bearing mutant egfrs, but not detectably in hbe <dig> and h <dig> cell lines bearing the wtegfr, either in the presence or absence of egf stimulation.
similar perinuclear mutant egfr accumulation was observed upon monensin treatment of cells grown in regular growth media without any growth factor deprivation or egf stimulation , and also in hbec cell lines stably expressing ectopic mutant egf receptors .
nsclc-associated mutant egfrs have been shown to attain sensitivity to hsp <dig> inhibitor 17--17-demethoxygeldanamycin  which targets the related receptor erbb <dig> to degradation by enhancing its lysosomal targeting  <cit> .
notably, the presence of monensin prevented the lysosomal targeting of mutant egfr and its degradation induced by 17-aag .
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  <cit> .
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 .
neither the overall egfr levels nor the overall level of egfr phosphorylation, as determined using anti-phosphotyrosine  and anti-phospho-egfr antibodies specific to py <dig> and py <dig>  showed a gross change upon monensin treatment .
mutant egfr colocalizes with markers of endocytic recycling compartment
enhanced colocalization of mutant egfrs with transferrin at 16°c together with perinuclear accumulation upon monensin treatment suggested that mutant egfrs preferentially transit through the endocytic recycling compartment.
therefore, we carried out confocal imaging studies to assess if the constitutively endocytosed mutant egfrs show colocalization with endocytic recycling compartment markers.
rab proteins are known to regulate various steps in endocytic traffic: rab <dig> regulates fast/direct recycling from the early endosomes to the plasma membrane, while rab <dig> regulates recycling from the deeper perinuclear recycling compartments  <cit> .
the newly identified ehd protein family also controls endocytic recycling, with ehd <dig> functioning in the endocytic recycling compartment and ehd <dig> in the early endosomes  <cit> .
the mutant egfr-expressing cell line hcc <dig> was transiently transfected with expression vectors coding for gfp-tagged rab <dig>  rab <dig>  ehd <dig> or ehd3; after  <dig> hr, the cells were fixed and immunostained with an anti-egfr antibody .
partial colocalization of mutant egfr with markers of early and recycling endosomes was observed , and notably, enlarged gfp-positive vesicles were observed surrounding the egfr-positive punctate structures , especially in cells transfected with rab4-gfp.
monensin treatment further increased the appearance of these enlarged vesicles for all of the early and recycling endosomal markers as well as colocalization between these markers and mutant egfr , consistent with the monensin-induced block of exit from the endocytic recycling compartments.
the confocal colocalization studies therefore further support the conclusion that mutant egfrs traffic through the endocytic recycling compartments.
src association with mutant egf receptors in the endocytic recycling compartment
constitutive localization of mutant egfrs in the endocytic recycling compartments  could allow preferential interaction of mutant egfrs with certain signaling pathways.
a particular egfr-relevant signaling partner in this regard is src, which is known to localize on endocytic vesicles including the endocytic recycling compartment  <cit> .
furthermore, mutant egfrs show increased constitutive association with src, and src-egfr interaction plays an important role in mutant egfr-induced oncogenic transformation  <cit> .
therefore, we examined the relative subcellular localizations of egfr and src in nsclc cell lines that were serum-starved and then left untreated or treated with egf for  <dig> min .
as observed above , egf-deprived h <dig> cells  showed predominantly surface-localized egfr staining, whereas hcc <dig> cell line showed constitutive localization of mutant egfr in intracellular vesicles.
anti-src staining showed a predominantly perinuclear vesicular staining, consistent with previous reports  <cit> .
while wtegfr was internalized upon egf stimulation of h <dig> cells as expected, we observed very little, if any, colocalization between egfr and src under these conditions.
in contrast, constitutively internalized mutant egfr in the hcc <dig> cell line exhibited enhanced colocalization with src when compared to wtegfr in the h <dig> cell line .
enhanced colocalization between phospho-egfr and phospho-src was also observed in the hcc <dig> cell line, indicating that constitutive active mutant egfr interacts with activated src in endosomal compartments .
similar results were seen with the h <dig>  hcc <dig> and h <dig> cell lines .
as monensin treatment increased the mutant egfr accumulation in the perinuclear endocytic vesicles , we examined the extent of egfr and src colocalization in cells treated with monensin.
treatments were carried out as in figure  <dig>  and cells were then immunostained for egfr and src .
monensin treatment increased the perinuclear accumulation of mutant but not wild-type egfr, similar to results in figure  <dig>  notably, src showed an enhanced colocalization with mutant egfrs that accumulated in perinuclear vesicles; quantification of the relative colocalization  confirmed the enhancement upon monensin treatment .
thus, mutant but not wild-type egfr displayed enhanced colocalization with src in endocytic vesicles, and such colocalization was further enhanced by inhibiting the exit of egfr from the endocytic recycling compartment with monensin.
monensin treatment enhances the mutant egfr-src association
in view of the increased colocalization of mutant egfr and src in monensin-treated cells, and recent findings that mutant egfrs constitutively complex with src  <cit> , we asked if monensin-induced block of egfr exit from the endocytic recycling compartment influences mutant egfrs and src association.
cells processed essentially as for confocal imaging in figure  <dig> were used to carry out co-immunoprecipitation analyses to assess egfr and src association .
in parallel with the increased mutant egfr and src colocalization seen in figure  <dig>  the amounts of src that co-immunoprecipitated with mutant egfrs, but not with wtegfr, were enhanced in the presence of monensin.
similar results were seen when cells were grown and treated with monensin in regular growth media .
discussion
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  <cit> .
in this respect, endosomal sorting plays a key role in the regulation of egfr signaling  <cit> .
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  <cit> .
the outcome of egfr signaling is intimately linked to its endocytic traffic, which is normally triggered by ligand-induced dimerization  <cit>  and phosphorylation-dependent as well as phosphorylation-independent recruitment of endocytic machinery components  <cit> .
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 .
mutant egfrs were found to localize in early and recycling endosomes based on colocalization with labeled transferrin  and gfp-tagged rab <dig>  rab <dig>  ehd <dig> and ehd <dig> proteins .
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  and increased its colocalization with markers of sorting and endocytic recycling compartments .
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 .
furthermore, monensin treatment increased the colocalization of mutant egfrs with src in the perinuclear endosomal compartment  and enhanced the biochemical association between mutant egfrs and src .
given the emerging evidence for a critical role of the constitutive engagement of src-mediated signaling pathways in mutant egfr-dependent oncogenesis  <cit> , 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  <cit> .
however, another study in an nsclc cell line reported that mutant egfr traffics into lysosomes upon egf stimulation  <cit> .
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  and block of their endocytic transit by low temperature incubation  or monensin treatment led to reduced degradation , 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  <cit> , 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  <cit> .
physiologically, ligands such as tgfα that promote egfr recycling rather than lysosomal degradation appear to engage the cbl and ubiquitin machinery more transiently  <cit> .
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  <cit>  as well as other factors  can regulate egfr recycling versus lysosomal degradation  <cit> .
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  are constitutively active and constitutively endocytosed .
recent studies have demonstrated that nsclc-associated kinase domain mutations promote constitutive dimerization of egfr  <cit> .
as dimerization is critical to egfr endocytosis and may promote internalization in a kinase-dependent  <cit>  or kinase-independent  <cit>  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 .
the intracellular localization of mutant egfr was also unaffected by src inhibitor pp <dig>  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  <cit> .
for example, inhibition of egfr internalization reduced the level of activation of akt and mapk downstream of the receptor  <cit> .
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  <cit> .
however, monensin treatment did not enhance erk, akt and stat <dig> phosphorylation levels .
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  <cit> .
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  <cit> , and src is overexpressed or hyperactive in nsclc as well as other cancers where egfr mutations or overexpression have been implicated in oncogenesis  <cit> .
importantly, src has been shown to localize to endosomes  <cit> , and recent studies have shown that src specifically localizes on recycling endosomes  <cit> .
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  <cit> .
confocal image analyses indeed support this possibility, as src and mutant egfrs show a detectable colocalization  ; moreover, this colocalization was further increased by inhibiting the exit of egfr from the endocytic recycling compartment using monensin .
also, a predominant pool of activated egfr colocalized with activated src , and src inhibitor slightly decreased the mutant egfr-src association , 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  <cit> .
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 .
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  <cit> , 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 h <dig> nsclc cell line showed increased internalization of egfr upon ligand stimulation when compared to the parental gefitinib-sensitive cell line  <cit> .
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  <cit> .
in our analyses, we observed a comparable pattern of subcellular localization and endocytic trafficking of gefitinib-sensitive  and gefitinib-resistant  egfr mutants .
similarly, both gefitinib-resistant h <dig> and gefitinib-sensitive h <dig> cell lines showed delayed internalization of labeled egf in comparison to the wtegfr-expressing cell line h <dig>  <cit> .
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 ; the perinuclear accumulation of egfr was dramatic in hcc <dig> and hcc <dig>  intermediate in h <dig>  and not readily apparent in h <dig>  similarly, quantitative assessments of egfr localization under steady-state conditions  suggested differences between different nsclc lines: the mutant egfr is evenly divided between tf-positive and lamp1-positive vesicles in h <dig>  hcc <dig> and hcc <dig> showed much more mutant egfr in lamp1-positive than in tf-positive vesicles; and gefitinib-resistant mutant egfr in h <dig> colocalized more with tf than with lamp <dig>  in addition, h <dig> cell line displayed more sensitivity to egf than other mutant egfr-expressing cell lines .
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 h <dig> cell line  <cit> , 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.
CONCLUSIONS
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.

 epidermal growth factor receptor is a prototype of receptor tyrosine kinases which control critical cellular responses to extra-cellular growth factors during development and tissue homeostasis. 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. as mutational activation of egfr imparts a higher sensitivity to inhibition by egfr-selective tyrosine kinase inhibitors, 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. 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. other studies have indicated a relatively selective activation of src downstream of mutant egfrs. in the context of src, use of src inhibitors and mutation of src-dependent phosphorylation sites within egfr 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. 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. additional mechanisms that function either in concert with cbl-dependent ubiquitin modification, such as sprouty sts-1/sts- and cortactin, or independently 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. notably, tgfα stimulation is associated with a more transient egfr-cbl association and egfr ubiquitinylation. egfr heterodimerization with erbb 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. 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. 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 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 to examine the cell surface versus intracellular localization of egfr, we carried out confocal immunofluorescence imaging analyses of a normal bronchial epithelial cell line hbe expressing wtegfr and nsclc cell lines expressing wtegfr or mutant egfrs. as anticipated for unstimulated wtegfr, egfr was essentially exclusively localized at the cell periphery in growth factor-deprived, unstimulated hbe and h cell lines, and egfr was only observed in punctate intracellular vesicles after egf stimulation of these cells. in contrast, all of the cell lines expressing mutant egfrs showed predominantly punctate egfr staining with a much lower level of peripheral cell surface staining when examined without egf stimulation; the intracellular punctate staining increased further upon egf stimulation. mutant egfrs ectopically expressed in hbec cell lines as egfr-gfp chimeras showed similar results, indicating that the constitutive intracellular localization of mutant egf receptors observed in figure was not due to cell type specificity. correlating with the immunofluorescence results, mutant egfrs displayed high levels of basal phosphorylation whereas the phosphorylation of wtegfr was observed only upon egf stimulation, as expected from previous biochemical analyses. to determine the identity of intracellular vesicles in which the mutant egfrs reside, we loaded cells with fluorescently-labeled transferrin, an early and recycling endosomal marker, followed by immunostaining for egfr and lamp the latter as a late endosomal/lysosomal marker. labeled transferrin loading for min allowed for an examination of both early and recycling endosomes. mutant egfrs in all of the cell lines examined colocalized both with transferrin and lamp albeit at different levels in the steady-state conditions. this data indicates that under steady-state conditions the mutant egfrs are located in multiple endosomal compartments within the cell. 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. 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 which could promote their entry into the endocytic recycling pathway; colocalization of mutant egfrs with transferrin 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. 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 h as expected, labeled transferrin exit out of the perinuclear endocytic recycling compartment was essentially complete within the min chase period; however, monensin treatment markedly delayed this process. to assess the impact of recycling inhibition on mutant versus wild-type egfr, we carried out concurrent egf stimulation and labeled transferrin chase in hbe and nsclc cell lines with or without pre-incubation in monensin. while the relatively low uptake of transferrin in the hbe cell line did not permit a clear assessment of transferrin accumulation upon monensin treatment, all of the nsclc cell lines, including the wtegfr-expressing cell line h showed a marked increase in perinuclear labeled transferrin staining in the presence of monensin, indicating an effective inhibition of cargo exit from the endocytic recycling compartment. importantly, monensin treatment induced the perinuclear accumulation of egfr in h hcc hcc and h cell lines bearing mutant egfrs, but not detectably in hbe and h cell lines bearing the wtegfr, either in the presence or absence of egf stimulation. similar perinuclear mutant egfr accumulation was observed upon monensin treatment of cells grown in regular growth media without any growth factor deprivation or egf stimulation, and also in hbec cell lines stably expressing ectopic mutant egf receptors. nsclc-associated mutant egfrs have been shown to attain sensitivity to hsp inhibitor 17--17-demethoxygeldanamycin which targets the related receptor erbb to degradation by enhancing its lysosomal targeting. notably, the presence of monensin prevented the lysosomal targeting of mutant egfr and its degradation induced by 17-aag. 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. neither the overall egfr levels nor the overall level of egfr phosphorylation, as determined using anti-phosphotyrosine and anti-phospho-egfr antibodies specific to py and py showed a gross change upon monensin treatment. mutant egfr colocalizes with markers of endocytic recycling compartment enhanced colocalization of mutant egfrs with transferrin at 16°c together with perinuclear accumulation upon monensin treatment suggested that mutant egfrs preferentially transit through the endocytic recycling compartment. therefore, we carried out confocal imaging studies to assess if the constitutively endocytosed mutant egfrs show colocalization with endocytic recycling compartment markers. rab proteins are known to regulate various steps in endocytic traffic: rab regulates fast/direct recycling from the early endosomes to the plasma membrane, while rab regulates recycling from the deeper perinuclear recycling compartments. the newly identified ehd protein family also controls endocytic recycling, with ehd functioning in the endocytic recycling compartment and ehd in the early endosomes. the mutant egfr-expressing cell line hcc was transiently transfected with expression vectors coding for gfp-tagged rab rab ehd or ehd3; after hr, the cells were fixed and immunostained with an anti-egfr antibody. partial colocalization of mutant egfr with markers of early and recycling endosomes was observed, and notably, enlarged gfp-positive vesicles were observed surrounding the egfr-positive punctate structures, especially in cells transfected with rab4-gfp. monensin treatment further increased the appearance of these enlarged vesicles for all of the early and recycling endosomal markers as well as colocalization between these markers and mutant egfr, consistent with the monensin-induced block of exit from the endocytic recycling compartments. the confocal colocalization studies therefore further support the conclusion that mutant egfrs traffic through the endocytic recycling compartments. src association with mutant egf receptors in the endocytic recycling compartment constitutive localization of mutant egfrs in the endocytic recycling compartments could allow preferential interaction of mutant egfrs with certain signaling pathways. a particular egfr-relevant signaling partner in this regard is src, which is known to localize on endocytic vesicles including the endocytic recycling compartment. furthermore, mutant egfrs show increased constitutive association with src, and src-egfr interaction plays an important role in mutant egfr-induced oncogenic transformation. therefore, we examined the relative subcellular localizations of egfr and src in nsclc cell lines that were serum-starved and then left untreated or treated with egf for min. as observed above, egf-deprived h cells showed predominantly surface-localized egfr staining, whereas hcc cell line showed constitutive localization of mutant egfr in intracellular vesicles. anti-src staining showed a predominantly perinuclear vesicular staining, consistent with previous reports. while wtegfr was internalized upon egf stimulation of h cells as expected, we observed very little, if any, colocalization between egfr and src under these conditions. in contrast, constitutively internalized mutant egfr in the hcc cell line exhibited enhanced colocalization with src when compared to wtegfr in the h cell line. enhanced colocalization between phospho-egfr and phospho-src was also observed in the hcc cell line, indicating that constitutive active mutant egfr interacts with activated src in endosomal compartments. similar results were seen with the h hcc and h cell lines. as monensin treatment increased the mutant egfr accumulation in the perinuclear endocytic vesicles, we examined the extent of egfr and src colocalization in cells treated with monensin. treatments were carried out as in figure and cells were then immunostained for egfr and src. monensin treatment increased the perinuclear accumulation of mutant but not wild-type egfr, similar to results in figure notably, src showed an enhanced colocalization with mutant egfrs that accumulated in perinuclear vesicles; quantification of the relative colocalization confirmed the enhancement upon monensin treatment. thus, mutant but not wild-type egfr displayed enhanced colocalization with src in endocytic vesicles, and such colocalization was further enhanced by inhibiting the exit of egfr from the endocytic recycling compartment with monensin. monensin treatment enhances the mutant egfr-src association in view of the increased colocalization of mutant egfr and src in monensin-treated cells, and recent findings that mutant egfrs constitutively complex with src, we asked if monensin-induced block of egfr exit from the endocytic recycling compartment influences mutant egfrs and src association. cells processed essentially as for confocal imaging in figure were used to carry out co-immunoprecipitation analyses to assess egfr and src association. in parallel with the increased mutant egfr and src colocalization seen in figure the amounts of src that co-immunoprecipitated with mutant egfrs, but not with wtegfr, were enhanced in the presence of monensin. similar results were seen when cells were grown and treated with monensin in regular growth media. discussion 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. 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. mutant egfrs were found to localize in early and recycling endosomes based on colocalization with labeled transferrin and gfp-tagged rab rab ehd and ehd proteins. 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 and increased its colocalization with markers of sorting and endocytic recycling compartments. 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. furthermore, monensin treatment increased the colocalization of mutant egfrs with src in the perinuclear endosomal compartment and enhanced the biochemical association between mutant egfrs and src. given the emerging evidence for a critical role of the constitutive engagement of src-mediated signaling pathways in mutant egfr-dependent oncogenesis, 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. 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 and block of their endocytic transit by low temperature incubation or monensin treatment led to reduced degradation, 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, 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. physiologically, ligands such as tgfα that promote egfr recycling rather than lysosomal degradation appear to engage the cbl and ubiquitin machinery more transiently. 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 as well as other factors can regulate egfr recycling versus lysosomal degradation. 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 are constitutively active and constitutively endocytosed. 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 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. the intracellular localization of mutant egfr was also unaffected by src inhibitor pp 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. 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. however, monensin treatment did not enhance erk, akt and stat phosphorylation levels. 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. 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. importantly, src has been shown to localize to endosomes, and recent studies have shown that src specifically localizes on recycling endosomes. 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. confocal image analyses indeed support this possibility, as src and mutant egfrs show a detectable colocalization; moreover, this colocalization was further increased by inhibiting the exit of egfr from the endocytic recycling compartment using monensin. also, a predominant pool of activated egfr colocalized with activated src, and src inhibitor slightly decreased the mutant egfr-src association, 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. 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 h 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 and gefitinib-resistant egfr mutants. similarly, both gefitinib-resistant h and gefitinib-sensitive h cell lines showed delayed internalization of labeled egf in comparison to the wtegfr-expressing cell line h. 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; the perinuclear accumulation of egfr was dramatic in hcc and hcc intermediate in h and not readily apparent in h similarly, quantitative assessments of egfr localization under steady-state conditions suggested differences between different nsclc lines: the mutant egfr is evenly divided between tf-positive and lamp1-positive vesicles in h hcc and hcc showed much more mutant egfr in lamp1-positive than in tf-positive vesicles; and gefitinib-resistant mutant egfr in h colocalized more with tf than with lamp in addition, h cell line displayed more sensitivity to egf than other mutant egfr-expressing cell lines. 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 h 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. 
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 lamp notably, mutant egfrs, but not the wild-type egfr, show a perinuclear accumulation and colocalization with recycling endosomal markers such as rab and ehd upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant egfrs preferentially traffic through the endocytic recycling compartments. while numerous studies have examined mutant egfr signaling, the endocytic traffic of mutant egfr within the nsclc milieu remains less clear. 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. endocytic trafficking of egfr is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. epidermal growth factor receptor controls a wide range of cellular processes, and altered egfr signaling contributes to human cancer.
 epidermal growth factor receptor 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 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 lamp notably, mutant egfrs, but not the wild-type egfr, show a perinuclear accumulation and colocalization with recycling endosomal markers such as rab and ehd 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. 