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Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes signaling to MAP in endosomes

Itziar Pinilla-Macuaa, Simon C. Watkinsa, and Alexander Sorkina,1

aDepartment of , University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved January 11, 2016 (received for review October 13, 2015)

Signaling from epidermal receptor (EGFR) to extracellular- endosomes and their subsequent sorting for degradation in ly- stimuli–regulated 1/2 (ERK1/2) is proposed to be sosomes, which results in signal attenuation. Numerous studies transduced not only from the cell surface but also from endosomes, demonstrated that EGFR remains -bound and capable of although the role of endocytosis in this signaling pathway is con- signaling in early endosomes until receptors are sequestered in troversial. Ras is the only membrane-anchored component in the multivesicular endosomes (reviewed in ref. 6). The hypothesis of EGFR–ERK signaling axis, and therefore, its location determines signaling to ERK1/2 from endosomes is under debate in the intracellular sites of downstream signaling. Hence, we labeled en- literature. Although the localization of receptor-proximal com- dogenous H-Ras (HRas) with mVenus fluorescent protein using plexes containing Grb2, Shc, and SOS in endosomes is unequivo- gene editing in HeLa cells. mVenus-HRas was primarily located at cally demonstrated in various experimental models (reviewed in ref. the plasma membrane, and in small amounts in tubular recycling 6), functional tests using inhibitors of endocytosis yielded contrast- endosomes and associated vesicles. EGF stimulation resulted in fast ing conclusions about the requirement of endocytosis for EGFR- but transient activation of mVenus-HRas. Although EGF:EGFR com- dependent activation of ERK1/2, ranging from the negative effects plexes were rapidly accumulated in endosomes together with the of endocytosis on ERK1/2 activation to an absolute requirement of endocytosis for normal duration and amplitude of ERK1/2 activity Grb2 adaptor, very little, if any, mVenus-HRas was detected in these – endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 (7 11). Because methods to specifically inhibit EGFR endocytosis remained high beyond the point of the physical separation of HRas without affecting other receptor properties are not available, un- derstanding the localization dynamics of the pathway constituents is from EGF:EGFR complexes and down-regulation of Ras activity. Para- critical for resolving these discrepancies. doxically, this sustained MEK1/2 and ERK1/2 activation was depen- Defining localization of Ras is the key to understanding the dent on the active EGFR kinase. Cell surface biotinylation and spatial organization of the EGFR–ERK pathway because Ras is selective inactivation of surface EGFRs suggested that a small fraction the most downstream pathway component that is membrane- of active EGFRs remaining in the plasmamembraneisresponsiblefor anchored but not bound to the receptor. Human cells express continuous signaling to MEK1/2 and ERK1/2. We propose that, under three main Ras isoforms, K-Ras (KRas), H-Ras (HRas), and N-Ras physiological conditions of cell stimulation, EGFR endocytosis serves (NRas), which are highly homologous. Their membrane-targeting – to spatially separate EGFR Grb2 complexes and Ras, thus terminating signals are located in the last 23–24 carboxyl-terminal amino acids, Ras-mediated signaling. However, sustained minimal activation of Ras known as the hypervariable domain (reviewed in refs. 12 and 13). by a small pool of active EGFRs in the plasma membrane is sufficient All Ras isoforms contain the “CAAX box” that is farnesylated (14). for extending MEK1/2 and ERK1/2 activities. NRas and HRas are also palmitoylated in the Golgi complex, which provides an additional membrane-anchoring signal (15, 16). KRas is EGF receptor | Ras | endocytosis not palmitoylated but uses a hexalysine (polybasic) motif to interact with the negatively charged at the plasma membrane as function as the molecular switch during signal (16). Our previous studies detected HRas and KRas in EGFR- Rtransduction from various extracellular stimuli to intracel- containing endosomes in PAE and A431 cells, although HRas lular signaling networks controlling , differen- tiation, motility, and (reviewed in refs. 1 and 2). A Significance multitude of receptors transmit signals through Ras to activate -activated protein kinases (MAPKs) and phosphotidyli- How endocytosis regulates intracellular signaling is a major nositol 3-kinase. Ras-mediated activation of extracellular-stim- unsolved question. In this study, we labeled Ras, which plays a uli–regulated 1/2 (ERK1/2) by epidermal growth central role in normal and oncogenic signaling, with the fluo- factor receptor (EGFR) is an extensively studied signaling pathway rescent protein by gene editing, and for the first time (to our that is centrally involved in regulation of normal and knowledge) examined the localization of endogenous Ras in EGFR-dependent tumorigenesis (reviewed in ref. 3). Ligand bind- living cells stimulated with (EGF). ing to EGFR triggers activation of its kinase and of Microscopy imaging of living cells demonstrated that, although residues in the receptor carboxyl terminus that serve as activated EGF receptors are rapidly internalized into endo- sites for the Src homology 2 (SH2) domain of the Grb2 somes, Ras is not present in these endosomes and mainly lo- adaptor (4). SH3 domains of Grb2 are constitutively associated with cated in the plasma membrane. Therefore, EGF receptors signal Son-of-Sevenless 1 and 2 (SOS1/2), the primary guanine nucleotide to MAP kinases through Ras exclusively from the plasma membrane. exchange factors of Ras. Binding of the Grb2–SOS complex to EGFR positions SOS in the proximity to membrane-anchored Ras, Author contributions: I.P.-M. and A.S. designed research; I.P.-M., S.C.W., and A.S. per- thus allowing Ras–SOS interaction and promoting the replacement formed research; I.P.-M. and S.C.W. contributed new reagents/analytic tools; I.P.-M., S.C.W., of GDP by GTP. GTP-loaded Ras recruits to the membrane and and A.S. analyzed data; and I.P.-M. and A.S. wrote the paper. activates Raf -threonine kinases, which leads to consequential The authors declare no conflict of interest. phosphorylation and activation of MEK1/2 and ERK1/2 (reviewed This article is a PNAS Direct Submission. in ref. 5). 1To whom correspondence should be addressed. Email: [email protected]. EGFR signaling is initiated at the cell surface, but ligand-in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. duced endocytosis leads to rapid redistribution of receptors to 1073/pnas.1520301113/-/DCSupplemental.

2122–2127 | PNAS | February 23, 2016 | vol. 113 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1520301113 Downloaded by guest on September 30, 2021 displayed substantially higher extent of endosomal localization compared with KRas (17). Other studies also demonstrated the localization and activation of HRas and other Ras isoforms on intracellular membranes (13, 14, 18–21). Most of the subcellular localization studies were, however, performed in cells transiently or constitutively overexpressing Ras (for example, see refs. 22 and 23). Localization of endogenous Ras isoforms has been rarely studied, owing to the lack of that efficiently detect Ras by immunofluorescence microscopy. In the present study, we analyzed the localization of endoge- nous HRas in living HeLa cells in which HRas was labeled with the fluorescent protein mVenus (mV-HRas) by gene editing. The rationale for focusing on HRas was that (i) HRas exhibits prominent colocalization with EGFR in endosomes when over- expressed (17); and (ii) H-Ras is frequently mutated in head-and- neck squamous cell carcinoma, tumors that are typically EGFR driven (24). Live-cell imaging demonstrated the predominant localization of mV-HRas in the plasma membrane and detected virtually no mV-HRas in EGFR-containing endosomes, suggesting that EGFR endocytosis serves to separate Ras from EGFR-proxi- mal activating complexes and therefore down-regulate Ras activity. Results and Discussion Kinetics of EGF–Ras–MAPK Pathway Activation in Gene-Edited Cells Expressing Endogenous Labeled HRas. To study the spatiotemporal regulation of the EGFR–ERK1/2 signaling pathway, we first examined the time course of activation of the main components of this pathway in serum-starved HeLa cells stimulated with EGF. This variant of HeLa cells was used in our previous studies of localization of Grb2 and MEK2 (25, 26). These cells express ∼35,000 EGFRs per cell, resembling EGFR expression levels in various types of normal epithelial cells. Measurements of the amount of GTP-loaded Ras revealed that Ras·GTP peaked in the first 2.5–5 min after EGF stimulation followed by fast in- activation in the following 5–10 min down to 10–15% of the peak activity (Fig. 1). In contrast, phosphorylation of the catalytic residues of MEK1/2 and ERK1/2 reached maximum at 5–10 min and slowly decayed during the following 45 min. c-Raf was rapidly phosphorylated at Ser338 upon EGF stimulation, but Fig. 1. Similar kinetics of EGF-stimulated Ras and MEK/ERK activities in parental the kinetics of c-Raf activity was difficult to monitor due to a HeLa and genome-edited HeLa cells expressing mVenus-HRas. (A) HeLa cells complex pattern of multiple phosphorylation/dephosphorylation were incubated with 4 ng/mL at 37 °C for indicated times. GTP-loaded Ras was events controlling this activity. The kinetics of Ras, MEK, and pulled down from cell lysates with GST-RBD. GST pulldowns and aliquots of the lysates were electrophoresed, and probed for Ras (pan-Ras ) in pull- ERK activities were similar when the cells were treated with downs, and phosphorylated MEK1/2 (pMEK) and ERK1/2 (pERK), and α-actinin EGF in the physiological range of concentrations (4 ng/mL; Fig. A A (loading control) in lysates. Western blots of a representative experiment are 1 ) and supraphysiological concentration (20 ng/mL; Fig. S1 ), shown. Graphs represent mean band intensities normalized to the amount of suggesting that in our experimental system, concentrations of α-actinin (±SEM; n = 4) expressed as percentage of the maximum high-signal EGF up to 20 ng/mL can be used to study the regulation of the intensity for each protein during the time course. (B) mVenus sequence was Ras–ERK activation pathway. Transient Ras activation followed inserted in-frame at the 5′-end of the coding sequence of the HRAS gene using a by the sustained ERK activation has been previously observed TALEN pair and a donor vector containing mVenus inserted between left and (27). Prolonged Ras activity was reported in cells substantially right homology arms (LHA and RHA) from the genomic HRAS sequence. (C) overexpressing Ras (28). HeLa/mV-HRas cells were stimulated with 4 ng/mL, and the activities of mV-HRas, Different activation kinetics of Ras and its downstream ef- MEK1/2, and ERK1/2 were measured as described in A. Western blots of a rep- fectors raises a question of how is the duration of EGFR-dependent resentative experiment are shown. Graphs represent mean band intensities activity of ERK controlled. In the same variant of HeLa cells, at normalized to the amount of α-actinin (±SEM; n = 4) expressed as percentage least 90% of EGFR complexes with Grb2 were shown to be lo- of the maximum high-signal intensity for each protein during the time course. cated in endosomes after 10–15 min of EGF stimulation, sug- (D) HeLa/mV-HRas cells transiently expressing CFP-EHD1 were imaged through 445-nm (CFP; red) and 515-nm channels (mVenus; green). Insets represent high gesting that EGFR is capable of signaling from endosomes (25). μ To examine whether these endosomal EGFRs can signal through magnification of the region marked by the white rectangle. (Scale bar: 10 m.) Ras to sustain ERK activity, we generated a gene-edited HeLa cell line by inserting mVenus fluorescent protein into the endogenous and parental HeLa cells were similar (Fig. 1 and Fig. S1). These locus of HRAS gene using activator-like effector nuclease (TALEN)-based method (further referred to as HeLa/mV- data suggest that a moderately increased expression of HRas does HRas cells) (Fig. 1B and Fig. S2A). Interestingly, despite a single- not affect the signal process. Given that this HRas allele editing (Fig. S2B), mV-HRas was expressed at ∼2.2-fold level is within the range of HRas expression levels in cultured higher level than HRas in parental cells (clone 3, Fig. S2C). The mammalian cells (29), HeLa/mV-HRas cells represent an ap- mechanism underlying an increased mV-HRas expression is un- propriate model to analyze the spatiotemporal regulation of known but most probably involves an increased rate of biosynthesis. EGFR–Ras–ERK signaling pathway. The kinetics of GTP loading of mV-HRas was essentially similar to that of total (pan) Ras in parental HeLa cells stimulated with 4 or Localization of mV-HRas in Cells Stimulated with EGF. Live-cell imaging 20 ng/mL EGF (Fig. 1C and Fig. S1B). Likewise, kinetics of MEK1/2 of mV-HRas by spinning disk confocal microscopy revealed highly CELL BIOLOGY andERK1/2phosphorylationinEGF-stimulatedHeLa/mV-HRas consistent pattern of HRas distribution within the cell population.

Pinilla-Macua et al. PNAS | February 23, 2016 | vol. 113 | no. 8 | 2123 Downloaded by guest on September 30, 2021 mV-HRas was mainly located in the plasma membrane (Fig. 1D). Many cells also contained mV-HRas in tubular structures that frequently span from and through the Golgi area and extend to and from the plasma membrane. These tubules were highly sensitive to chemical fixations, which precluded analysis in fixed cells. In living cells, mV-HRas tubules were positive for markers of recycling endosomes, such as EHD1 (Fig. 1D)andRab11(Fig. S2D). In some cells, one or two bright vesicular-shape structures of mV-HRas were seen associated with and moving along tubular recycling compart- ments. Some vesicular structures containing mV-HRas were colo- calized with or surrounded by compartments positive for GRASP55- mCherry, a Golgi apparatus protein (Fig. S2D). Treatment of cells with EGF or EGF conjugated to rhodamine (EGF-Rh) did not lead to detectable changes in mV-HRas lo- calization. EGF-Rh fluorescence overlapped with mV-HRas in the plasma membrane during the first few minutes of cell stim- ulation (Fig. 2 A and B). Transiently expressed Grb2-CFP could also be detected together with EGF-Rh and mV-HRas in the plasma membrane at early time points (Fig. S3A). To compare dynamics of EGF-Rh and mV-HRas in the plasma membrane, the cell bottom membrane of HeLa/mV-HRas cells incubated with EGF-Rh was imaged using total internal reflection fluo- rescence microscopy (TIR-FM). Following rapid binding, EGF- Rh concentrated in numerous diffraction-limited spots (Fig. S4), with a pattern reminiscent of EGF:EGFR clustering in clathrin- coated pits (30). mV-HRas was diffusely distributed throughout the membrane with a few clusters representing a minor fraction of mV-HRas in the plasma membrane (Fig. S4). Only 2–3% of EGF-Rh spots overlapped with mV-HRas spots during the first 5 min of the time course (Fig. 2C and Fig. S4), suggesting that mV-HRas is not corecruited with EGF:EGFR complexes into clathrin pits and endocytic vesicles. These data are consistent

Fig. 2. Localization of mVenus-HRas in cells stimulated with EGF-Rh. (A)HeLa/ mV-HRas cells were incubated with 4 ng/mL EGF-Rh at 37 °C for indicated times, and live-cell imaging was performed through 515-nm (mVenus; green) and 561-nm (rhodamine; red) channels. The image acquisition time was 1.2 s for mVenus throughout the time course, whereas for rhodamine it was 600 ms (0–5 min), 400 ms (10 min), and 200 ms (15 min) to avoid rhodamine signal saturation in endosomes at later time points. (Scale bar: 10 μm.) (B) Insets show high-magnification images of regions indicated by white rectangles in A to demonstrate an overlap of EGF-Rh and mV-HRas fluorescence in the plasma membrane (a and b) and no overlap of EGF-Rh and mV-HRas in endosomes (c–e). (C) The cell bottom surface of HeLa/mV-HRas cells was continuously im- aged during stimulation with 4 ng/mL EGF-Rh at 37 °C using TIR-FM. EGF-Rh was added at a 10-s time point. Typically, specific cell-bound fluorescence of EGF-Rh was detected at ∼40-s time point. The percentage of EGF-Rh–positive spots colocalized with mV-HRas spots relative to the total number of EGF-Rh spots is plotted against time. Mean values (±SD; n = 4) are presented. Corre- sponding representative images are shown in Fig. S4.(D) HeLa/mV-HRas cells were incubated with 4 ng/mL EGF-Rh as in A,andz stacks of 18–21 confocal images were acquired through 515- and 561-nm channels using the same image acquisition parameters as in A. The percentage of mV-HRas colocalized with Fig. 3. Localization of fluorescent EGF and Grb2 but not mV-HRas in early EGF-Rh relative to the total cellular mV-HRas fluorescence was calculated in endosomes. (A) HeLa/mV-HRas cells transiently expressing Grb2-CFP were multiple 3D images. Mean values (±SD) are presented. (E) Parental HeLa cells incubated with 4 ng/mL EGF-A647 for 15 min at 37 °C. Images were acquired transiently expressing YFP-HRas or HeLa/mV-HRas cells were incubated with from living cells at 37 °C. Merge images of Grb2-CFP (blue) and mV-HRas 4 ng/mL EGF-Rh for 15 min at 37 °C, and z stacks of confocal images were ac- (green) in cells before stimulation, and after EGF-A647 (red) stimulation are quired through 515- and 561-nm channels. Representative images of YFP-HRas shown. Insets represent high-magnification images of individual are shown in Fig. S5. Total amounts of YFP-HRas or mV-HRas per cell were and a merge image of the region indicated by white rectangle to show high calculated from 3D images using segmentation. Mean YFP-HRas expression extent of Grb2-CFP and EGF-A647 colocalization. (B) HeLa/mV-HRas cells levels were calculated as fold difference to the mean expression level of transiently expressing CFP-Rab5 were incubated with 10 ng/mL EGF-A647. mV-HRas (× mV-HRas). Molar stoichiometry (515/561 ratio) of mV-HRas or Images were acquired from living cells at 37 °C. Merge images of CFP-Rab5 YFP-HRas to EGF-Rh in endosomes was calculated in, respectively, HeLa/mV- (blue) and mV-HRas (green) in cells before stimulation and after EGF-A647 HRas cells or parental cells expressing low (mean expression, 0.6× mV-HRas), (red) stimulation are shown. Insets represent high-magnification images of intermediate (“med,” mean expression, 10.7× mV-HRas), and high (mean each individual protein and a merge image of the region indicated by white expression, 55.4× mV-HRas) levels of YFP-HRas. Mean 515/561 ratio values rectangle to show high extent of CFP-Rab5 and EGF-A647 colocalization. (±SD) obtained from 100 to 240 endosomes at each condition are presented. (Scale bars: 10 μm.)

2124 | www.pnas.org/cgi/doi/10.1073/pnas.1520301113 Pinilla-Macua et al. Downloaded by guest on September 30, 2021 Fig. 4. EGFR kinase activity is required for sustained MEK and ERK activation. (A) Schematics of the ex- perimental protocol used in B–F.(B) HeLa/mV-HRas cells were incubated with 4 ng/mL EGF at 37 °C for 15 min. DMSO (vehicle) or PD158780 (50 nM) was then added to cells, and the cells were further in- cubated at 37 °C for indicated periods of time in the same medium (treatment chase). Cell lysates were probed by Western blotting with antibodies to EGFR pY1068, pMEK1/2, pERK1/2, and α-actinin (loading control). (C–E) Graphs show mean values of band intensities normalized to the amount of α-actinin (±SEM; n = 8) exemplified in B and presented as percentage of the mean value at time “0” (after the 15-min incubation with EGF but before the chase treatment). (F) HeLa/mV-HRas cells were incubated with EGF for 15 min, and then with DMSO or PD158780 for additional 5 min as described in B. The amount of mV-HRas·GTP was measured using the GST-RBD pull- down assay as in Fig. 1. Bar graphs show mean band intensities (±SEM; n = 5). Paired t tests were performed. *P < 0.05, **P < 0.01, and ***P < 0.001.

with previous observations of different endocytic routes of HRas levels of YFP-HRas (Fig. 2E). Similarly, significant localization and EGFR: clathrin-independent, ARF6-dependent endocytosis of YFP-HRas in EGF-Rh–containing endosomes could be ob- of HRas (31), and clathrin-mediated endocytosis of EGFR in served in COS1 cells expressing high but not low levels of YFP- HeLa cells stimulated with low EGF concentrations (32). HRas (Fig. S6). Furthermore, the expression level of endogenous Continuous endocytosis (5–15 min) resulted in a clear sepa- mV-HRas was not sufficient to detect GTP-loaded mV-Ras by ration of EGF-Rh:EGFR complexes, which were accumulated in imaging of various types of fluorescent-protein–tagged Ras·GTP vesicular endosomes, from mV-HRas, which was located in the (22,33).YFP-HRasexpressionthatis>20-fold higher than plasma membrane and tubular compartments. Quantitative analysis that of mV-HRas was necessary for detection of EGF-induced of 3D images demonstrated that <1% of total cellular mV-HRas HRas activation using the same sensors. Overall, our experiments in overlaps with EGF-Rh at the 15-min time point (Fig. 2D). Visual HeLa/mV-HRas cells demonstrate preferential targeting of the analysis of individual EGF-Rh–containing endosomes demon- majority of endogenous HRas molecules to the plasma mem- strated no obvious mV-HRas localization in most of these endo- brane. The relative proportion of endogenously labeled mV- somes, although faint mV-HRas fluorescence overlapping with HRas associated with intracellular membranes was lower than some endosomes could be detected. Based on the ratio of the ap- what was typically observed in cells overexpressing HRas. For parent quantum yields of YFP and rhodamine in our imaging sys- example, the fluorescence intensity of tubular compartments tem (25), calculations of the molar ratio of mVenus and rhodamine containing overexpressed YFP-HRas was comparable to that in showed that ∼2 molecules of mV-HRas were colocalized with 100 the plasma membrane (16, 31). Limited localization of mV-HRas in molecules of EGF-Rh in endosomes at the 15-min time point (Fig. vesicular endosomal compartments in cell periphery and Golgi area 2E). For comparison, the molar ratio of Grb2-YFP to EGF-Rh was observed in contrast to what was reported in cells over- under the same experimental conditions was previously found to be expressing HRas (17, 31, 34, 35) (Figs. S5 and S6). Certainly, 2:1 (25). Likewise, in gene-edited HeLa cells, transiently expressed differences in localization of gene-edited endogenous and ex- Grb2-CFP was highly colocalized with EGF-Alexa647 (EGF-A647) ogenous HRas can be due to different cells types and levels of but not with mV-HRas in endosomes (Fig. 3A). GFP-tagged SOS1 overexpression of the latter. was also present in endosomes containing EGF-A647 in HeLa cells Altogether, the data in Figs. 2 and 3 and Figs. S2–S6 suggest (Fig. S3B). Grb2-CFP and GFP-SOS1 could not be detected in the that endocytosis of activated EGFR separates EGFR–Grb2–SOS plasma membrane and tubular endosomes where mV-HRas was complexes from HRas, leading to down-regulation of HRas activity. located after 10–15 min of EGF stimulation (Fig. 3A and Fig. S3B). In fact, the time course of EGF-Rh/mV-HRas colocalization (Fig. Fluorescent EGF but not mV-HRas was highly colocalized with 2D) was highly similar to the time course of Ras activity (Fig. 1C). markers of early/intermediate endosomes (Rab5 and EEA.1) (Fig. The possibility that other Ras isoforms are recruited to EGFR- 3B and Fig. S3C). containing endosomes cannot be formally ruled out. To directly To test whether the amount of HRas present in EGFR-containing examine KRas (4B form) localization in our variant of HeLa endosomes depends on the expression level of HRas, YFP-HRas cells, YFP-KRas and CFP-KRas were transiently expressed in or CFP-HRas were transiently expressed in parental HeLa or parental HeLa and HeLa/mV-HRas cells, respectively. Regard- HeLa/mV-HRas cells, respectively. YFP-HRas and CFP-HRas less of the expression level, YFP-KRas and CFP-KRas were were clearly detected in endosomes containing EGF-Rh when mostly located in the plasma membrane; very rarely, examples of highly overexpressed (Fig. S5). Quantifications revealed that, in colocalization of KRas and EGF-Rh in endosomes could be cells overexpressing YFP-HRas, the stoichiometry of HRas and detected (Fig. S7). Similarly, transient expression of GFP-NRas

EGF in endosomes is significantly higher (∼35:100) than in revealed virtually complete separation of endosomal EGF-Rh CELL BIOLOGY HeLa/mV-HRas cells or HeLa cells expressing low/moderate from NRas that was located in the plasma membrane and

Pinilla-Macua et al. PNAS | February 23, 2016 | vol. 113 | no. 8 | 2125 Downloaded by guest on September 30, 2021 concentrated in the pericentriolar area (Fig. S8). Given that time courses of EGF-induced activity of total Ras and mV-HRas were essentially similar (Fig. 1 and Fig. S2), indicative of a similar activation kinetics of all Ras species, the involvement of KRas and NRas in endosomal signaling by EGFR is unlikely. Finally, it is also possible that small amounts of mV-HRas present in EGFR-containing endosomes are below the detection limit of our imaging system, although this system is capable of single-molecule imaging (36). Image acquisition times as long as 1–1.2 s were used to obtain a maximum signal-to-noise ratio in mV-HRas images, while avoiding the cross-bleed of the rhodamine fluorescence. Given that copy numbers of HRas and other Ras isoforms per cell are relatively low (37, 38), and that a very weak mV-Ras fluorescence in tubular compartments was detected (for example, see Fig. 1D), the amount of undetectable Ras in EGFR- containing endosomes is expected to be extremely small.

Sustained MEK and ERK Activities Require EGFR Kinase Activity. Rapid decay of Ras activity (Fig. 1) and lack of mV-HRas in EGFR–Grb2–SOS-containing endosomes (Fig. 3A) suggested that the sustained phosphorylation of MEK1/2 and ERK1/2 in the absence of upstream activating signals could be due to slow dephosphorylation of these kinases by (39, 40). To test the hypothesis that the sustained activity of MEK1/2 and ERK1/2 is EGFR independent, EGFR kinase activity was blocked by PD158087 in cells incubated with EGF for 15 min (Fig. 4A). As expected, EGFR phosphorylation at Tyr1068 (major Grb2 ) was reduced by 70% following expo- sure to PD158087 (Fig. 4 B and C). Incomplete dephosphory- lation of the receptor is likely due to a limited accessibility of pTyr1068 to phosphatases, for example, due to sequestration of a Fig. 5. A small pool of surface EGFR contributes to a sustained signaling – pool of active EGFRs in multivesicular endosomes. Surprisingly, through the Ras ERK pathway. (A) Schematics of the experimental protocol used in B and C.(B) HeLa/mV-HRas cells were incubated with 4 ng/mL EGF for inhibition of EGFR kinase also resulted in dephosphorylation of − MEK1/2 and ERK1/2, demonstrating that sustained phosphory- 2.5 or 15 min at 37 °C, and then incubated in either DMEM (pH 7.4) ( AW) or an acidic buffer (pH 4.5) (+AW) for 1 min at 4 °C. The cells were then biotinylated lation of these kinases does require the EGFR kinase activity. and lysed. Biotinylated proteins were pulled down by Avidin-agarose. Pulldowns MEK1/2 dephosphorylation was significantly faster than that of B–E and aliquots of cell lysates were electrophoresed and probed with antibodies to ERK1/2 (Fig. 4 ). This delay is likely due to time- and signal- EGFR pY1068 and total EGFR (1005). Bar graphs on the Right represent mean dependent expression of ERK1/2 phosphatases (40). Although band intensities (±SEM; n = 3) presented as percentage of the band intensities of mV-HRas activity in cells incubated with EGF for 15 min is only pulldowns recovered from cells that were incubated with EGF for 2.5 min and 15–20% of the peak maximum, PD158087 treatment further not treated with the acidic buffer (lane 1). (C) Serum-starved HeLa/mV-HRas cells reduced this activity (Fig. 4F). were incubated with EGF at 37 °C, and treated with the acidic buffer (+AW) or How do activated EGFRs, which are highly concentrated in incubated DMEM (−AW) as described in B. Cells that were not treated with acid endosomes, transduce signals to MEK and ERK through Ras, wash, were further incubated with 4 ng/mL EGF for 5 min at 37 °C, whereas acid which is not present in these endosomes? One possibility is that wash-treated cells were incubated in DMEM containing 0.1% BSA for 5 min Ras activity in the plasma membrane is maintained through at 37 °C. The amounts of EGFR pY1068, pMEK1/2, and pERK1/2 in lysates ERK-mediated stabilization of the SOS–Ras interaction (28). were measured as described in Fig. 4B, and the amount of mV-HRas·GTP was However, such positive would not be EGFR dependent measured as in Fig. 4D. Mean band intensities (±SEM; n = 6) are plotted as because levels of pERK1/2 remain high and not sensitive to percentage of that intensity of the bands corresponding to cells that were “ ” not treated with the acidic buffer. Paired t test was performed. *P < 0.05, PD158087 for at least 5 min in our treatment chase experi- < < ments (Fig. 4B). Another possibility is that a small pool of active **P 0.01, and ***P 0.001. EGFRs remaining at the cell surface after 15 min of continuous – endocytosis is sufficient for sustaining signaling through the Ras same pH 4.5 treatment of cells incubated with EGF for 15 min ERK pathway. Technically, small pools of fluorescently tagged inactivated only ∼20% of surface EGFRs. It is possible that re- proteins are more difficult to detect in the plasma membrane ceptors remaining on the cell surface after 15-min EGF stimulation than in endosomes because of the diffuse distribution of the fluo- rescence in the plasma membrane in contrast to highly concentrated form high-affinity complexes with EGF, which are resistant to low fluorescence of proteins in endosomes. Biochemical measurements pH and require a 37 °C incubation for dephosphorylation, or are in the same variant of HeLa cells have previously demonstrated that inaccessible to phosphatases due to association with clathrin lattices about 5–10% of 125I-EGF remains at the surface after 15 min in- (42). Nevertheless, these data indicate that a small pool of active cubation of cells with 4 ng/mL 125I-EGF (25). To test whether this EGFRs is present in the plasma membrane after 15 min of small pool of EGFRs remaining in the plasma membrane are ac- continuous endocytosis. tive, surface biotinylation method was used. EGFR phosphorylated The acidic wash treatment of the cells incubated with EGF for at Tyr1068 was detected in the biotinylated fraction recovered from 15 min and subsequent incubation for 5 min at 37 °C reduced cells treated with 4 ng/mL EGF for 15 min at 37 °C (Fig. 5). This Ras·GTP level and MEK1/2 phosphorylation by 40–45% com- surface-active EGFR pool was ∼20%ofthatincellsincubatedwith pared with cells that were continuously exposed to EGF (Fig. EGF for 2.5 min, conditions of the spatial overlap of EGF-Rh 5C). The extent of inactivation of Ras and MEK1/2 paralleled that and mV-HRas (Fig. 2 A and B). Treatment of cells preincubated of EGFR dephosphorylation (Fig. 5C). Dephosphorylation of with EGF for 2.5 min with the mild acidic buffer (pH 4.5) at 4 °C, ERK1/2 in these experiments was insignificant (Fig. 5C)because which releases surface-bound EGF without affecting cell viability longer than 5-min “treatment chase” time was needed to de- (41), reduced the amount of biotinylated pTyr1068 EGFRs by phosphorylate ERK1/2 (Fig. 4). Similar experiments in cells in- 80%, confirming efficient EGF dissociation (Fig. 5 B and C). The cubated with EGF for 2.5 min demonstrated strong dependence

2126 | www.pnas.org/cgi/doi/10.1073/pnas.1520301113 Pinilla-Macua et al. Downloaded by guest on September 30, 2021 of Ras activity on surface EGFR signaling at this time point cells expressing high levels of EGFR and treated with high EGF (Fig. 5), whereas deactivation of MEK1/2 and ERK1/2 was not concentrations, conditions causing clathrin-independent endo- observed during an onset of their activation at early times (2.5– cytosis of EGFR and massive activation of pinocytotic processes, 7.5 min) after EGF stimulation. Altogether, the data in Figs. 4 which may lead to “mixing” of different endosomal compart- and 5 support the hypothesis that a small pool of surface EGFR ments and thus substantial colocalization of active EGFR and remaining after 15 min of continuous endocytosis may maintain Ras in endosomes. Future studies of the localization of endog- low levels of plasma membrane Ras·GTP to sustain MEK and enous Ras in an expanded panel of cells with different levels of ERK activity. EGFR expression will provide full understanding of the intra- Many studies arguing for or against the idea of the endosomal cellular sites of Ras signaling. signaling to ERK were based on the detection of signaling pro- teins in endosomes or the use of general endocytosis inhibitors. Materials and Methods Although it is unequivocally proven that a substantial fraction of The sources of EGF, antibodies, inhibitors, chemicals, and DNA plasmids are EGFR–Grb2–SOS complexes remain intact in endosomes, our listed in Supporting Information. Parental HeLa, HeLa/mV-HRas, and COS1 studies suggest that, at least in cells with low EGFR levels, the cells were maintained in DMEM and 10% (vol/vol) fetal bovine serum. Before main function of endocytosis is to separate proximal receptor experiments, cells were serum-starved for 16 h in DMEM. Transient trans- complexes from membrane-anchored Ras. Another important fections, TALEN design and generation of HeLa/mV-HRas gene-edited cells, implication from our experiments is that signaling by a small biochemical measurement of Ras activity, cell surface biotinylation, and number of active EGFRs (1,000–2,000 per cell) leading to a Western blotting detection of phosphorylated EGFR, MEK, and ERK are relatively low level of Ras activity is sufficiently amplified to described in Supporting Information. Live-cell fluorescence microscopy using maintain MEK and ERK activities for a long time. Our findings spinning disk confocal microscopy, TIR-FM imaging, and methods of image are complementary to previous observations that inhibition of analysis were performed as previously described (25, 30, 44, 45). See Sup- endocytosis does not affect ERK1/2 activation (8–10, 43). On the porting Information for details. other hand, the inhibitory effects of dynamin mutants on EGFR- dependent ERK activation observed in HeLa and other cells (for ACKNOWLEDGMENTS. We are grateful to Drs. D. Bar-Sagi, J. Donaldson, example, in refs. 7 and 29) could be due to the effects on the E. Galperin, A. Linstedt, and O. Weisz for the gifts of reagents, to Ms. Nancy Zurowski (University of Pittsburgh) for help with cell sorting, and to localization of the downstream components of the ERK pathway Dr. A. Grassart (University of California, Berkeley) for his advice on TALEN rather than on EGFR endocytosis per se. An additional scenario methodology. This work was supported by National Institute that may lead to EGFR–Ras endosomal signaling is possible in Grant CA089151.

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