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Endocytosis Separates EGF Receptors from Endogenous Fluorescently Labeled Hras and Diminishes Receptor Signaling to MAP Kinases in Endosomes

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Endocytosis Separates EGF Receptors from Endogenous Fluorescently Labeled Hras and Diminishes Receptor Signaling to MAP Kinases in Endosomes Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes Itziar Pinilla-Macuaa, Simon C. Watkinsa, and Alexander Sorkina,1 aDepartment of Cell Biology, 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 growth factor receptor (EGFR) to extracellular- endosomes and their subsequent sorting for degradation in ly- stimuli–regulated protein kinase 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 ligand-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 phospholipids at the plasma membrane as GTPases 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 cell proliferation, differen- tiation, motility, and apoptosis (reviewed in refs. 1 and 2). A Significance multitude of receptors transmit signals through Ras to activate mitogen-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 protein kinase 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 cell growth and knowledge) examined the localization of endogenous Ras in EGFR-dependent tumorigenesis (reviewed in ref. 3). Ligand bind- living cells stimulated with epidermal growth factor (EGF). ing to EGFR triggers activation of its kinase and phosphorylation of Microscopy imaging of living cells demonstrated that, although tyrosine residues in the receptor carboxyl terminus that serve as activated EGF receptors are rapidly internalized into endo- docking 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 serine-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 antibodies 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
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