The handle http://hdl.handle.net/1887/82070 holds various files of this Leiden University dissertation.
Author: Bakker, J.M.
Title: Manipulating endosomal systems: the molecular mechanisms of transport decisions and Salmonella-induced cancer
Issue Date: 2019-12-12
Manipulating endosomal systems:
the molecular mechanisms of transport decisions and Salmonella-induced cancer
Jeroen Bakker
ISBN: 978-90-903-2558-3 Copyright: Jeroen Bakker
Cover: Manipulation of endosomes by a regulatory mechanism. The colors represent the fluorescent labels that are used to detect endosomes marked by different
endosomal proteins during confical microscopy experiments. Design: Jeroen Bakker.
The research on which this thesis is based was performed at the department of Cell Biology II, Netherlands Cancer Insttute, Amsterdam, the Netherlands, as well as at the department of Chemical Immunology, Leiden University Medical Center, Leiden, the Netherlands.
Financial support for printing this thesis was provided by the Leiden University Medical Center (LUMC)
Printed by: Printenbind
Manipulating endosomal systems:
the molecular mechanisms of transport decisions and Salmonella-induced cancer
Proefschrift
Ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Prof. Mr. Carl Stolker, Rector Magnificus
& voorzitter college van bestuur volgens besluit van het College voor Promoties te verdedigen op donderdag 12 december 2019
om 15:00 uur
Door
Jeroen Maarten Bakker
Geboren te Amsterdam
Op 6 augustus 1985
Prof. Dr. J.J.C. Neefjes Co-Promotor:
Ass. Prof. Dr. I. Berlin Promotiecommissie:
Prof. Dr. P. ten Dijke Prof. Dr. J. Borst
Prof. Dr. A. Sonnenberg Prof. Dr. T.H.M. Ottenhoff Prof. Dr. C. Jalink
Universiteit van Amsterdam
Prof. Dr. W.H. Molenaar
Table of contents:
Scope of this thesis 6
Chapter 1: 7
An introduction to endocytosis.
Chapter 2: 21
The EGFR odyssey – from activation to destruction in space and time.
“Journal of Cell Science, Dec. 2017, 130 (24), p4087-4096f”
Chapter 3: 45
SKIP-HOPS recruits the GAP TBC1D15 to mediate Rab7-to-Arl8b identity switch for controlled transport of late endosomes.
“Manuscript in preparation”
Chapter 4: 93
The interplay between endocytosis and cell manipulation by pathogens.
Chapter 5: 103
Salmonella Manipulation of Host Signaling Pathways Provokes Cellular Transformation Associated with Gallbladder Carcinoma.
“Cell Host and Microbe, Jun. 2015, 17(6), p763-774”
Chapter 6: 127
Supplementary information - Salmonella Manipulation of Host Signaling Pathways Provokes Cellular Transformation Associated with Gallbladder Carcinoma.
“Cell Host and Microbe, Jun. 2015, 17(6), p763-774”
Chapter 7: 149
Summary and Discussion
Nederlandse Samenvatting 160
Curriculum Vitae 164
List of publications 165
Acknowledgment 167
endocytosis from 2 different angles. The first part of this thesis focusses on the molecular systems by which a cell controls positioning of intracellular vesicles and how this affects handling of their associated cargoes, while the second part describes the manipulation of the host cell by Salmonella bacteria and the potential harm this interference could do.
In chapter 1 I will introduce the general mechanisms behind endocytosis and the role these processes play in the life of a cell. To further elaborate these mechanisms chapter 2 uses the example of EGFR trafficking to showcase the multiple molecular pathways involved in receptor endocytosis. In addition to this, chapter 3 describes a novel regulatory system involved in a crucial event in endosomal maturation: GTPase exchange. In more detail, chapter 3 describes a handover system that exchanges the late endosomal Rab GTPase Rab7 upon activation of Arl8b, by recruitment of Rab7GAP through the HOPS complex. This exchange is important in the proper segregation of Arl8b associated late endosomes and the retrieval of its cargo from the Rab7-positive endosomal compartment.
In chapter 4 I will introduce the second part of this thesis. I will explain how pathogens use the molecular mechanisms of its host cell, including endosomal transport, to facilitate their own growth and survival. I will also highlight the dangers of these invasive mechanisms for the host by showing the role bacterial infections can play in host maladies, such as cancer. Chapter 5 and 6 show a first report of a causal link between salmonella and cancer. This section focusses on how salmonella’s usage of the host alters intracellular signaling events in pretransformed cells, with the potential to cause cancer. In vivo, ex vivo and in vitro systems are used to show the, non- immune related, oncogenic capacities of Salmonella and the molecular events that lie behind this effect.
Finally, in chapter 7 I will encapsulate all observations from this thesis with a summary and a discussion that places the findings of this thesis in perspective with the aim to provide deeper insights in the meaning of these results and how it can implicate further research on these topics.
An introduction to endocytosis
Chapter 1
An introduction to endocytosis
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In order to maintain homeostasis, the eukaryotic cell uses a multitude of ways to regulate its internal functions. Various organelles perform, sometimes cell-type specific, functions to facilitate processes such as cell growth, proliferation, differentiation, energy production and migration. A key process that connects the workings of all of these organelles is endocytosis (Schmid et al., 2014). During endocytosis cargo molecules are engulfed by a lipid bilayer membrane, forming an endosome, that then mediates transport of cargo in the cells. Through this system, the cell is able to take up molecules including activated receptors and nutrients from the plasma membrane, exchange cargo between organelles or introduce newly synthesized molecules for transport towards their site of function. An opposite mechanism, called exocytosis, also takes place where intracellular cargo is released in the surrounding space after vesicle fusion with the plasma membrane. By organizing transport, a major role of the endosomal system lies in the degradation of cargo molecules. Endocytosis results in a gradual acidification of organelles and the acquisition of hydrolytic enzymes. As a result, various cargo undergo degradation in the later compartments of the endocytic track, ending up in lysosomal compartments.
Through lysosomal degradation the cell is able to acquire building blocks for new protein, lipids and carbohydrate synthesis, acquire other essential cellular compounds, regulate the availability of nutrients during starvation, as well as control the activity of enzymes and proteins entering the endosomal system. In addition, the acidic lysosomes can help in the control of potentially harmful entities such as pathogens and may eliminate unnecessary or dysfunctional molecules and protein aggregates by the process of autophagy (Di Fiore and von Zastrow, 2014; Lim and Zoncu, 2016).
Altogether, the endosomal system functions as a highly diverse cellular system that consist of numerous vesicle subtypes, all of which work together to perform a wide and complex array of functions essential for the broad biology of cells.
The molecular players that regulate endocytosis
The endosomal system consists of numerous vesicles occupying various stages of maturation, which can be discriminated based on the different molecular landscapes present on their membranes. Most notable, at different stages of endosomal maturation, various members belonging to the Rab, Arf and Arf-like (Arl) families of small GTPases are associated with specific endosomal membranes (Stenmark, 2009;
Numrich and Ungermann, 2014). These GTPases are switchable molecules that alternate between an active GTP-bound and inactive GDP-bound state. Inactive GTPases are mainly cytosolic and without function. However, when activated, they become competent to associate with select endosomal membranes and recruit specific effector molecules to mediate key endosomal processes, including transport and fusion. In order to facilitate timely recruitment of GTPases to specific membranes, two types of molecules regulate the GTP/GDP switch. Guanine Exchange Factors (GEFs) facilitate the exchange of GDP for GTP, thereby activating the GTPases. In an opposing fashion, GTPase Activating Proteins (GAPs) promote hydrolysis of GTP to GDP, which renders the GTPase inactive and results in its release from membranes
An introduction to endocytosis
GTPases to spatially and temporally discrete segments of the endosomal system, these molecules play a vital role in regulation of endosomal transport, fusion and maturation (Stenmark, 2009).
After uptake from the plasma membrane, newly formed early endosomes (EE) become marked by the GTPase Rab5 (Woodman, 2000). At this stage of endocytosis, extensive recycling back towards the plasma membrane can occur (Goldenring, 2015).
By balancing internalization and recycling of proteins residing on the plasma membrane, the cell is able to regulate their surface levels and thus their interactions with the outside world. Later in the endocytic pathway, internalized vesicles undergo step-wise maturation initiated when these nascent endosomes fuse through the help of the tethering factor Early Endosome Antigen 1 (EEA1) (Simonsen et al., 1998). At this stage, the cell has to decide whether to recycle cargo or send it further down the endocytic route towards degradation in lysosomes. In the latter case, a new GTPase termed Rab7 is recruited to the maturing endosomal membrane through a GTPase hand-over mechanism, which at the same time removes Rab5, resulting in a Rab7 positive late endosome (LE) (Poteryaev et al., 2010; Nordmann et al., 2010; Huotari and Helenius, 2011).
Characteristic to the divide between the early and late endosomal populations is their cellular localization. Upon maturation, endosomes move away from the periphery of the cell and towards the perinuclear region through a process that relies on GTPase function (Wandinger-Ness and Zerial, 2014; Neefjes, 2017). When activated, GTPases interact with effectors that bind specialized motor proteins (Figure 1, box 1). These motors can ‘walk’ over the microtubule cytoskeleton, thereby dragging their associated vesicles along. In order to move late endosomes towards the perinuclear area, Rab7, in combination with its effector molecule Rab-interacting Lysosomal Protein (RILP), recruits the Dynein motor (Jordens et al., 2001). Since motor proteins can only move in one direction over the polar-organized microtubules, Rab7-RILP-Dynein activity directs vesicles towards a region called the Microtubule Organizing Center (MTOC), close to the nucleus. In order to move late endosomes in the opposite direction towards the plasma membrane, Rab7 is able to recruit a different effector called FYVE and coiled-coil domain containing 1 (FYCO1), which mobilizes Kinesin-1 (Pankiv et al., 2010). This member of the kinesin motor family transports organelles to the plus end of microtubules and therefore takes endosomes away from the MTOC towards the cell’s periphery. This enables transport of cargoes that pass through the late endosomal compartment on their way to the plasma membrane, as illustrated by the immune receptor Major Histocompatibility Complex II (MHCII). Here, in the acidic late endosomal environment, MHCII acquires peptides to be presented at the cell surface to the immune system. Once loaded with a cognate peptide, MHCII needs to be transported to the plasma membrane in order to instigate the immune response (Neefjes et al., 1990; Neefjes et al., 2011). Although transport events, such as described for MHCII, move cargo in a net outward direction, in reality, fast loading and unloading of transport complexes ensures endosomal transport to occur fast and in a bi-directional and stop-and-go fashion. This high mobility increases the chance of interactions between endosomes themselves and between endosomes and other
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Figure 1: The endocytic route in a nutshell
At the plasma membrane a plethora of cargo, including pathogens, nutrients and activated receptors, are targeted for internalization in the endocytic track. Upon internalization through
An introduction to endocytosis
organelles, allowing high levels of regulation and increased endosomal functioning (Bonifacino and Neefjes, 2017).
Deciding the fate of endocytosed cargo: degradation or recycling?
The final stage of minus end-directed cargo transport is its entry into the lysosomal compartment (Lys). Not only cargo retrieved from the plasma membrane, but also cytoplasmic material taken up through the process of autophagy is delivered to this compartment for degradation (Figure 1, box 4) (Luzio et al., 2007). In the first step towards degradation, membrane-bound molecules are internalized from the limiting endosomal membrane into intraluminal vesicles (ILVs) in a sequence of events mediated by proteins belonging to the endosomal sorting complexes required for transport (ESCRT) machinery (Henne et al., 2011). This process is initiated by the recognition of ubiquitin-tagged cargoes on the endosomal membrane by specific ESCRT molecules, resulting in the recruitment of the full ESCRT machinery complex.
This complex then initiates the uptake of membrane cargoes into ILVs, resulting into the formation of so-called Multi-vesicular Bodies (MVBs). This process halts protein Once removed from the cytoplasm and taken up in the LE/Lys compartment, cargo is (continue from previous page)
membrane budding and scission from the membrane the cargo enters the early endosomal compartment (EE). At this stage, extensive recycling back to the plasma membrane takes place. When not selected for fast recycling, cargo can be sent further down the endosomal pathway where maturation results in the formation of the late endosomal (LE) and lysosomal (lys) compartments. Upon maturation, endosomes move away from the peripheral region where EEs reside towards the perinuclear region. In order to move across the microtubule cytoskeleton, endosomes recruit effector molecules through their activated GTPases on the cytosolic site of the vesicular membrane. Effector molecules subsequently recruit various proteins including motor proteins, which allow the endosomes to move along actin or microtubules (box 1). A net recruitment of inward directed transport complexes results in the formation of the perinuclear cloud of late endosomes close to the microtubule organizing center (MTOC). Although inward transport moves endosomes and their cargo in the direction of lysosomal degradation, cargoes can be extracted from the endosomes for recycling. In order to avoid further degradative processing, newly formed vesicles are loaded with other regulatory protein complexes. New GTPases are recruited sometimes at the cost of existing GTPases (box 2). These newly formed vesicles are able to recycle cargo back towards the plasma membrane and avoid endosomal regulation that primes them towards degradation.
During endosomal maturation, the number of contacts with other endosomes increases, which further stimulates maturation through endosomal tethering and fusion. Also, contacts with other cellular organelles occurs, most notable the endoplasmic reticulum (ER). The ER membrane harbors many regulatory proteins that control positioning and movement of LEs. Through the Rab7 associated cholesterol sensor ORP1L ER-contact sites are formed dependent on the levels of cholesterol in the endosomal membrane (box 3). When levels of cholesterol are high, the sensor domain of ORP1L binds to cholesterol, resulting in a protein formation that allows recruitment of the transport complexes. When levels of cholesterol are low, the conformation of ORP1L changes, which allows its FFAT domain to interact with the ER resident molecule VAP-A. The resulting ER tethering stops endosomal movement and allows for ER mediated endosomal regulation.
Finally, endosomal cargo reaches the lysosomal compartment (box 4). Due to import of protons, the endosomes acidify. This promotes the activity of endosomal degradative enzymes, which break down endosomal cargo, as a final step in endosomal transport.
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set for degradation. Through proton pumps and chloride ion channels, protons (H+) are imported inside the LE/Lys lumen. The resulting low pH (4,5-5,0) is ideal for activation of lysosomal enzymes responsible for proteolysis (Hu et al., 2015). Following degradation, cells either clear themselves from the remnants or use them as crucial nutrients for cell maintenance during starvation.
Not all molecules entering the endosomal system are targeted for degradation. These obviously include the lysosomal enzymes. To avoid degradation through bulk flow towards the lysosomal compartment, cells possess mechanisms to salvage specific cargoes throughout the endocytic pathway through a process generally termed recycling (Figure 1, box 2) (Taguchi, 2013; Goldenring, 2015). To this end, selected cargoes are sorted inside vesicles budding from the maturing endosome. These buds or tubules recruit transport motor machinery for transport away from the mother endosome, often towards the cell periphery and/or the plasma membrane. In order to avoid the Rab5-to-Rab7 conversion poised towards degradation, recycling GTPases must take over. During the early stages of endocytosis extensive recycling takes place through recruitment of Rab11 and Rab35 amongst others (Ullrich et al., 1996;
Chaineau et al., 2013). However, recycling GTPases, such as ADP-ribosylation factor- like protein 8b (Arl8b), can mediate cargo transport away from the LE/Ly compartment and thereby provide the possibility to salvage materials just prior to the final stage of endocytosis (Hofmann and Munro 2006; Rosa-Ferreira and Munro, 2011). How acquisition of the LE/Lys recycling GTPase Arl8b occurs, along with the concomitant removal of the previous GTPase Rab7 is described in Chapter 3 of this thesis.
Lipids: intrinsic timers of endocytosis
In order to regulate all steps of endocytosis, it is important for cells to control the molecular landscape on endosomal membranes. This is accomplished through recruitment of GTPases, and by extension their effector molecules, at the right place and time along the endocytic route. Crucial to this regulation is the lipid content of the endosomal membrane. The membrane itself is formed by a bilayer of phospholipids, which consist of a hydrophilic ‘head’ and one or two hydrophobic alkyl ‘tails’. During endosomal maturation, the lipid content of this membrane bilayer continuously changes and functions as a molecular ‘clock’ to determine which complexes will be preferentially recruited at any given time. A key membrane lipid involved in this temporal control is phosphatidylinositol (PI) (Jean and Kiger, 2012; Schink et al., 2016).
This molecule can be phosphorylated by PI kinases on one -or multiple- of the 3-, 4- and 5-hydroxyl groups of the inositol head group, resulting in 7 possible phosphorylation combinations. These PI forms are present at different stages of the endosomal track, where they mediate direct and indirect recruitment of key proteins to endosomal membranes. Exchange of the PI content in the membrane provides the basis for the cascade of regulatory proteins involved in membrane dynamics throughout endocytosis. For example, early in endocytosis PtdIns(4,5)P2 promotes vesicle internalization through direct recruitment of the µ2 subunit of the AP2 adaptor complex, which mobilizes the clathrin coat for clathrin-mediated endocytosis (Rohde
An introduction to endocytosis
plasma membrane, PtdIns(4,5)P2 is dephosphorylated to PtdIns4P, resulting in the release of AP2 and uncoating of the vesicle (Cremona et al., 1999). Further down the endocytic pathway, rising levels of PtdIns3P on the endosomal membrane attract factors that instigate recruitment of Rab7 and subsequent removal of Rab5, effectively targeting endosomes for inward transport and increasing maturation (Zoncu et al., 2009; Poteryaev et al., 2010). Changing PI content on the membrane has also been found to facilitate other endosomal processes, such as recycling and exocytosis, by targeting specific protein machinery to the appropriate endosomal membranes at the right time (Schink et al., 2016).
In addition to PI, cholesterol (another membrane lipid) is also involved in the regulation of endosomal trafficking. Through the Rab7-associated cholesterol sensor Oxysterol- Binding Protein-Related Protein 1L (ORP1L), late endosomes are able to respond to varying levels of cholesterol on endosomes (Figure 1, box 3). When cholesterol levels are high, the sensory ORD domain in ORP1L recognizes and binds to cholesterol in the endosomal membrane. The resulting configuration allows Rab7 to attract the machinery for minus-end microtubule-based transport consisting of the multi-subunit Homotypic fusion and vacuole Protein sorting (HOPS) tethering complex and the
p150glued subunit of the dynein motor (van der Kant., 2013). Conversely, in the absence
of cholesterol, the ORD domain remains untethered to the endosomal membrane, exposing its FFAT motif to bind VAP tethering molecules on the endoplasmic reticulum (ER). This leads to the release of the minus end-directed transport machinery and formation of a membrane contact site (MCS) between the endosome and ER (Rocha et al., 2009; van der Kant et al., 2013; Wijdeven et al, 2016). Due to increasing levels of cholesterol during endosomal maturation, ORP1L functions as a timer for endosomal transport and fusion towards the endo-lysosomal system. Hypothetically, this also results in the retention of low cholesterol, ER tethered vesicles and their cargo keeping them available for further regulation and potential recycling. Recent advancements in our understanding of the interactions between endosomes and the ER have shown that the ER plays a much larger role in regulating endosomal transport than previously thought. Importantly, the positioning within the cell dictates the behavior of endosomes. While the peripheral pool of endosomes is highly mobile, the endosomal ‘cloud’ located closer to the nucleus is far more stationary, a situation which favors tethering, fusion and maturation. The ER plays a pivotal role in keeping this organization in place. By regulating the ubiquitination landscape on the late endosome through the ER-located ubiquitin ligase RNF26, the ER delegates retention, but also the recruitment of multiple regulatory transport complexes towards the endosomal membrane (Jongsma et al, 2016; Neefjes et al., 2017). Combined, this indicates that based on lipid content, especially by cholesterol, endosomal positioning, fusion and movement are placed under control of regulatory systems such as the ER.
Endocytosis and disease
Endocytosis plays a critical role in many vital cellular processes, ranging from protein homeostasis and control of nutrient availability to regulation of signaling cascades.
Consequently, failure to regulate endocytosis could result in numerous diseases
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associated with these processes (Maxfield, 2014). Degradation of cargo molecules is an important function of the endocytic pathway, and when flaws occur in this process, stacking of unwanted proteins or (glyco)lipids can take place in the lysosomal compartment. The resulting lysosomal storage diseases can have a severe impact on the health of the cell (Ferreira and Gahl, 2017). This is best exemplified by the diseases associated with erroneous processing of cholesterol, in itself a regulator of endocytosis. In mammalian cells, intracellular cholesterol can be derived either through synthesis in the ER or uptake from LDL/HDL particles via Low-Density Lipoprotein (LDL) receptors (Goldstein and Brown, 2009). Due to increasing levels of fat and cholesterol in the Western diet, endosomal biology is also affected by large amounts of cholesterol. Various diseases affecting endosomal lipid handling (including Gaucher and Niemann Pick disease) all result in neurological diseases and early mortality.
Increased cholesterol levels and their associated cardiovascular disease, make the failure to maintain a healthy cholesterol-balance one of the leading causes of death worldwide (Röhrl and Stangl, 2013; Maxfield, 2014).
Intracellular handling of cholesterol is affected not only through imbalanced dietary intake, but can also be deregulated in genetic diseases. An extensively studied example of such lysosomal storage diseases is Niemann Pick disease C. Luminal endosomal proteins Niemann Pick disease protein 1 and 2 (NPC1 and NPC2) work together to transfer the cholesterol residing in the LE to the ER. Mutations in NPC1 and NPC2 genes then results in elevated cholesterol storage in the lysosomal compartment. This phenotype clinically manifests itself through a wide spectrum of symptoms, depending on the cell types and tissues affected (Xu et al., 2007;
Gelsthorpe et al., 2008; Neefjes and van der Kant, 2014).
Although cholesterol is not able to pass the blood-brain barrier, nerve cells can also succumb to endocytosis related diseases (Perret et al., 2015; Solé-Domènech et al., 2016). One major example is Alzheimer’s disease (AD), which is caused by plaque formation upon mishandling of (amongst others) the Amyloid Precursor Protein (APP), resulting in neurodegeneration and loss of nerve functioning and viability (Hu et al., 2015). Newly synthesized APP passes through the ER and Golgi and is transported through the endosomal system to the plasma membrane where it is reportedly involved in synapse formation and stability (Montagna et al., 2017). At various stages of endocytosis and at the plasma membrane, specific secretases cleave APP, generating Ab peptides that are prone to aggregation and constitute the main component of AD- related plaques (Toh and Gleeson, 2016). Recent studies have shown the role the endosomal system in the handling of APP and its contribution to Alzheimer’s disease (Woodruff et al., 2016; Kimura and Yanagisawa, 2017).
Besides affecting storage and processing related processes, defects in endocytosis have also been extensively reported in oncogenesis (Mellman and Yarden, 2013). Like healthy cells, tumor cells rely on the endosomal system to maintain homeostasis.
Additionally, tumor cells require a functional degradation system, which enables them to cope with stress and nutrient starvation through the process of autophagy (Mathew et al., 2007). Besides its function in cell maintenance, endocytosis also plays a more
An introduction to endocytosis
turnover of signaling receptors. In order to keep proliferative signaling in check, activated receptors are endocytosed and targeted for lysosomal degradation to allow temporal signaling. The balance between receptor activation and endosomal degradation tunes the cellular response to proliferating stimuli. When this balance shifts away from the latter, oncogenic signaling pathways, including RAS, mitogen- activated protein kinase (MAPK), mammalian target of rapamycin (mTOR) and epidermal growth factor receptor (EGFR), can be impacted to stimulate tumor formation through increased growth and differentiation signaling (Sorkin and von Zastrow, 2009; Schmid, 2017; Bakker et al., 2017).
Concluding remarks
The endocytic system is a highly complex and dynamic system that allows the cells to regulate a wide variety of processes. By transport of cargo, it affects the activity of catalytic molecules or mediates their degradation. It also allows the cells to respond to extracellular cues through handling of activated receptors and to cope with stress and nutrient deprivation. Although the molecular players are known of many endosomal processes, how these systems are activated at the right place and time is poorly understood. Chapter 2 of this thesis will further dissect the function of the endocytic pathway in relation to the EGFR, the primary transmembrane tyrosine kinase signaling molecule that is processed by endosomal uptake after ligand binding to terminate signaling. In Chapter 3, I will discuss in detail a newly identified mechanism for cargo retrieval from the LE/Lys compartment for recycling to the periphery. These chapters illustrate the complex temporal and spatial control of endosomal processes in cells.
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The EGFR odyssey – from activation to destruction in space and time
Chapter 2
The EGFR odyssey – from activation to destruction in space and time
Jeroen Bakker*, Menno Spits*, Jacques Neefjes and Ilana Berlin‡
Department of Chemical Biology, Leiden University Medical Center LUMC, Einthovenweg 22, 2333 ZC, Leiden, The Netherlands.
* These authors contributed equally to this work
‡ Corresponding author, email: i.berlin@lumc.nl Journal of Cell Science, Dec. 2017, 130 (24), p4087-4096f
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Abstract
When cell surface receptors engage their cognate ligands in the extracellular space, they become competent to transmit potent signals to the inside of the cell, thereby instigating growth, differentiation, motility and many other processes. In order to control these signals, activated receptors are endocytosed and thoroughly curated by the endosomal network of intracellular vesicles and proteolytic organelles. In this Review, we follow the epidermal growth factor (EGF) receptor (EGFR) from ligand engagement, through its voyage on endosomes and, ultimately, to its destruction in the lysosome.
We focus on the spatial and temporal considerations underlying the molecular decisions that govern this complex journey and discuss how additional cellular organelles – particularly the ER – play active roles in the regulation of receptor lifespan.
In summarizing the functions of relevant molecules on the endosomes and the ER, we cover the order of molecular events in receptor activation, trafficking and downregulation, and provide an overview of how signaling is controlled at the interface between these organelles.
Introduction
Multicellular life necessitates communication between distantly located cells in a manner that is straightforward to initiate, decode and act upon. To serve these universal needs, cell surface receptors have evolved to recognize and respond to environmental cues with exquisite specificity and precision. In mammalian cells, some of the most vital cellular signaling pathways, including proliferation and differentiation, fall under the purview of growth factor receptors. Imbedded in the plasma membrane, these proteins extend ligand-interacting sensory platforms into the extracellular space and receptor tyrosine kinase (RTK) response modules into the cytosol. This arrangement couples environmental inputs received via growth factor binding to signaling cascades transduced inside the cell upon kinase activation. Because stimulatory ligands for these receptors are produced at a distance, their activation is inducible on demand. Crucially, once the receptors become turned ‘on’, their signals must be terminated in order for cells to regain equilibrium and maintain responsiveness to future inputs. This balance between activation and downregulation is managed largely by the uptake of receptors from the cell surface into the vesicular network of the endocytic pathway, where timing and directionality of transport modulate signal duration and determine receptor fate. Adding further complexity to the matter, receptors such as epidermal growth factor (EGF) receptor (EGFR) signal not only at the cell surface, where ligand engagement occurs, but continue signaling on endosomes for a comparable period (Haugh et al., 1999; Leonard et al., 2008; Foley et al., 2012; Francavilla et al., 2016). EGFR has also been reported to localize to the nucleus, where it is suggested to function as a transcription factor that is associated with cancer disease progression (Kamio et al., 1990; Brand et al., 2013). From ligand encounters to receptor degradation in the lysosome, in this Review, we discuss how EGFR navigates the endosomal system, toggling its signaling switch in cellular space and time.
The EGFR odyssey – from activation to destruction in space and time
What happens at the cell surface (doesn’t always stay there) EGFR – the model RTK
EGFR is the first identified member of the receptor tyrosine kinase (RTK) family (Burgess et al., 2003; Bublil and Yarden, 2007) and, in accordance with its plethora of functions, is expressed on the surface of numerous cell types (Chen et al., 2016).
When in its active or ‘on’ state, EGFR transduces signals to the cell interior that instigate key processes of life, such as growth, differentiation, proliferation and motility (Ceresa and Peterson, 2014; Li et al., 2017). Given these profound effects, the association of EGFR with cancer is self-evident and exemplified by the vast number of studies that link deregulated expression and degradation of EGFR, as well as its activating mutations, with transformation (Shan et al., 2012; Tomas et al., 2014).
Because many of the basic principles of EGFR biology are shared by its lesser-studied family members and beyond, EGFR represents the model growth factor RTK.
Activate me
EGFR can be activated by a number of ligands, of which EGF is most extensively studied (Cohen, 1962; Cohen and Carpenter, 1975; Harris et al., 2003; Singh et al., 2016). These ligands are produced as transmembrane precursors whose juxtacrine, paracrine and/or endocrine origins vary depending on the biological cues that instigate activity of EGFR. Typically, EGF production is locally controlled, as opposed to being delivered systemically, such as in the case of hormones, which makes it possible for different organs to conduct their own EGF-mediated programs (Singh and Harris, 2005; Conte and Sigismund, 2016). Once released into the extracellular milieu, EGF and related ligands begin the search for their cognate receptors, thereby setting in motion cellular programs of survival and growth (Massague and Pandiella, 1993; Sahin et al., 2004; Li et al., 2015; Chen et al., 2017). Specificity of EGFR activation is mediated through the establishment of defined contacts between the ligand and the binding groove of the receptor located on its extracellular face (Bajaj et al., 1987; Lax et al., 1988; Ferguson et al., 2003; Jorissen et al., 2003; Zhu et al., 2017). Variations in sidechain features between different ligands, as well as post-translational modifications present on the extracellular EGFR domain, determine the strength of engagement (Azimzadeh Irani et al., 2017). Solid-state nuclear magnetic resonance (NMR) experiments have demonstrated that, in the absence of ligand, the intracellular region of EGFR exists in a rigid conformation, while the extracellular domain remains highly dynamic. Ligand binding sharply restricts this flexibility, providing a stable platform for ligand mediated dimerization – a key event in receptor activation and initiation of downstream signaling (Ogiso et al., 2002; Kaplan et al., 2016). Within the receptor dimer, rotation of the transmembrane segment transduces a conformational change to the intracellular kinase domains, resulting in their asymmetric positioning, which in turn promotes cross-phosphorylation of cytoplasmic receptor tails (Honegger et al., 1989; Moriki et al., 2001; Kourouniotis et al., 2016; Purba et al., 2017).
Depending on the type and degree of phosphorylation, the latter can now recruit specific signaling complexes and thus have the potential to initiate a wide variety of
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downstream signaling cascades associated with EGF-dependent responses (Foley et al., 2012; Wagner et al., 2013; Ceresa and Peterson, 2014; Li et al., 2017).
Ligand or not
In the absence of ligand, most EGFR molecules remain in their monomeric form and are therefore inactive. However, because the arrival of any external signals is difficult to anticipate, EGFR has evolved to be intrinsically poised towards the ‘on’ state, occasionally giving rise to auto-activation (Ferguson et al., 2003; Burgess et al., 2003;
Ceresa and Peterson, 2014). Therefore, while maintaining acute responsiveness to ligands, cells must also guard themselves against aberrant or excessive activation of EGFR. These needs are accommodated through continuous surface sampling and the differential intracellular routing of receptors (Fig. 1). Although inactive receptors continuously travel through the endocytic compartment (Fig. 1, step 1+route 1), slow internalization and rapid recycling rates ensure their accumulation on the cell surface.
Upon ligand binding (Fig. 1, step 2), this equilibrium shifts rapidly (Herbst et al., 1994;
Burke and Wiley, 1999; Wiley, 2003; Ceresa and Peterson, 2014; Tomas et al., 2014), causing activated receptors to spend extended periods of time traveling the endocytic route (Fig. 1, step 3+route 3). In this case, signaling continues until receptors are either recycled back to the cell surface or taken up into proteolytic lysosomes, leading to their demise. Understanding how cells control the duration of legitimate ligand-mediated responses, while keeping unwarranted activation at bay in many ways encompasses the crux of signaling pathways. It appears that cells have taken the ‘divide and conquer’
approach to solving this problem by segregating the receptor ‘on’ and ‘off’ states in cellular space and time. How this is orchestrated to afford proper regulation of EGFR lifespan is discussed in the following sections.
Receptor endocytosis and the peripheral–perinuclear divide Endosomes – signaling hubs or traps for destruction?
Although key steps in ligand engagement and nucleation of signaling cascades take place at the cell surface, once activated, EGFR molecules actually spend most of their remaining lifetime in the cell interior, traversing the vesicular network of the endosomal system. Under conditions of low ligand availability, activated EGFR is typically subjected to clathrin-mediated endocytosis (CME) (Sigismund et al., 2005; Robinson, 2015). Although inactive EGFR can move into preformed clathrin-coated pits, phosphorylated receptor accelerates CME by attracting the adaptor protein 2 complex (AP-2) (Rappoport and Simon, 2009), which in turn recruits large amounts of clathrin, resulting in receptor clustering and rapid expansion of the budding vesicle (Sorkin et al., 1996; Tomas et al., 2014; Robinson, 2015). Accumulation of receptors in the bud further enhances cross-phosphorylation initiated by ligand binding (Ibach et al., 2015), thereby amplifying low-intensity signals. At the same time, phosphorylation of the β2 subunit of AP-2 by EGFR helps to initiate internalization, directing EGFR into the endocytic pathway (Fingerhut et al., 2001; Huang et al., 2003; Traub, 2009). The resulting endosomes dwell in the peripheral cytoplasm (Fig. 1, step 3A); here,
The EGFR odyssey – from activation to destruction in space and time
Fig. 1.
Destinations of activated EGFR – from the cell periphery to the perinuclear ‘cloud’.
Ligand-free monomers of EGFR, residing primarily on the cell membrane (1a), can be spontaneously internalized and recycled (Route 1). Even in the absence of stimulation, stochastic dimerization (1b) and auto-activation (1c) of EGFR may occur. The latter is kept in check by endocytosis, inactivation and recycling through the Rab11 recycling endosomes (REs) (Route 1). Ligand binding promotes receptor dimerization (2a, 2b), leading to activation and phosphorylation of the cytoplasmic tails (2b) that mediate recruitment of various adaptor proteins (such as Grb2) (3a, 3b) for downstream signal transduction cascades. The intracellular fate of EGFR depends on the extent of its activation. Under conditions of ‘low’
(legend continues on next page)
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are available to spare receptors from the degradation (Watanabe and Boucrot, 2017) that takes place in the perinuclear region of the cell, where proteolytic lysosomes abound (Johnson et al., 2016). While, at first, EGFR was considered to predominantly transduce signals at the plasma membrane, recent studies have shown that receptor endocytosis does not interfere with its signaling capabilities (Vieira et al., 1996; Sousa et al., 2012; Conte and Sigismund, 2016). Interestingly, it appears that for certain signaling pathways, such as activation of ERK1/2 proteins (also known as MAPK3 and MAPK1, respectively) downstream of EGFR, intracellular localization of signal transduction (i.e. at the plasma membrane versus on endosomes) correlates to the resulting transcriptional response (Sousa et al., 2012; Wu et al., 2012). In this way, spatial compartmentalization of signaling complexes fine-tunes their biological outcomes.
Fast and furious with ubiquitin
When the canonical endocytic route described above is saturated owing to increasing abundance of ligand, ‘fast’ clathrin independent endocytosis (CIE) can take over, rapidly routing receptors toward degradation (Sigismund et al., 2005) (Fig. 1, step 3B).
The decision to rapidly traffic endosomes carrying activated EGFR for degradation appears to be triggered by receptor ubiquitylation, as ubiquitylation-impaired EGFR overwhelmingly travels through the recycling-promoting CME route (Sigismund et al., 2005). Ubiquitylation of EGFR is mediated by the E3 ubiquitin ligase Cbl (Huang et al., 2006), which is targeted to the phosphorylated EGFR by the adaptor growth factor receptor-bound protein 2 (Grb2) (Batzer et al., 1994; Levkowitz et al., 1999; Jiang et al., 2003). Once ubiquitylated, EGFR can be recognized by the ubiquitin-dependent adaptors of the endosomal sorting complexes required for transport (ESCRTs) and sequestered into the intraluminal vesicles (ILVs) of the multivesicular body (MVB) (Henne et al., 2011). This physically removes the signaling tail of EGFR from the cytosol, effectively terminating the downstream signaling cascade (Eden et al., 2009).
Receptor ubiquitylation exhibits a sigmoidal response to increasing concentrations of EGF, ensuring that under conditions of low ligand availability, activated EGFR will not be marked for destruction (Sigismund et al., 2013). Precisely what sets up this barrier to degradation is not entirely clear. One suggested mechanism postulates that high (continue from previous page)
stimulation, the AP-2 adaptor is recruited for clathrin-mediated endocytosis (CME) (3a), resulting in EGFR-containing early Rab5-positive signaling endosomes (3a, 3b). As these endosomes mature, they travel to the perinuclear region, where ligand-activated (and auto- activated) EGFR encounters increasing phosphatase activity and is inactivated prior to being recycled (Route 2). By contrast, ‘high’ levels of EGFR activation result in extensive receptor phosphorylation and ubiquitylation by the E3 ligase Cbl, which causes diversion of EGFR, which in this case is preferentially internalized by clathrin-independent endocytosis (CIE), away from recycling and towards degradation in the lysosome (Ly) located in the perinuclear ‘cloud’.
This occurs via the Rab7-positive late endosome (LE), where ubiquitylated EGFR is directed from the limiting endosomal membrane into intraluminal vesicles (ILVs), giving rise to a multivesicular body (MVB) (Route 3). Subsequent late endosome–lysosome fusion delivers EGFR for degradation.
The EGFR odyssey – from activation to destruction in space and time
levels of receptor phosphorylation trigger simultaneous recruitment of Grb2 and Cbl2, resulting in efficient ubiquitylation (Sigismund et al., 2013). It is thought that a productive association of Cbl with the receptor is achieved above a certain threshold of phosphorylation, which couples ubiquitylation to the intensity of ligand-induced stimulus. In contrast, lower levels of stimulus offer fewer phosphorylated binding sites that are preferentially occupied by signaling molecules, such as Ras and phospholipase C (PLC)γ (Chardin et al., 1993; Haugh et al., 1999; Henriksen et al., 2013; Sigismund et al., 2013; Tomas et al., 2014). Thus, by segregating peripheral signaling and recycling pathways from perinuclear degradation in accordance with the degree of stimulation, cells can maximize life sustaining inputs and effectively cope with overstimulation.
Recycling goes deep
Receptors that are only moderately activated, either owing to low ligand availability or because they are activated in a ligand independent manner, are still internalized into endosomes, but their reduced signaling potential does not require degradation. Upon entry into the early endosomal compartment, these receptors are recycled in vesicles characterized by the presence of the GTPase Rab11 (Ullrich et al., 1996; Baumdick et al., 2015). This pathway takes receptors through the perinuclear region, where they become increasingly exposed to the tyrosine-protein phosphatase nonreceptor 1 (PTP1B; also known as PTPN1) that resides at the ER. PTP1B dephosphorylates EGFR at ER–endosome contact sites, ensuring that receptors transported back to the plasma membrane are no longer active. This mode of regulation results in an inverse spatial relationship between cellular kinase (peripheral) and phosphatase (perinuclear) activities (Fig. 1), which are facilitated by the interactions between endosomes and the ER (as discussed below). In contrast, fully activated EGFR molecules are redirected away from recycling vesicles and traffic toward the late endosomal compartment for degradation (Sabet et al., 2015). Prior to their degradation, these molecules also encounter the ER-associated phosphatase PTP1B (Eden et al., 2012b), which disables further signaling downstream. Additionally, in response to the intensity of incoming signals, the cell varies the number of signaling vesicles, which helps to maintain a relatively consistent amount of activated EGFR molecules per endosome (Villasenor et al., 2015). This, in turn, keeps the dephosphorylation rate constant and enables the cell to maintain robust responses to the dynamic extracellular environment without becoming vulnerable to overstimulation. The existence of multiple regulated means to abrogate signaling responses (i.e. dephosphorylation and degradation) underscores both the flexibility and rigor of the systems that function to keep cellular signaling cascades in check. Moreover, this complex regulatory framework exemplifies how spatiotemporal regulatory capabilities of the endocytic compartment elegantly serve the greater interests of the cell. How the trafficking and transport of EGFR is orchestrated in molecular terms is discussed in the next section.
