The interplay between clathrin-coated vesicles and cell signalling

Seminars in Cell & Developmental Biology 18 (2007) 459–470

Review

The interplay between clathrin-coated vesicles and cell signalling Ian G. Mills ∗ Cancer Research UK, Cambridge Research Institute, Robinson Way, Cambridge CB2 ORE, UK Available online 6 July 2007

Abstract Internalization of cargo proteins and lipids at the cell surface occurs in both a constitutive and signal-regulated manner through clathrin-mediated and other endocytic pathways. Clathrin-coated vesicle formation is a principal uptake route in response to signalling events. Protein–lipid and protein–protein interactions control both the targeting of signalling molecules and their binding partners to membrane compartments and the assembly of clathrin coats. An emerging aspect of membrane trafficking research is now addressing how signalling cascades and vesicle coat assembly and subsequently disassembly are integrated. © 2007 Elsevier Ltd. All rights reserved. Keywords: Clathrin; Kinase; Endocytosis; Ubiquitin; Signalling

Contents 1.

2.

3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Defining clathrin-coated vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Structure and function and adaptor proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Cargo selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1. Motif-mediated endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2. Signal-mediated endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CCV formation in response to receptor tyrosine kinase (RTK) signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. EGFR sorting and clathrin-mediated endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Post-translational modifications to endocytic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell cycle control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocytic proteins with alternate signalling functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Defining clathrin-coated vesicles Endocytosis is characterized by the internalization of molecules from the cell surface into intracellular membrane compartments. Trafficking can be divided into two main pathways—the classic, clathrin-mediated endocytic pathway

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and the non-classic, clathrin-independent route which is often lipid-raft dependent [1–5]. Clathrin-coated vesicles are named after the protein that self-polymerises into a lattice around these vesicles as they bud from the major sites of formation within the cell: the plasma membrane, trans-Golgi network (TGN) and endosomes [6–8]. Clathrin is a central organiser which concentrates cargo adaptors and this produces a diverse protein and lipid load in the nascent vesicle. In addition clathrin polymerisation produces a curved lattice which stabilises the membrane bud as it forms [9,10]. Structurally, each clathrin molecule consists of an N-terminal domain with a ␤-propeller structure and binds to peptide motifs between the blades of this propeller

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[11]. Multiple blades of the propeller allow for a wide range of protein interactions with varying specificities. This creates the potential for a versatile array of cargo adaptors and membrane attachment proteins to assemble within the coat [12–17]. The main cargo adaptor proteins characterised to date are the classical adaptor protein (AP) complexes, AP1, AP2, AP3 and AP4 [18–22]. Most AP complexes adapt and/or link clathrin to selected membrane cargo and lipids, and they also bind accessory proteins that regulate coat assembly and disassembly, including AP180, epsins and auxilin [23]. ‘Alternative adaptors’ can also function in clathrin-mediated vesicle budding. Most adaptors seem to be associated with budding from the plasma membrane. This is possibly due to the repertoire of cargo and the range of constitutive and regulated/signalling events occurring at this surface. A critical question for researchers is to what extent is there a redundancy in the repertoire of adaptors involved in CCV formation at the plasma membrane. RNAi knockdown of the AP-2 adaptor subunits ␣ and ␮2 significantly inhibited endoctyosis of the transferrin receptor whereas endocytosis of both EGF receptors and chimeric CD4/LDL receptors were sustained in AP2-depleted cells [24]. This line of evidence implies that for the constitutive clathrin-mediated uptake of receptors, such as the transferrin receptor, there may be greater dependency on the ‘core’ AP2 adaptor and that for the regulated or signal-responsive uptake of other classes of receptor the ‘alternative’ or ‘monomeric’ adaptors may play a more significant part. Arrestins, epsins, disabled-2 and ARH (autosomal recessive hypercholesterolemia protein) can also be classified as alternative adaptors as they also link cargo and membranes to the clathrin lattice [16,17,23,25,26]. Since multiple adaptors are found in individual coated pits, it is not necessary for all adaptors to have a direct clathrin interaction. Alternative adaptors also function at other sites of clathrin-coated vesicle formation elsewhere within the cell and these include the GGA adaptors (Golgi-localized, ␥-ear containing, ADP-ribosylation factor-binding proteins) which are associated with TGN clathrin coats and bind cargo, membranes, clathrin and accessory factors [27]. During the course of this review I will focus primarily on what is known about the structure, function and regulation of AP adaptors and accessory adaptor proteins in the context of endoctyosis. 1.2. Structure and function and adaptor proteins The large subunits of adaptor complexes have domains called ears or appendages. As shown by electron microscopy these domains are attached to the main trunk of the AP complex by a flexible linker known as a hinge. The first appendage structure to be solved was from the AP2 ␣ subunit [28,29]. It contained two subdomains, a platform with ␣-helical and ␤-sheet content and a support ␤-sandwich subdomain. These two subdomains are present in most appendage domains, whereas ␥-adaptin and the structurally related GGA adaptors have only the ␤-sandwich subdomain [30,31]. At first it was thought that the platform subdomain contained the only ligand-binding site, but ligand binding to both the ␤ sandwich subdomains of ␥ and GGA appendages has prompted a re-evaluation of the possibility that

each subdomain has a distinct binding site for ligands. This has led to the structural resolution of binding sites on both the platform domain and a ‘side’ site for the beta appendage domain of the AP2 adaptor complex [32]. Clathrin can also bind to the beta appendage domain. This has led to a model in which accessory proteins can be displaced as the coated pit matures [32,33]. In cases where we know the functions of the appendage domain binding partners we can say that they are involved in congregating the proteins necessary to polymerise, invaginate and promote fission of the nascent vesicle [23]. For example, AP2 complexes bind to epsin1, which is involved in driving membrane bending in addition to promoting the polymerisation of clathrin [16]. The AP1 appendage domains bind a homologue of this protein, epsinR, which also binds clathrin and membranes but has a different lipid specificity, consistent with the different membrane localization [34]. AP appendage domains also recruit synaptojanin1, a lipid phosphatase that will deposphorylate the lipids in the coated vesicle to which many of the coat attachment proteins bind [14,35]. This should make way for coat disassembly. Proteins that interact with the appendage domains do so by short peptide motifs found in regions lacking tertiary structure, which we call ‘motif domains’. Appendage binding motifs in these domains are often found in multiple copies, especially where they have a low appendage affinity. Thus there are eight DPW motifs (required for AP2 appendage interaction) in the motif domain of epsin1. The unstructured nature and thus greater fluidity of this domain increases the chance of an individual motif finding its interaction partner. The multiple copies of motifs and their low affinity mean that these binding partners have higher avidity for appendage domains when concentrated by clathrin polymerisation. When the clathrin cage is disassembled and the adaptors dispersed, these low affinity interactions will readily fall apart. 1.3. Cargo selection 1.3.1. Motif-mediated endocytosis Clathrin coats are versatile in their cargo selection and recruitment. Classical adaptors in clathrin-coated vesicles have many ways to recognise cargo. The ␮-subunit is specialised to recognise Yxx motifs and each AP complex has a different ␮-subunit [36]. Interestingly the crystal structure of the core of the AP2 adaptor found that the putative Yxx binding site was obstructed by the adjacent ␤2 subunit [37]. Based on this phosphorylation of the ␮2 subunit together with binding to PI(4,5)P2 has been proposed to be required to release the block on the Yxx binding site and allow cargo binding [38,39]. This is one example of the regulatory effects of kinases on AP2 recruitment to membranes, cargo selection and AP2-clathrin coassembly which will be further described later. It implies that even constitutive endoctyosis, based on the presence of an appropriate amino acid motif in the cytoplasmic tail of a receptor, may in fact be subject to regulation. Transferrin and LDL receptors do not appear to compete for sorting into coated pits and one role of alternative adaptors (e.g. arrestins, Disabled-2 (Dab2), GGAs and hepatocyte

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growth factor-regulated tyrosine kinase receptor substrate (Hrs)) appears to be as receptor-specific components of the sorting machinery. For example an alternative sorting motif, FxNPxY, found in the LDL receptor family is recognised by Disabled-2 (Dab2), which also binds to AP2 and can mediate clathrin assembly. A role for these alternative adaptors in regulating receptor uptake has however proved difficult to establish. Other classes of signalling receptors including receptor tyrosine kinases (RTKs) and G protein-coupled receptors have ‘customised’ their uptake mechanisms. 1.3.2. Signal-mediated endocytosis There are at least 10 binding partners of the AP2 complex, six for the AP1 ␥-appendage, one for the AP3 complex. This diversity may reflect additional levels of regulation affecting CCV formation from different cellular membranes. Clathrinmediated endocytosis, as a part of synaptic vesicle recycling in brain extracts, is perhaps one the best-studied routes for clathrinmediated trafficking and the vast majority of AP2 ligands were identified in this context. It is therefore also the best understood example of regulated clathrin-mediated endocytosis. In the synapse, vesicle retrieval is stimulated by calcium activation of calcineurin, which results in the dephosphorylation of many of these AP2 accessory components [40]. This calcium regulation means that AP2-dependent endocytosis only occurs in response to exocytosis at the synapse. It also means that there may be partial preassembly and recruitment of endocytic proteins to endocytic zones and thus the process is primed to occur immediately following exocytosis. Whilst much of this diversity is absent at other sites within the cell at which coated vesicle assembly occurs, it is retained for clathrin-mediated endocytosis in a broad spectrum of non-neuronal tissues. Here the responses may be principally to cytokines or growth factors such as epidermal growth factor (EGF) or transforming growth factor beta (TGF-␤) rather than neurotransmitters. Here too phosphorylation, as well as ubiquitination, appear to be critical modifications that regulate the protein–protein interactions required for cargo selection and coat recruitment although research into the dynamics of the assembly and disassembly of clathrin-coated vesicles is in its’ infancy relative to the significant body of structural data on coat proteins and adaptor complexes. 2. CCV formation in response to receptor tyrosine kinase (RTK) signalling The RTK family controls the proliferation, differentiation, cell survival, migration and adhesion of a wide range of cell types. The mechanisms that underlie the trafficking of this class of receptor are best understood for the EGFR system. EGF binding induces the dimerization of EGFR and the autophosphorylation of tyrosine residues in the cytoplasmic domain of EGFR [41]. These phosphorylation sites are also then docking sites for Src-homology-2 (SH2)- or phosphotyrosine-binding-domaincontaining downstream effector proteins. These proteins form a signalling network that regulates the biological response to the ligand [42]. It has long been assumed that clathrin-dependent endocytosis is the main pathway that is involved in the down-

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regulation of cell-surface receptors such as EGFR. However, studies on several cell-surface receptors have made it clear that many receptor systems are endocytosed through non-clathrin pathways [43,44]. Cells therefore use multiple internalization pathways to control cell surface receptors, and this is crucial for regulating cell signalling, receptor turnover and the magnitude, duration and nature of signalling events. 2.1. EGFR sorting and clathrin-mediated endocytosis The EGFR is an extensively studied model of cell-signalling receptor internalization through clathrin-mediated endocytosis that occurs within minutes of ligand stimulation. The internalization of EGFR is mediated by a RING-finger E3 ubiquitin ligase called Cbl that binds to activated EGFR, as well as to many other receptors. Binding promotes the ubiquitination of the receptor which targets EGFR for endocytosis and subsequent sorting to the multivesicular body (MVB) [45–48]. Cbl can also promote endocytosis together with CIN85 (Cbl-interacting protein of 85 kDa) and endophilins that possess BAR (‘Bin, amphiphysin, Rvs’) domains, which induce membrane curvature and help in the fission of clathrin-coated buds from the membrane [49–51] (Fig. 1). Consequently Cbl is an important component in EGFR trafficking. EGFR can however also directly regulate endocytosis as well as acting as a cargo and is known to phosphorylate both clathrin itself, inducing the redistribution of clathrin to the cell periphery, as well as the accessory adaptor protein Eps15. The phosphorylation of Eps15 by the receptor is essential for EGFR internalization [52], and the monoubiquitination of both Eps15 and epsin1 is also regulated by EGFR [53,54]. Since both Eps15 and epsin are required for EGFR internalization, they probably function as a complex during EGFR internalization. Monoubiquitination may however ultimately trigger the release of these accessory adaptors from the clathrin coat since epsin-1 loses its capacity to bind to PtdIns(4,5)P2 and to interact with AP2 and clathrin upon modification. The timing of monoubiquitination versus the release of the nascent CCV from the plasma membrane is still uncertain and it will be important to characterise this more precisely in order understand the dynamics of coat formation and vesicle release versus the subsequent membrane fusion of these vesicles with early endosomes. Upon internalization into the early endosomes, receptors can be sorted to the recycling endosome, from which they travel back to the cell surface (Fig. 1). An alternative fate sees EGFR, together with Eps15, form a ternary complex with signal transduction adaptor molecule (STAM) and Hrs [55]. This assembles on the surface of early endosomes and together with a flat clathrin coat clusters and directs EGFR into intralumenal vesicles of multivesicular endosomes resulting in the termination of signalling (Fig. 1) [56,57]. The invagination of endosomal membranes to generate multivesicular bodies is driven by Tumour-Suppressor Gene 101 (TSG101) and the multiprotein endosomal sorting complex required for transport (ESCRT) complexes [58,59]. Protein components of these complexes are highly homologous to use vacuolar protein sorting (Vps) class E proteins in S. cerevisiae. At each stage cargo recognition and transfer is based on the ability of components of these complexes to bind to the

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Fig. 1. The trafficking of epidermal growth factor receptor. At the cell surface EGF-bound growth factor receptors are autophosphorylated and may move out of lipid rafts prior to internalization into clathrin-coated pits. Src activation by EGFR can result in tyrosine phosphorylation of both clathrin and dynamin increasing the pool of clathrin at the cell surface for incorporation into pits and the GTPase-dependent scission activity of dynamin to release nascent CCVs (not shown). Activated EGFR recruits the E3 ubiquitin ligase, Cbl, which ubiquitinates EGFR on multiple sites and complexes with CIN85 and Endophilins. EGFR also phosphorylates and induces the ubiquitination of epsin and EGFR-pathway-substrate-15 (Eps15). Epsin interacts with adaptor protein-2 (AP2), clathrin and phosphatidyl 4,5-bisphosphate. Binding to the phosphoinositide lipid induces helix formation and insertion to initiate membrane curvature. Recruitment of coat proteins then braces the deformed membranes (not shown). Eps15 and epsin may also bind to monoubiquitinated EGFR through their ubiquitin-interacting motifs (UIMs). In early endosomes, hepatocyte growth factor-regulated tyrosine kinase receptor substrate (Hrs) binds to phosphatidylinositol 3-phosphate (PtdIns3P) through a ‘Fab1, YOTB, Vac1, EEA1’ (FYVE) domain, and forms a ternary complex with EGFR via signal transduction adaptor molecule (STAM) and Eps15 which interacts through its UIM with the receptor. Ubiquitinated Hrs is also phosphorylated after EGFR activation. From the early endosomes, EGFR is either recycled back to the cell surface or is incorporated into multivesicular bodies (MVBs) prior to trafficking to late endosomes/lysosomes to be degraded (adapted by permission from Macmillan Publishers Ltd. [135], copyright 2005).

ubiquitin tags conjugated to internalized receptors. Ubiquitinated Hrs is also phosphorylated in response to the activation of EGFR and other RTKs, and this may regulate the composition of endosomal machinery and so the fate of the EGFR

although functional effects on Hrs are yet to be precisely defined [60]. All of these data indicate that clathrin-mediated endocytosis together with phosphorylation, multiubiquitination and the specialized composition of endocytic membranes function coop-

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eratively to control the fate of EGFR and many other receptor systems that traffic through the clathrin pathway. The repertoire of signalling molecules with which EGFR interacts depends on its’ subcellular distribution and endocytosis. Whilst this suggests that trafficking controls the molecular response to RTK signalling, and in particular EGFR activation, it has been difficult to prove this categorically. Early studies using an EGFR variant defective for internalization clearly demonstrated that cell-surface EGFR could induce mitotic responses and the transformation of cells at low doses of EGF [61]. These studies also showed that the inhibition of endocytosis does not block biological effects such as cell proliferation [62,63]. Endosomally localized EGFR associates with many, if not all, of its downstream effectors, including Shc (SH2-domaincontaining transforming protein) and Grb2 and this leads to the recruitment of mamalian son-of-sevenless (mSOS) and to the endosomally localized activation of Ras, Raf, MEK1 and the entire mitogen-activated protein kinase (MAPK) cascade (MEK1 stands for ‘MAPK and extracellular signal-regulated kinase (ERK) kinase-1’) [64,65]. Furthermore upon blocking cell-surface EGFR activation and EGFR recycling back to the cell surface, internalized activated receptors signal from endosomes and promote cell survival [66]. RTKs can clearly signal from endosomes. These studies raise an important point. Is endosomal signalling a passive event that is initiated at the plasma membrane and maintained during trafficking, or is it biologically relevant? Some pathways are dependent on endocytosis for activation whilst others appear to be less so. Blocking EGFR internalization and analysing signalling has revealed that MAPK activation is dependent on endocytosis, whereas the activation of phospholipase C␥ (PLC␥) and Shc is not [62]. These differences may depend on whether signalling scaffolds need to nucleate around endocytosed/activated RTKs in endosomal compartments. For example p14, a protein that is localized to endosomal compartments, recruits the MP1 (MEK1 partner) MAPK scaffolding protein to endosomes and is important for the robust EGFdependent activation of ERK, but not of another kinase known as p38 [67]. Consistent with the idea that unique signalling activities arise from the endosomal compartment, active Rap1, a Ras-related GTPase, has been reported to be preferentially enriched on endosomes, possibly through an endosomally localized Ras guanine nucleotide-exchange factor [68]. This moves some of the focus on signalling firmly from clathrin-coated vesicles to early endosomes but this is in part because the dynamic assembly and disassembly of clathrin coats makes it very difficult to attribute a signalling event to the presence of an activated RTK in a CCV itself. Given that the magnitude and kinetics of signal activation in RTK pathways are important in disease – in particular, in cancer – integrating trafficking with signalling provides an elegant mechanism for tuning signalling responses. Where does clathrin-mediated endocytosis fit into this? It both alters the repertoire of signalling events triggered by the activated receptor by changing its’ subcellular distribution and then through the role played by accessory adaptor proteins like Eps15 in both CCV and MVB formation ultimately contributes to the downregulation of signalling.

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Interfering with this integration by disrupting RTK turnover is a key molecular pathology that underlies many cancers. This is highlighted by Cbl, which was first identified as v-Cbl. v-Cbl is the protein product of the transforming gene of the Cas NS-1 murine leukaemia virus, and it is a dominant-negative mutant that consists of only the EGFR-binding domain of Cbl. It therefore blocks receptor downregulation, promotes recycling and mediates EGF-induced transformation [69]. The amplification of ErbB2 (HER2), which is downregulated slowly compared to EGFR (ErbB1), is also important in a subset of breast cancers. The crucial role that trafficking has in this oncogenic activity is highlighted by the emphasis on developing therapeutic antibodies to target overexpressed ErbB family receptors in breast cancer and at other organ sites [70]. Perhaps the most wellknown example of such an antibody is a humanized version of the L26 antibody (Herceptin) targeting ErbB2-overexpressing breast cancers. There is however some controversy as to whether this works in all cells by promoting receptor internalization or may in some cell-lines alternatively function by inhibiting Akt activation and promoting PTEN recruitment to the membrane [71–73]. In a sense this encapsulates the difficulties of separating signalling and trafficking effects. More recently it has been proposed that multiple antibodies targeting different epitopes on the same protein may more effectively promote clustering and endocytic clearance of receptors [70]. Receptor trafficking is therefore an important target for the design of new therapeutics. These studies highlight the importance of linking signalling to receptor trafficking in the RTK system, and this is a link that might be far more intimate than was first imagined, especially as a growing number of reports are showing direct regulatory interactions between signalling modules and components of the endocytic machinery [74]. 2.2. Post-translational modifications to endocytic proteins In addition to ubiquitination, phosphorylation changes have been shown to be critically important in the regulation of clathrin-mediated endocytosis. The interplay between signalling enzymes and coat assembly was first illustrated in the synapse in which clathrin-coated vesicles are abundant and readily purified. At the synapse calcium-responsive desphorylation of proteins including dynamin, amphiphysin, AP180 and synaptojanin by calcineurin was shown to promote an increase in endocytosis following neurotransmitter release [75]. The kinases responsible for maintaining their phosphorylation in the resting neuron are believed to include casein kinase II, protein kinase C and others. Many of these are not tissue-specific and there is no reason to believe that phosphorylation–dephosphorylation cycles affecting clathrin coat components are not important feedback responses to other signalling events elsewhere. The kinase(s) responsible for phosphorylation of ␣- and ␤2-adaptins are unknown, but two distantly related kinases, cyclin-G-associated kinase (GAK, also known as auxilin-2) and adaptor-associated kinase 1 (AAK1) have been shown to phosphorylate the ␮2subunit in vitro [76,77]. Phosphorylation by AAK1 of ␮2 on threonine 156, a site that is known to be important for the function of this subunit in vivo, markedly increases the affinity of

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Fig. 2. The regulation of clathrin-mediated endocytosis by vesicle-associated kinases. Although the precise timing of action of CCV-enriched kinases is unclear based on their functions in driving cargo recruitment and promoting coat disassembly it is possible to speculate as to where they may act. The phosphorylation of threonine-156 of the ␮2 subunit of the adaptor protein-2 (AP2) complex is thought to be required prior to the binding of tyrosine-based cargo motifs in receptor proteins. This phosphorylation event is thought to occur through the action of cyclin G-associated kinase (GAK) and/or adaptor-associated kinase 1 (AAK1). At the same time in neurons many of the accessory proteins including amphiphysin, dynamin and synaptojanin required for CCV assembly and disassembly are dephosphorylated by calcineurin in a membrane potential-dependent manner to make them available for incorporation into clathrin coats (not shown). In growth factor responsive cells, tyrosine phosphorylation of dynamin and clathrin may promote coat formation (not shown). The ␮2 subunit of AP2 is then believed to remain phosphorylated through the lifespan of the clathrin coat (estimated to be around 30 s following formation [134]). Dissociation of the clathrin coat then triggers release of GAK and/or AAK1 from the vesicle and dephosphorylation of ␮2. Casein kinase 2 (CK2) is also incorporated into the CCV but is inactive when membrane-bound. Upon clathrin uncoating, CK2 is released from this inhibition and is able to phosphorylate a wide range of peripheral membrane coat components including clathrin light chain (LCa/b) and the ␣ and ␤ subunits of AP2 adaptors transiently inhibiting immediate coat reassembly and a futile cycle of these factors on and off the same membranes. Subsequent dephosphorylation of these components in the cytosol then restores the available pool of clathrin and AP2 for subsequent rounds of CCV formation.

AP2 complexes for tyrosine-based internalization motifs (Fig. 2) [38,77]. More recently a systematic study was carried out in which high-throughput RNA interference and automated image analysis were used to knockdown all of the known kinases in the human genome and explore the effects of doing so on caveolae/lipid raft-mediated endocytosis and clathrin-mediated endocytosis. An assay for Simian Virus 40 (SV40) uptake was used as a measure of caveolae/lipd raft internalization and vesicular stomatitis virus (VSV) internalization was used as a surrogate for clathrin-mediated endocytosis with HeLa cells as the chosen model [78]. Pelkmans et al., demonstrated that discrete subgroups of kinases were important in regulating each trafficking route. Eighty kinases specifically blocked VSV infection, whilst 43 kinases were specifically required for SV40 infection [78]. There are clearly significant questions arising

from this work not least of which being the extent to which these effects were direct, in other words due to substrates of these kinases being clathrin-coated vesicle or lipid raft constituents, or indirect. This approach has, however, provided some interesting start points for more mechanistic studies and underlined the potential complexity of the interplay between trafficking and signalling. The question of substrate specificity on the other hand has so far been approached stochastically. Dynamin has been described as a substrate for the ERK-MAP kinase cascade, principally ERK2 leading to a reduction in GTPase activity at the synapse. Src-dependent phosphorylation of dynamin at tyrosines-231 and -597 has been reported to be required for the internalization of G-protein coupled receptors including the beta2-adrenergic receptor, as well as being a substrate for a truncated and oncogenic variant of EGFR through Src kinase activation [79].

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However dynamin is required for both clathrin-dependent and -independent endocytosis so this does not provide a discriminator between trafficking routes. A more intriguing recent observation is that the activation of Ephrin B2 receptors, one of a class of receptors associated with a variety of processes including cell shape, adhesion and separation, and movement via cell attraction and repulsion, results in the phosphorylation of synaptojanin 1 and regulates endocytosis [80]. Synaptojanin has long been known as a dephosphorylation target facilitating membrane retrieval following neurotransmitter release [81]. The finding that synaptojanin is tyrosine phosphorylated within its’

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proline-rich domain in response to Ephrin B2 activation resulting in endocytosis suggests for the first time a direct connection between the kinase activity of a receptor and the phosphorylation status of a coat protein [80]. This phosphorylation event inhibits an interaction with endophilin which is required to stimulate the 5 -lipid phosphatase activity of synaptojanin, and phosphorylation consequently results in a local increase in phosphoinositide 4,5-bisphosphate within the plasma membrane [80]. This increases the binding of endocytic adaptors and promotes endocytosis of the AMPA receptor, an effect ablated by phenylalanine mutants of synaptojanin 1. Is synaptojanin the

Table 1 Kinases, SUMO and ubiquitin ligases associated with coat proteins Enzyme

Coat binding partner

Coat substrate

Functions

AAK1

␮2 adaptin

Facilitates receptor and membrane binding in endocytic trafficking

Undefined

Promotes ligand-induced degradation of EGFR through a ubiquitin-binding domain [107]

Casein kinase 2

Clathrin heavy chain and appendage domain of ␣-adaptin [77,105] Clathrin heavy chain via an LIDFG motif. Activated EGFR through a C-terminal domain [106] Clathrin heavy chain via an LIDFG motif. Regulated by Cdc42 [108] Undefined

Cbl (E3 ubiquitin ligase)

Cargo-activated EGFR [109]

Clathrin light chain ␣ adaptin, ␤2 adaptin, ␮2 adaptin, amphiphysin, AP180, dynamin, synaptotagmin Eps15/Epsin1 [54,110–114]

Cdk1

RalBP1-POB1 complex [85]

Epsin 1 (site: S357) [86]

Cdk5

Amphiphysin [115]

Dyrk1a/Minibrain kinase

Undefined

Ephrin B2 receptor

Undefined

Amphiphysin (site: S272, S276, S285) [115]. Dynamin (site: S774, S778 [89], T780 [90]) Amphiphysin (site: S293) [116]. Dynamin 1 (site: S857) [117]. Synaptojanin [118] Synaptojanin [80]

ERK/MAP kinase ERK/MAP kinase

Undefined Undefined

Dynamin [119] Amphiphysin [120]

GAK

Clathrin heavy chain and appendage domain of ␣-adaptin, phospholipids [14,121,122] Clathrin heavy chain through an LLDSD motif [123]

␮2 adaptin [76]

Rho kinase

Undefined Undefined Undefined Dynamin (coiled coil GTPase effector domain -GED domain) [97] Undefined

Dynamin (site: S795) [125] Synaptojanin [81] Synaptotagmin (site: T112) [126] K376 in an 11-residue peptide of dynamin but so far no known in vivo or full-length substrates [97] Endophilin A1 (site: T14) [127]

Src

Undefined

Clathrin heavy chain (site: Y1477) [128] Dynamin (sites: Y231, Y597) [79]

ACK1

ACK2

GRK2

Protein kinase C Protein kinase C Protein kinase C PIAS-1 (E3 SUMO ligase)

Src

Agonist-induced phosphorylation of receptor cargo (GPCRs)

Inhibits transferrin receptor endocytosis when overexpressed by competing with AP2 for clathrin binding [108] Inhibitory effects on clathrin binding and interactions with AP2 and clathrin [76] Initiates endocytosis through cargo ubiquitination but may ultimately contribute to the exclusion of accessory adaptors from the coat Inhibits the association with POB1 and the appendage domain of ␣ adaptin [84,86] Phosphorylation inhibits the association of amphiphysin with ␤-adaptin and of dynamin with amphiphysin [90] Reduces binding of amphiphysin to endophilin. Reduces binding of dynamin to SH3 domain-containing proteins including amphiphysin Promotes AMPA receptor endocytosis through the tyrosine phosphorylation of synaptojanin [80] ERK2 inhibits GTPase activity Inhibits the association between AP2 and amphiphysin Cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Blocks receptor-mediated endocytosis upon transient overexpression Promotes GPCR endocytosis. Clathrin regulates kinase and cargo recruitment and regulates receptor phosphorylation [124] Blocks phospholipid binding [125] Inhibits endocytosis Potentially inhibitory Sumo1 and Ubc9 inhibit lipid dependent oligomerisation of dynamin and endocytosis [97] Inhibition of receptor endocytosis by blocking an interaction with CIN85 [127] Facilitates membrane binding [128] Promotes GPCR endocytosis [79]

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sole protein to be tyrosine phosphorylated by a receptor and is the Ephrin B2 receptor the sole RTK to do so? This study suggests that RTK activation may promote receptor endocytosis through additional routes to the ubiquitination of coat proteins.

periods in which endocytosis does not occur. Many vesicle coat components are ideally suited to these roles. By applying fractionation and microscopy to cell populations enriched at the various stages of the cell cycle further insights will be gathered into the extent to which coat proteins play a role.

3. Cell cycle control 4. Endocytic proteins with alternate signalling functions Clathrin-mediated endocytosis is arrested during mitosis as are other endocytic steps [82,83]. An RNAi screen for kinases implicated in the regulation of clathrin-mediated endocytosis suggested that MAP2K1 (MEK1) and CNK (also PLK3 or Polo-like kinase-3) play a role in regulating transferrin receptor internalization [78]. Cyclin-dependent kinases (Cdks) have in the past been reported to phosphorylate accessory adaptor proteins resulting in an inhibition of interactions with the core endocytic proteins such as clathrin and AP2 adaptors (Table 1). Epsin and Eps15 were reported to be phosphorylated during mitosis by Cdk1 blocking their interaction with the appendage domain of ␣-adaptin and their participation in vesicle formation, and thus inhibiting endocytosis [84]. This phosphorylation event was subsequently reported to be mediated by components of the Ral signalling pathway, implicated in Ras-dependent oncogenesis, and specifically RLIP/RalBP and POB1 downstream molecules of the small GTP-binding protein Ral [85,86]. RalBP1, POB1, epsin and Eps15 form a complex with ␣-adaptin of AP2 in Chinese Hamster Ovary Cells [86,87]. RalBP1 however also interacts with active p34cdc2.cyclinB1 (cdk1) and phosphorylation of epsin at serine-357 and of POB1 at serine411 is believed to block the interaction with AP2 adaptors and inhibit endocytosis [86]. EGF and insulin activate Ral and so in non-mitotic phases of the cell cycle this would be predicted to promote assembly of a RalBP1-POB1-Epsin-Eps15 complex to drive ligand-stimulated receptor endocytosis. Cdk5 has also been implicated in the regulation of synaptic vesicle endocytosis [88–90]. During mitosis with endocytosis inhibited it remains possible that coat components may serve alternate roles. Recent papers implicated clathrin in mitosis using an RNAi approach [91,92]. In the course of cell cycle progression it is clear that membrane fission and fusion events are essential for the assembly and disassembly of organelles and chromosomal segregation. Multiprotein scaffolds are required for this during

In beginning to explore the role of phosphorylation by signalling kinases in the regulation of coat assembly an alternate way of identifying candidates, in addition to RNAi screens, is to identify coat proteins that are both adaptors for clathrin/cargo and for kinases themselves (Table 1). A well-studied example of a protein involved in both signalling and endocytosis is ␤arrestin which binds to activated GPCRs and terminates their interaction with heterotrimeric G proteins [93]. Additionally ␤-arrestin interacts with clathrin, AP2 adaptors and phosphoinositides participating in coated pit assembly and GPCR endocytosis [93]. Beta-arrestin also associates with Src and components of the MAP kinase pathway where its’ role is as an activator of signaling [93]. Perhaps the most exciting molecule as a hub for the recruitment of signalling enzymes to the coated pit may be dynamin itself. Amongst the SH3 domain-containing binding partners of this protein are components of the Ras and JNK signalling pathways, and WASP and Rho family guanine nucleotide exchange factors [94,95]. The association between a member of the family of E3 small ubiquitin-like modifier (SUMO) ligases and dynamin requires further study in that no endocytic adaptor substrate has yet been identified, although cargo in the form of the kainite receptor was recently reported to be sumoylated [96,97]. Whether these interactions are compatible with the formation of a dynamin oligomer at the neck of a coated pit and the recruitment of signalling enzymes into the coat is so far undetermined however they may help to narrow the list of testable candidates. Whilst clearly at a very early stage our understanding of the effects of signal transduction on clathrin coat assembly is also of potential value in the context of alternate roles for clathrin and accessory adaptor proteins. In the course of the last few years a number of papers have been published reporting the involvement of proteins such Eps15, Eps15R, Huntingtin interacting protein 1

Table 2 Alternate functions of coat proteins Coat protein

Binding partner

Cell-line/tissue

Functions

Beta-arrestin 2 [129] Clathrin heavy chain [99]

CRM1? [129] p53 [99]

HeLa MCF-7 and H-1299 cells [99]

Clathrin triskelia [91,92]

Epsin 1 [130]

Mitotic spindle—clathrin-binding proteins within the complex yet to be identified [91,92] CRM1 [131], PLZF [130]

NRK cells transfected with GFP-clathrin light chain [92]. HEK-293 cells transfected with GFP-clathrin heavy chain and siRNA CV-1 [131], CHO [130]

Eps15 [132]

CRM1 [131,132]

HeLa [132], CV-1 [131]

HIP1 [133]

Androgen receptor [133]

LNCaP [133]

Nucleocytoplasmic shuttling of JNK3 [129] Stabilises a p53-p300 complex on promoters enhancing p53 transcriptional activity [99] Proposed to act as a molecular brace between microtubules in a kinetochore fibre increasing fibre stability and facilitating mitosis (‘bridging’ hypothesis) Nucleocytoplasmic shuttling [131], transcriptional regulation [130] Nucleocytoplasmic shuttling [131,132], transcriptional regulation [131] Binds to androgen-responsive PSA promoter and enhances AR transcriptional activity [133]

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(HIP1), cyclin G-associated kinase (GAK), dynamin and most recently clathrin heavy chain as transcriptional co-regulators. This implies the existence of a nuclear sub-population of adaptors capable of incorporation into a transcriptional complex on a DNA scaffold as opposed to a coat complex on a membrane scaffold (Table 2). In the case of dynamin 2 and clathrin heavy chain there is the suggestion that these proteins act as transcriptional coregulators for p53 [98,99]. Given that we do not at present have a clear global picture of how and when post-translational modifications occur on coat proteins and what their effects may be on coat assembly, it is difficult to combine these two relatively recent strands of research. Clearly further work is needed to understand how the same proteins can perform conceptually similar functions in very different subcellular locations. Yet if phosphorylation can drive the disassembly of an endocytic complex as discussed above perhaps it can promote the assembly of other complexes.

not need to be appreciably enriched within a particular compartment to play an active role and so the traditional approaches to characterise vesicles of biochemical fractionation coupled with microscopy may not be adequate to uncover all the players. Strikingly the signalling kinases reported to phosphorylate endocytic proteins (Table 1) seem to promote clathrin coat formation whereas the CCV-enriched kinases, such as AAK1, although they may facilitate cargo binding through ␮2 phosphorylation, may also promote uncoating and be inhibitory. The other potent argument for exploring the interplay between signalling and clathrin-mediated endocytosis arises from our ability to transform fibroblastic cells and induce tumours through the overexpression of coat proteins such as Eps15 and HIP1 [102–104]. Future work should focus on a better understanding of the dynamics of coat assembly, on the contribution of vesicle coat proteins to the positive and negative regulation of signalling and finally on the impact of this on disease progression.

5. Concluding remarks

Acknowledgement

Clathrin-coated vesicle formation has been meticulously dissected at a structural level and to some degree at a functional level. Such is the array of independent or semi-redundant molecular routes for the assembly of a clathrin coat that the field is now seeking, through an evolutionary analysis of the conservation of these proteins across organisms, to narrow down the minimum/core machinery for coat formation and to rationalise this information with network theories [15]. It is however most likely that these multiple routes to obtain a clathrin coat simply reflect the vast array of different extracellular stimuli that can provoke signalling and transcriptional responses as well as changes in the rates of CCV formation and the nature of the cargo. If this is true we need to know more about how the cell senses and regulates the dynamics of cargo recruitment and coat assembly since otherwise applying a reductionist approach based on an evolutionary assessment of the conservation of trafficking components across organisms will be confounded by the fact that they sense and respond to very different physiological challenges. Furthermore we are not yet at the point of commenting on whether receptors internalized into CCVs can maintain signalling cascades in transit between the plasma membrane and endosomes. This is important because these receptors are clearly active at the cell surface and within early endosomes. Incorporation into CCVs therefore either represents a transient ‘off’ stage in which receptor tails are occluded from the cytoplasmic kinases and signalling adaptors by the clathrin coat and trafficking adaptors, or some of the signalling machinery is incorporated as a complex with the receptor tail when the receptor is internalized and the signalling relay is maintained. The ability of Reps1 to interact with both Eps15 and AP2 adaptors as well as signalling adaptors such as Grb2 and CRK in response to EGFR activation hints at the latter but there has been no systematic examination of these two hypotheses as yet [85,86,100]. The presence of an active and enriched classII wortmannin-resistant PI 3-kinase in CCVs is a further pointer that kinases associated more traditionally with signalling events may be present and functional in these vesicles [101]. However clearly signalling enzymes do

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[90]

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