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European Journal of Cell Biology 86 (2007) 533–547 www.elsevier.de/ejcb

Nuclear functions of endocytic proteins
Iwona Pilecka, Magdalena Banach-Orlowska, Marta MiaczynskaÃ
International Institute of Molecular and Cell Biology, Laboratory of Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw, Poland Received 6 March 2007; received in revised form 19 April 2007; accepted 19 April 2007

Abstract
An increasing number of proteins appear to perform multiple, sometimes unrelated functions in the cell. Such moonlighting properties have been recently demonstrated for proteins involved in clathrin-mediated endocytosis. Some clathrin adaptors and endosomal proteins can undergo nucleocytoplasmic shuttling, which is often based on intrinsic sequence motifs and requires active transport mechanisms. Endocytic proteins can associate with nuclear molecules, changing their localization and/or activity and may modulate the levels and specificity of gene transcription. It is not clear how the nuclear and cytoplasmic pools of endocytic proteins are interconnected, or whether these molecules act as nuclear second messengers upon extracellular stimuli, but alike in endocytosis, they seem to form multi-component scaffolding platforms in the nucleus. Added to their endocytic functions, the nuclear roles of Eps15, Epsin1, CALM, HIP1, Dab1/2, b-arrestins, APPL1/2 and the components of ESCRTs clearly increase the complexity of signaling networks affecting cellular growth, proliferation and homeostasis. r 2007 Published by Elsevier GmbH.
Keywords: Endocytic adaptors; Clathrin-associated sorting protein; Endosomal sorting complex required for transport; Nucleocytoplasmic shuttling; Gene transcription

Introduction
Nucleocytoplasmic shuttling of proteins is a wellestablished mechanism of communication between the nucleus and the cytoplasmic compartments of eukaryotic cells. Classical examples are multiple signaling
Abbreviations: CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; CHMP, chromatin-modifying protein/charged multivesicular body protein; CLASP, clathrin-associated sorting protein; EAP, RNA polymerase II elongation factor (ELL)-associated proteins; ESCRT, endosomal sorting complex required for transport; LMB, leptomycin B; MVB, multivesicular body; NES, nuclear export signal; NLS, nuclear localization signal ÃCorresponding author. Tel.: +48 22 597 0725; fax: +48 22 597 0726. E-mail address: miaczynska@iimcb.gov.pl (M. Miaczynska). 0171-9335/$ - see front matter r 2007 Published by Elsevier GmbH. doi:10.1016/j.ejcb.2007.04.004

molecules, which translocate to the nucleus to modulate gene expression in response to extracellular stimuli (Xu and Massague, 2004). Recently, other classes of proteins initially considered exclusively cytosolic have been described to undergo nucleocytoplasmic transport. Among them, transmembrane growth factor receptors and their ligands, as well as adhesion receptors appear to play non-schematic, alternative roles in the nucleus (reviewed in (Aplin and Juliano, 2001; Bryant and Stow, 2005; Johnson et al., 2004)). Moreover, the ability to relocalize to the nucleus has been demonstrated for an increasing number of proteins present on clathrincoated vesicles (CCVs) and endosomes. Accumulating evidence suggests that many endocytic adaptors may have a dual function: in membrane sorting during endocytosis and in regulating gene expression in the

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nucleus. Here we attempt to summarize the present knowledge on the nuclear transport and functions of proteins commonly recognized as endocytic proteins. Clathrin-dependent endocytosis enables the uptake of extracellular molecules and their subsequent intracellular transport through a series of vesicle compartments (endosomes; Fig. 1). The internalization step involves clustering of membrane receptors and their ligands in clathrin-coated pits (CCPs). These invaginate and pinch off, forming CCVs, that subsequently lose their clathrin coat and fuse with early endosomes. There the cargo is sorted and delivered either back to the plasma membrane in the recycling process or to the multivesicular bodies (MVBs)/late endosomes and lysosomes for degradation. Proper trafficking of internalized cargo is vital for cell growth and survival, and is controlled by various classes of proteins responsible for vesicle budding, fusion and motility, as well as cargo sorting. The latter task is largely ascribed to an army of endocytic adaptor proteins which can be operationally divided into two groups: (1) clathrin-associated sorting

proteins (CLASPs) involved in accumulation of cargo in the invaginating CCP at the plasma membrane, and (2) proteins acting on the level of early and late endosomes, including the effectors of Rab GTPases and subunits of endosomal sorting complexes required for transport (ESCRTs). For a schematic overview and distribution of endocytic proteins, see Fig. 1. (1) CLASPs localize to CCPs and CCVs and are characterized by their ability to simultaneously bind multimeric adaptor protein AP-2, clathrin, phosphoinositides, and the endocytic cargo. Due to their multidomain structure, they are able to regulate the assembly of CCPs at the plasma membrane by synchronizing cargo selection and clathrin lattice polymerization. The particular functions of monomeric adaptors in cargo-specific sorting and CCP formation have been reviewed in detail elsewhere (Maldonado-Baez and Wendland, 2006; Mousavi et al., 2004). Here we will concentrate on alternative, non-endocytic functions of Eps15, Epsin1, CALM, HIP1, Dab1 and Dab2, and b-arrestins. (2) Distinct domains on endosomes are occupied by specific Rab GTPases that direct cargo sorting and trafficking, recruiting a large number of effectors (Grosshans et al., 2006; Zerial and McBride, 2001). Among Rab5 partners, the two related proteins APPL1 and APPL2, present on a subpopulation of early endosomes, migrate to the nucleus and associate with nuclear complexes (Miaczynska et al., 2004). The three multimeric ESCRTs that are responsible for the ubiquitin-dependent sorting of proteins at the MVB are conserved from yeast to mammals. However, in addition to their role in MVB biogenesis, cargo sorting and viral budding, some of ESCRT subunits acquired nuclear functions that are unrelated to membrane trafficking (for review, see (Slagsvold et al., 2006)). Here, we will describe nuclear roles of TSG101 (the mammalian homologue of the class E vacuolar protein-sorting molecule Vps23p and a part of ESCRT-I), EAPs (subunits of ESCRT-II), and chromatin modifying proteins/charged multivesicular body proteins (CHMPs) (subunits of ESCRT-III).

It matters where you are: nucleocytoplasmic transport
Compartmentalization of mammalian cells provides a control mechanism over the complex arrays of signaling networks. Subcellular localization of proteins is vital to their functions, and an obvious prerequisite for a nuclear function of a protein is its physical presence in the cell nucleus. Transport across the nuclear envelope

Fig. 1. Distribution of relevant CLASPs and endosomal proteins along the endocytic pathway. See text for description.

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occurs through the nuclear pore complex (NPC). Small proteins (up to ca. 50 kDa) undergo free diffusion across the meshwork formed by nucleoporins (Stewart et al., 2001). Bigger proteins can be actively transported in and out of the nucleus by dedicated importins and exportins, which belong to the importin b family of transport receptors (Gama-Carvalho and Carmo-Fonseca, 2001; Harel and Forbes, 2004). They circulate between the nucleus and cytoplasm, recognize cargo molecules, and transfer them from one side of the nuclear envelope to the other in an energy-dependent process guided by a gradient of RanGTP. Importins, such as IMPb/ IMPa, recognize nuclear localization signals (NLS), while exportins, such as CRM1 (chromosomal region maintenance 1), recognize leucine-rich nuclear export signals (NES) within cargo molecules. Formation of a complex between CRM1 and NES-harboring proteins can be blocked by an antifungal antibiotic leptomycin B (LMB), inhibiting export and causing nuclear accumulation of target proteins (Ossareh-Nazari et al., 1997).

to a transcription factor promyelocytic leukemia zinc finger (PLZF), and overexpression of PLZF forces nuclear localization of Epsin1 (Hyman et al., 2000). An armadillo domain of p120-catenin, structurally related to ENTH, was demonstrated to interact with Kaiso transcription factor, homologous to PLZF (Daniel and Reynolds, 1999). Nuclear import of CALM may depend on another transcriptional regulator, CALM interactor expressed in thymus and spleen (CATS) which upon overexpression promotes nuclear localization of CALM (Archangelo et al., 2006). Similarly, a partial relocation of TSG101 to the nucleus has been observed upon overexpression of its nuclear interacting partners: AATF (apoptosis-antagonizing transcription factor; Burgdorf et al., 2004) and Epstein–Barr virus R transactivator (Rta; Chua et al., 2007).

How do endocytic adaptors exit the nucleus?
Certain proteins undergoing nucleocytoplasmic shuttling may appear cytosolic under steady-state conditions. Only after nuclear export is inhibited by LMB their nuclear accumulation can be observed. This approach was undertaken for many endocytic adaptors, revealing that their nuclear export is mediated by CRM1 (Table 1). b-Arrestin2 has a classical NES, which is absent within the corresponding sequence of b-arrestin1 due to a single residue substitution. Accordingly, b-arrestin2 is excluded from the nucleus, while b-arrestin1 is both nuclear and cytoplasmic (Scott et al., 2002). Interestingly, a heterodimer composed of both b-arrestins is transported out of the nucleus (Storez et al., 2005). The native arrestins are small enough (44–46 kDa) to diffuse through the nuclear pore. However, there are indications that b-arrestins complexed to other proteins exit the nucleus via the LMBsensitive pathway (Song et al., 2006). Also Dab1 accumulates in the nucleus in the presence of LMB, due to two CRM1-specific NESs. In the steady state, full-length Dab1 is mainly located in the cytoplasm, while its N-terminal fragment (amino acids 1–271) fused to GFP is present both in the nucleus and in the cytoplasm (Honda and Nakajima, 2006). The classical leucine-rich NES was identified in Eps15 as the last 6 amino acids, and its functionality has been confirmed by deletion mutagenesis and by the export of GFP fused to this sequence (Poupon et al., 2002; Vecchi et al., 2001). In contrast to Eps15, a related Eps15R exhibits a pronounced nuclear staining at steady state (Poupon et al., 2002). Eps15R lacks a classical leucine-rich NES and does not seem to interact with CRM1, that might explain its constitutive nuclear localization. CALM and Epsin1 despite the lack of a canonical NES interact with CRM1 in a Ran-dependent manner (Vecchi et al., 2001). Similarly to Epsin1 or Eps15, a nuclear localization of CALM was observed

How can endocytic adaptors enter the nucleus?
Certain endocytic proteins were demonstrated or predicted to harbor classical NLS, while some appear to enter the nucleus via interactions with proteins other than importins (Table 1). HIP1 contains a classical NLS at its C-terminus, and a mutation of a conserved arginine residue within this motif (R1005E) leads to the reduced nuclear localization of HIP1 (Mills et al., 2005). A bipartite nuclear localization signal has been identified in the N-terminus of Dab1 (Honda and Nakajima, 2006). This sequence is not conserved in a related adaptor Dab2. Also APPL2 has a predicted NLS motif. It is not present in APPL1, but since APPL proteins can form heterodimers (Nechamen et al., 2007), this could provide the means of nuclear entry for both proteins. b-Arrestins are actively imported to the nucleus and their N-terminal domain is indispensable for nuclear localization (Wang et al., 2003b). Eps15 is imported to the nucleus in an NLS-independent fashion, and this process requires EH (Epsin homology) domains present at the N-terminus (Poupon et al., 2002). EH domains participate in interactions with Hrb (HIV-1 Rev-binding) protein, which is related to nucleoporins, harbors phenylalanine–glycine repeats characteristic of this group (Fridell et al., 1995) and exhibits nucleoporinlike staining pattern (Fritz et al., 1995). Thus, it is conceivable that Hrb facilitates nuclear translocation of Eps15 and possibly of Eps15R, for which no import mechanisms have been described but which also interacts with Hrb. Epsin1 is another example of a protein that uses a ‘‘transporter’’ to enter the nucleus. It binds via an ENTH (Epsin NH2-terminal homology) domain

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Table 1. Protein

Features of nuclear transport and general functions of endocytic proteins found in the cell nucleus Nuclear import Nuclear export ND General function in the nucleus

APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper motif) APPL2 

No classical NLS  Nuclear localization enhanced upon
EGF stimulation (Miaczynska et al., 2004) Potential NLS (PKKKENE) within the N-terminal BAR domain 

ND  Binds NuRD complex
(Miaczynska et al., 2004)

ND 

ND  Binds NuRD complex
(Miaczynska et al., 2004) Nuclear scaffold for p300 and CREB (Kang et al., 2005)

b-Arrestin1

Translocates to the nucleus upon stimulation of k- and d-opioid receptors (Kang et al., 2005) Translocates to the nucleus upon stimulation of hOR17-4 (Neuhaus et al., 2006)

No effect of LMB (Scott et al., 2002): no classical NES LMB-sensitive (Kang et al., 2005): NES at Cterminus (L395/L397) (Scott et al., 2002)

b-Arrestin2 

Activation of transcription in
Gal4 assay (Neuhaus et al., 2006)  Translocates MDM2 and JNK3 to the cytoplasm (Song et al., 2006)  Binds to ERK (Kobayashi et al., 2005) and Akt (Beaulieu et al., 2005) Activation of transcription in Gal4 assay (Vecchi et al., 2001) via its TAD domain (amino acids 408-572) (Archangelo et al., 2006)

CALM (clathrin assembly protein lymphoid myeloid) 

No classical NLS  Interaction with CATS transcription
factor (Archangelo et al., 2006) 

No classical NES
(Vecchi et al., 2001)  LMB-sensitive (Vecchi et al., 2001)  Interaction with CRM1(Vecchi et al., 2001) ND

CHMPs 

Bipartite NLS in CHMP1  Localized in the nuclear matrix
(Stauffer et al., 2001) Bipartite NLS (Honda and Nakajima, 2006)

Chromatin remodeling and stable gene silencing (Stauffer et al., 2001) ND

Dab1 (Disabled 1)

LMB-sensitive: two NESs (Honda and Nakajima, 2006) ND

Dab2 (Disabled 2, splice variants: p67 and p96) 

Potential NLS, nuclear localization
may be affected by phosphorylation (Tseng et al., 1999)  Nuclear distribution is cell-cycle dependent (Mishra et al., 2002) 

Transcriptional activator (p67
4 p96) (Cho et al., 2000) 

Activator of TGFb-dependent
transcription (Hocevar et al., 2005)

Eps15 (EGFR phosphorylation substrate 15) 

No classical NLS  N-terminal domain containing EH
motif responsible for nuclear localization (Poupon et al., 2002)  No stimuli known to induce nuclear localization (tested: UV, H2O2, heat shock, EGF stimulation) (Vecchi et al., 2001) 

LMB-sensitive:
classical C-terminal leucine-rich NES  Interaction with CRM1 (Vecchi et al., 2001; Poupon et al., 2002)

Activation of transcription in Gal4 assay (Vecchi et al., 2001)

Eps15R (Eps15related) 

No classical NLS  Constitutive import (Poupon et al.,
2002) 

No classical NES  No interaction with
CRM1 (Poupon et al., 2002)

ND

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Table 1. (continued ) Protein Epsin1 Nuclear import Nuclear export General function in the nucleus ND 

No classical NLS  Interaction with PLZF transcription
factor (Hyman et al., 2000) 

LMB-sensitive: no
classical NES found  Interaction with CRM1 (Vecchi et al., 2001) ND

HIP1 (huntingtininteracting protein 1) 

Classical NLS at the C-terminus
(Mills et al., 2005)  Nuclear localization constitutive, enhanced upon androgen stimulation (Mills et al., 2005)

Activation of transcription: 

in Gal4 assay (Mills et al.,
2005) 

in luciferase assay: from
ARE-containing promoters (Mills et al., 2005) ND General transcriptional corepressor (Hittelman et al., 1999; Sun et al., 1999; Watanabe et al., 1998)

TSG101 (tumor susceptibility gene 101) 

Nuclear localization is cell-cycle
dependent (Xie et al., 1998; Zhong et al., 1998)  Nuclear localization increased through binding to nuclear proteins: AATF (Burgdorf et al., 2004), Daxx (Muromoto et al., 2004), and Rta (Chua et al., 2007)

ND, not determined.

exclusively in LMB-treated cells, suggesting rapid and efficient nuclear export of these molecules. In addition, the clathrin adaptor a-adaptin, but not b-, m2-, or g-adaptins, accumulates in the nucleus upon treatment with LMB (Vecchi et al., 2001). Also another classical component of CCPs, endophilin A2, seems to shuttle in and out of the nucleus in an LMB-sensitive, cell cycle-dependent manner (Cheung et al., 2004).

Regulation of nucleocytoplasmic shuttling
At present, the knowledge concerning the molecular mechanisms controlling the nucleocytoplasmic shuttling of endocytic proteins is rather limited. Only in a few cases the effects of cellular stimulation have been studied in more detail. There are instances where the nucleocytoplasmic shuttling of endocytic proteins seems to be constitutive: for example the localization of Dab1 is not changed upon phosphorylation by Fyn tyrosine kinase or by Reelin treatment (Honda and Nakajima, 2006). Similarly, the nuclear localization of Eps15 is not affected by any of the tested stimuli: epidermal growth factor (EGF), serum, UV irradiation, oxidative stress, or heat shock (Vecchi et al., 2001). Mechanistically,

oligomerization of Eps15 may regulate its nuclear localization, as a mutant lacking the coiled-coil domain required for oligomerization accumulated in the nucleus after a short treatment with LMB (30 min versus 16 h in the case of wild-type Eps15). This could indicate that oligomerization of Eps15 delays its nuclear import possibly by masking the NLS (Poupon et al., 2002), a control mechanism described for several proteins (reviewed by Poon and Jans (2005)). Regarding Eps15 nuclear export, it is tempting to speculate that this process might be regulated directly or indirectly by ubiquitination since the Eps15 NES is located within the ubiquitin-interacting motif (UIM; Polo et al., 2002). In the case of Eps15R and Dab2, nuclear access seems to be regulated by alternative splicing. The three shorter variants of Eps15R are equally abundant in CCPs but show stronger nuclear staining than the full-length form (Poupon et al., 2002). The alternatively spliced p67 form of Dab2 is found both in cytoplasm and the nucleus, while the full-length p96 form is mainly cytoplasmic (Cho et al., 2000). p67 lacks the amino acid residues 230–447 with clathrin-binding sites, poorly binds to AP-2, and does not localize to vesicles (Morris and Cooper, 2001). It is, thus, likely that incorporation into endocytic complexes excludes the binding to nuclear import factors.

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In some instances, an increased nuclear accumulation of endocytic proteins can be observed upon cell stimulation. HIP1 interacts directly with androgen receptor (AR) and accumulates in the nucleus upon androgen stimulation (Mills et al., 2005). AR also translocates to the nucleus in response to androgen and it is likely that the nuclear import of HIP1 is facilitated by the interaction with AR. b-Arrestins translocate to the nucleus in response to the activation of G proteincoupled receptors (GPCRs). This effect is clearly specific for certain receptors, as b-arrestin1 translocates to the nucleus in response to d- and k-opioid receptor, but not m-opioid or b-adrenergic receptors (Kang et al., 2005). Similarly, stimulation of hOR17-4, but not other odorant receptors, induces nuclear translocation of b-arrestin2 (Neuhaus et al., 2006). In unstimulated cells, the relative distribution of b-arrestin2 between the nucleus and cytoplasm varies among cell types (Song et al., 2006). Another example of regulated shuttling is APPL1, which upon EGF treatment or oxidative stress dissociates from endosomal membranes and transiently translocates to the nucleus (Miaczynska et al., 2004). Nuclear translocation can be cell cycle dependent, as shown with many proteins regulating cell proliferation, like in the case of TSG101 and Dab2. TSG101 is found on endosomes, in the cytoplasm and in the nucleus during interphase, and colocalizes with the mitotic apparatus during mitosis (Xie et al., 1998). It accumulates in the nucleus in G1 and S phases, but its expression level remains constant throughout the cell cycle (Zhong et al., 1998). Dab2 is found in the nucleus of pre/postmitotic cells, in contrast to the cytoplasmic localization during interphase (Mishra et al., 2002).

Nuclear functions of clathrin adaptors
HIP1 has been shown to stimulate transcription in a Gal4-based reporter assay and to induce expression of luciferase from a promoter containing androgen-responsive elements (ARE; Mills et al., 2005). Furthermore, HIP1 interacts with and enhances the transcriptional activity of AR and coimmunoprecipitates with DNA regions containing AREs. A model based on these observations envisages that upon androgen stimulation HIP1 associates with AR and translocates to the nucleus where it binds to the promoters of androgen-responsive genes, thus co-activating their expression (Mills et al., 2005; Vecchi and Di Fiore, 2005). Of interest, HIP1 reduces the rate of AR degradation which could potentially prolong the cellular responses to androgen. Finally, in addition to its role in androgen-regulated gene expression, HIP1 also acts as a coactivator for estrogen and glucocorticoid receptors (Mills et al., 2005). The transcriptional activity of Eps15 was shown in a Gal4-based reporter assay. However, no biological function of Eps15 in the nucleus has been proposed (Vecchi et al., 2001). Knowing that Eps15 is tyrosinephosphorylated in response to several growth factors (van Delft et al., 1997), one might speculate that Eps15 could – in addition to its role in endocytosis – act as a signal transducer which directly enters the nucleus to exert a biological function. Interestingly, Eps15 and Eps15R synergize with Hrb through a direct interaction via their EH motifs and enhance the function of viral regulatory protein Rev in the CRM1-dependent export pathway (Doria et al., 1999; Salcini et al., 1997). Although it was postulated initially that Eps15/ Eps15R-Hrb binding takes place in the cytoplasm (Doria et al., 1999), it is possible that these interactions occur also in the nucleus. Nucleocytoplasmic shuttling of Eps15/Eps15R might be responsible for regulation of the Rev export pathway, initially identified to transport unspliced viral mRNA. Moreover, Eps15 and Hrb seem to increase Rev stability. Since Rev protein is less stable in the cytoplasm (Kubota et al., 1996), the reimport of Rev to the nucleus by Eps15 and Hrb could protect it from degradation. The role of Eps15 may not be restricted to the regulation of Rev trafficking, but also controlling localization and stability of other proteins employing the route operationally defined as ‘‘the Rev export pathway’’. Transcriptional activity of CALM protein in a Gal4 reporter assay (Vecchi et al., 2001) has been attributed to its transcriptional activator domain (TAD; Archangelo et al., 2006). CALM directly interacts with CATS, a nuclear transcription factor expressed in lymphoid organs (Archangelo et al., 2006). CATS could potentially bring CALM to the nucleus, where it might regulate transcription of its target genes. Alternatively,

Nuclear functions of endocytic proteins
In the majority of presented cases the exact biological relevance of nucleocytoplasmic shuttling of endocytic proteins remains unclear. Typically, the most common nuclear function of endocytic proteins seems to be regulation of gene expression. As transcriptional coactivators and corepressors they may act as scaffolds recruiting the basal transcription apparatus or functioning in chromatin modification/remodeling. They may also bind directly to DNA, or operate as coregulators of known transcription factors, such as steroid receptors. Accordingly, for many proteins presented in this review, potential transcriptional activity has been demonstrated in Gal4 reporter assays (Keegan et al., 1986; Table 2). In most cases the target genes for regulation by endocytic proteins are unknown, as no relevant studies were undertaken. However, for a few proteins such as HIP1, b-arrestin, Dab2, and TSG101, the nuclear functions have been described in more detail.

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Table 2. targets Protein

Nuclear interaction partners and the related functions of endocytic proteins in the nucleus, including the transcriptional Binding partner p300, CREB - ATP-dependent helicase DHX8, GAPDH-2, Histone H2A.X, KIF14, RCC-1 - ERK2 - ERK2 Specific function in the nucleus Increases acetylation of histone H4 (Kang et al., 2005) - Potentially regulates gene expression after fertilization (Neuhaus et al., 2006) - Stimulates activity of retinoid acid receptors (Piu et al., 2006) - Enhances nuclear localization of activated ERK without affecting the total amount of phosphoERK upon activation of b2-adrenergic receptor (Kobayashi et al., 2005) - Relocalizes MDM2 from the nucleus to cytoplasm and stabilizes p53 upon GPCR stimulation (Wang et al., 2003a) - Relocalizes JNK3 from the nucleus to cytoplasm, increasing cytoplasmic activity of JNK3 (McDonald et al., 2000) - Negatively regulates NF-kB activation (Gao et al., 2004) - Inhibits AP-1 and NF-kB transcriptional activity (Wang et al., 2006) - Inhibits NF-kB activity (Witherow et al., 2004) - Impairs translocation of ERK2 to the nucleus and inhibits Elk-1-driven transcription downstream of angiotensin II type 1a receptors (Tohgo et al., 2002) Unknown – possibly related to CATS-dependent transcription (Archangelo et al., 2006) - Chromatin remodeling and stable gene silencing (Stauffer et al., 2001) Affected genes and promoters c-fos, p27 - ND

b-Arrestin1 b-Arrestin2

- 3X-DR1-Luc reporter gene - ND

- MDM2

- p21

- JNK3

- ND

- IkBa b-Arrestins - TRAF6 - IkBa - ERK2

- IL-1b, IL-6, IL-8 - Ccl2, Tnf, Nfkbiz, IL-6, IL-8 - NF-kB-responsive luciferase reporter - Elk-1-responsive luciferase reporter

CALM CHMPs

CATS - Chromatin remodeling proteins: SSRP1, SMARCA4/ BRG1 (Tsang et al., 2006), Pcl, BMI1 (Stauffer et al., 2001) - HIPK2, UBE2I, PIAS2 - TAK1 kinase - Smad2/3

Reporter genes HIS3 and LACZ in Gal4 assay - ND

- Potential function in the nuclear sumoylation machinery (Tsang et al., 2006) - Increases TGFb-mediated gene transcription (Hocevar et al., 2005) - Restores TGFb-mediated phosphorylation, translocation, and transcriptional responses of Smads (Hocevar et al., 2001) - Inhibits TPA-induced AP-1 activity (Tseng et al., 1999) - Uncouples MAPK activation and c-Fos expression (He et al., 2001) - Inhibits accumulation of b-catenin and decreases canonical Wnt pathway (Hocevar et al., 2003) - Unknown – possibly related to coactivation of steroid receptors Regulate the inhibitory activity of ELL (suppress negative regulation of polymerase activity in promoter-specific transcription) (Shilatifard, 1998)

- ND - Fibronectin - Fibronectin, PAI-1

Dab2

- ND (activity stimulated by phosphorylation of Ser24) - ND - Axin and Dvl-3 - ARIP3/PIASxa (Cho et al., 2000) EAPs RNA polymerase II elongation factor ELL

- TPA-responsive element from the promoter of collagenase gene - c-Fos - TCF/LEF-1-responsive promoters - ND AdML promoter

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Table 2. (continued ) Protein Epsin1 HIP1 TSG101 Binding partner PLZF transcription factor Androgen receptor (AR) - ND Specific function in the nucleus Unknown – possibly related to PLZF-dependent transcription (Hyman et al., 2000) Co-activates AR-driven transcription (Mills et al., 2005) - General transcriptional suppressor (Watanabe et al., 1998) Affected genes and promoters ND Androgen-responsive genes containing AREs - CAT reporter plasmids with estrogen response element, thymidine kinase promoter, b-globin promoter, SV40 promoter (Watanabe et al., 1998) - Glucocorticoid receptorresponsive MMTV promoter - Glucocorticoid receptorresponsive MMTV promoter - Reporter genes regulated by estrogen receptor- and thyroid hormone receptorresponsive elements - AR-responsive MMTV promoter - p21 (Ruland et al., 2001)

- AF-1 domain of glucocorticoid receptor - Daxx transcriptional corepressor of nuclear hormone receptors - p300 transcriptional cofactor

- Reduces glucocorticoid transcriptional activation (Hittelman et al., 1999) - Cooperates with Daxx in repression of glucocorticoid receptor transcriptional activity (Muromoto et al., 2004) - General transcriptional repressor function independent of p300 binding (Sun et al., 1999)

- Monoubiquitinated AR and AATF transcription factor - Monoubiquitinated MDM2

- Rta viral transcription factor
ND, not determined.

- Prevents polyubiquitination of AR, enhancing its transcriptional activity (Burgdorf et al., 2004) - Inhibits degradation of MDM2 and reduces protein levels of transcription factor p53 (Li et al., 2001) - Increases transactivating activity of Rta (Chua et al., 2007)

- Rta-responsive EpsteinBarr virus late genes

CALM could compete with CATS for some regulatory elements in DNA. In the case of Epsin1, the Gal4 reporter assay revealed no direct transcriptional activity (Vecchi et al., 2001). Instead, nuclear presence of Epsin1 could somehow affect transcription of specific genes due to its interaction with PLZF, a DNA-binding transcriptional repressor (Hyman et al., 2000). b-Arrestins are quite exceptional among CLASPs, since their nuclear functions, including regulation of gene transcription, have been quite intensively studied in various systems. A recent review has been dedicated to this topic (Ma and Pei, 2007). Briefly, b-arrestins bind to a number of signal transducers and nuclear proteins and some of these interactions are specific for only one type of b-arrestins (summarized in Table 2). Traditionally, b-arrestins have been characterized as regulators of GPCR signaling through their effects on internalization and desensitization of receptors. By now, involvement of b-arrestins in several other signaling pathways has been studied, including those involving NF-kB (Gao et al., 2004; Wang et al., 2006; Witherow et al., 2004),

mitogen-activated protein kinases (MAPK; Kobayashi et al., 2005; Tohgo et al., 2002) and retinoid acid receptors (Piu et al., 2006). Two mechanisms of b-arrestin action are particularly worth mentioning. First, b-arrestin1 seems to function in scaffolding transcription factors upon GPCR stimulation, as it binds to the promoter regions of the c-fos and p27 genes, recruiting histone acetyltransferase p300 to the transcription factor CREB (Kang et al., 2005). This enhances specific acetylation of histone H4, leading to chromatin reorganization and increased gene expression. The second mechanism of action employed by b-arrestins is related to their ability to shuttle to and from the nucleus, typically resulting in redirection of their binding partners out of the nucleus. For example, overexpressed b-arrestin relocates the ubiquitin protein ligase MDM2 to the cytoplasm, leading to increased activity of p53 (Wang et al., 2003a). Even though shuttling of Dab1 in and out of the nucleus is well documented (Honda and Nakajima, 2006), there is no described nuclear function of this protein outside the canonic Reelin signaling pathway. On

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the other hand, Dab2 acts as an adaptor in several signaling pathways, affecting gene transcription downstream of a variety of cellular stimuli (Table 2). The fusion of a Gal4 DNA-binding domain with a short splice variant p67, and to a lesser extent with p96, displays intrinsic transcriptional activity, with the proline-rich C-terminal region of Dab2 being essential for the transcriptional activation (Cho et al., 2000). The function of Dab2 has been characterized in the context of the transforming growth factor b (TGFb) pathway, where Dab2 transiently associates with signal transducers Smad2 and Smad3 in a ligand-dependent manner (Hocevar et al., 2001). Interestingly, nuclear translocation and transcriptional activation of Smad2/3 is dependent on TGFb receptor endocytosis (Penheiter et al., 2002). Dab2 is required for TGFb-mediated activation of Jun N-terminal kinase (JNK), which leads to expression of fibronectin and increased cell motility (Hocevar et al., 2005). In addition, Dab2 serves as a negative regulator of transcriptional complex AP-1, formed by c-Fos and c-Jun and associated with cell proliferation and tumorigenicity. In this case, both p96 and p67 forms of Dab2 can inhibit the AP-1 activity induced by TPA (12-O-tetradecanoylphorbol-13-acetate) measured in luciferase reporter gene assays (Tseng et al., 1999). Another pathway in which Dab2 was demonstrated to act is Wnt signaling. While the overexpression of Dab2 increases JNK activation in a non-canonical pathway stimulated by Wnt-5A, it negatively regulates the canonical Wnt signaling. Through the interactions with Dvl-3 and axin, Dab2 was found to stabilize the b-catenin destruction complex upon Wnt-3A stimulation (Hocevar et al., 2003). Mouse embryonic fibroblasts derived from Dab2 conditional null animals exhibit increased nuclear levels of b-catenin, as well as higher basal and Wnt-induced reporter activity (Hocevar et al., 2003). Finally, Dab2 has at least two partners that are nuclear proteins with established functions in gene transcription: ARIP3 (androgen receptor-interacting protein 3; Cho et al., 2000) and mDaIP2 (mouse disabled 2-interacting protein 2; Choi et al., 2005). SUMO protein ligase ARIP3, also known as PIASxa, has no intrinsic transcriptional activity, but it binds to AR and enhances its activity (Kotaja et al., 2000; Moilanen et al., 1999). mDaIP2, a mouse orthologue of human Nostrin (nitric oxide synthase trafficker), binds to both forms of Dab2 and is located in the nucleus and cytoplasm (Choi et al., 2005). One study suggests that mDaIP2 binds to its own promoter region and represses its activity, thus acting as autorepressor (Kim et al., 2005).

Nuclear functions of endosomal proteins
APPL proteins are implicated in the regulation of cell proliferation because their depletion by RNAi reduced

the proliferation rate of HeLa cells. This observation can be functionally connected to their binding to the nucleosome remodeling and histone deacetylase complex NuRD/MeCP1 (Miaczynska et al., 2004). The NuRD complex acts as repressor of gene expression and is involved in a number of cellular and developmental processes (Ahringer, 2000; Feng and Zhang, 2001). In contrast to the endocytic adaptors presented so far, the involvement of ESCRT complexes in nuclear activities has been studied well before their endocytic function was discovered. For example, there are several known functions of TSG101 in addition to its endocytic role (schematically shown in Fig. 2). TSG101 not only inhibits transcription in the Gal4 assay, but also suppresses the activity of herpes simplex virus (HSV)encoded transcriptional activator protein (VP16) and the basal promoter activity of commonly used thymidine kinase, rabbit b-globin, and large T antigen of simian virus 40 (SV40) promoters (Watanabe et al., 1998). TSG101 was shown to suppress the transcriptional activity of the nuclear hormone receptor superfamily, including receptors for estrogen, androgen, glucocorticoid, vitamin D, retinoic acid, and thyroid hormone (Hittelman et al., 1999; Sun et al., 1999; Watanabe et al., 1998). Protein pattern searches identified multiple DNA-binding motifs characteristic of transcription factors in the TSG101 sequence, including a leucinezipper motif in its coiled-coil region (Li and Cohen, 1996). Indeed, the coiled-coil domain of TSG101 is required for its activity as a general transcriptional suppressor. Conversely, the N-terminal region fused to the Gal4 DNA-binding domain activates transcription (Sun et al., 1999). The N-terminal region containing the UEV domain (homologous to the ubiquitin-conjugating enzyme domain) is required for efficient activation of androgen receptor-mediated transcription (Burgdorf et al., 2004). Interestingly, TSG101 seems to prevent polyubiquitination of the androgen receptor, thereby locking it in the monoubiquitinated form that is transcriptionally active. Thus, one functional activity of TSG101 – binding to monoubiquitinated proteins – seems to affect distinct processes depending on cellular localization: transcriptional activity of nuclear hormone receptors, sorting of internalized cargo in late endosomes, and budding of viruses from the plasma membrane (Fig. 2). Another non-endocytic function of TSG101 concerns its participation in the p53 pathway. Homozygous TSG101À/À embryos die at day E6.5 and show decreased proliferation (Ruland et al., 2001). The mutated embryos display an elevated p53 protein level and increased transcription of a p53 effector, the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1 (Ruland et al., 2001). This phenotype could be explained by the observation that TSG101 stabilizes MDM2 by inhibition of its ubiquitination and degradation, which leads to the downregulation of p53 and

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Fig. 2. Endocytic and nuclear functions of TSG101. Domains in TSG101: UEV, ubiquitin E2 variant; Pro, proline-rich; CC, coiledcoil. Other abbreviations: AR, androgen receptor; GR, glucocorticoid receptor. See text for detailed description.

stimulation of the cell cycle (Li et al., 2001). An interesting example of the role of TSG101 in gene transcription comes from its activity in the replication of Epstein–Barr virus. Interaction with Rta, a transactivator of viral lytic genes, causes accumulation of TSG101 in the nuclei and recruitment to the viral promoters (Chua et al., 2007). TSG101 contributes to efficient binding of Rta to these promoters, and augments the transactivating activity of Rta. Depletion or mutation of TSG101 leads to decreased yield of virus particles. This activity of TSG101 is quite different from its role in the release of new viral particles from the host cell, as it has been shown for HIV virus (Garrus et al., 2001). The ESCRT-II subunits are conserved from yeast to mammals, and apparently in addition to their importance in membrane trafficking they have acquired

nuclear functions. The mammalian orthologues of the yeast ESCRT-II complex have been first described as the ELL-associated proteins (EAPs). ELL is the RNA polymerase II elongation factor, that binds to Pol II and has a double role in the process of gene transcription: it stimulates the rate of elongation by suppressing transient pausing at multiple sites along the DNA, and also acts as negative regulator of polymerase activity by inhibiting promoter-specific initiation of transcription by Pol II. The complex named ‘‘Holo-ELL’’ was purified from rat liver and consisted of ELL together with three subunits of ESCRT-II: EAP20/hVps25, EAP30/hVps22/hSnf8, and EAP45/hVps36/hVac3 (Shilatifard, 1998). The Holo-ELL heterotetramer increases the catalytic rate of transcription elongation in vitro. Interestingly, binding of EAPs suppresses the transcriptional inhibitory activity of ELL (Kamura

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et al., 2001; Schmidt et al., 1999; Shilatifard, 1998). In addition, Snf8p was isolated from a screen for yeast mutants defective in derepressing expression of glucoserepressed genes (Yeghiayan et al., 1995). Based on this evidence, it is clear that the ESCRT-II complex can affect expression of certain genes by controlling the rate of promoter-specific initiation of transcription. Similarly to ESCRT-II, mammalian ESCRT-III subunits (CHMPs) were initially characterized in the nuclear context. CHMP, upon discovery of endosomal functions, acquired an additional synonym: charged multivesicular body protein, which is used interchangeably. In a systematic screen for human CHMP protein partners, 19 out of 45 novel interactions involve nucleusrelated proteins (Tsang et al., 2006). For example, SSRP1 and SMARCA4/BRG1, binding to CHMP1B and CHMP5, respectively, are involved in chromatin remodeling. Of interest, several CHMP proteins participate in a network involved in nuclear sumoylation processes, binding to SUMO-conjugating enzyme UBE2I, E3 SUMO ligase PIAS2, and homeodomaininteracting protein kinase 2 (HIPK2; Tsang et al., 2006). Sumoylation of HIPK2, the nuclear protein kinase that acts as corepressor for homeodomain transcription factors, is involved in DNA damage-induced transcriptional silencing (Roscic et al., 2006). Mammalian Vps46 (CHMP1) controls chromatin structure and cell cycle progression (Stauffer et al., 2001). CHMP1 binds to Polycomb like protein that, together with other members of the Polycomb group (PcG), maintains the chromatin condensation and gene silencing during development. CHMP1 contains a predicted bipartite NLS and is distributed both in the cytoplasm and in the nuclear matrix. In contrast to the single 32-kDa form found in the cytoplasm, CHMP1 in the nucleus exists predominantly as a 35-kDa form and is likely subjected to covalent posttranslational modification(s) (Stauffer et al., 2001). Overexpressed CHMP1 localizes to distinct subnuclear regions that contain nuclease-resistant condensed chromatin. It has been proposed that CHMP1 plays a role in mitotic chromosome condensation, acting at a transition zone between active and inactive chromatin domains. Exogenous CHMP1 decreases mitotic index and BrdU incorporation, inhibiting the transit of cells through the S-phase without increase in apoptosis (Stauffer et al., 2001).

various organisms, including yeast, Drosophila, and mammals. In general, the endocytic regulators described here are involved in regulation of transcription at two levels: (1) Chromatin remodeling – which allows initiation of transcription by ‘‘loosening’’ nucleosomes, permitting the entry of DNA binding transactivators, Pol II, and the basal transcription machinery to the regulatory regions of the promoters (Falbo and Shen, 2006; Kumar et al., 2004). The examples of proteins operating at this level are: APPL1/2 and ESCRT-III proteins, which bind to chromatin remodeling complexes. (2) Regulation of transcription initiation – the step where transcription factors facilitate recognition of promoter-specific DNA sequences. Most endocytic proteins act as transcriptional coregulators by binding to known transcription factors (Table 2). This association can affect the activity (e.g. TSG101), stability (e.g. HIP1), or localization (e.g. b-arrestins) of transcription factors. A key question to be answered with respect to the nuclear roles of endocytic proteins is to what extent these diverse functions are interconnected, thus serving to coordinate various cellular processes, or whether they are largely independent. So far the limited experimental evidence indicates that both possibilities may be valid depending on a particular protein or function. On the one hand, functional transcriptional regulation that is a feature of many endocytic proteins may represent a powerful channel for communicating information from the extracellular environment to the nucleus. Extracellular signals that influence the relative rates of nuclear import and export of the signal transducer proteins can control gene expression. In some cases, as it is with b-arrestins and APPL, ligand stimulation that initiates endocytosis also results in nuclear translocation. It is likely that in a physiological situation the nuclear translocation and a localized activity of endocytic proteins are affected by agonistdependent endocytosis of cell surface receptors. On the other hand, molecules of the same protein may have unrelated distinct functions depending on whether they are found in cytoplasmic, membrane-bound or nuclear compartments. The nuclear translocation of Epsin1, Eps15, CALM and a-adaptin is not affected upon inhibition of endocytosis (Vecchi et al., 2001). Mutated b-arrestin that is impaired in binding to CCVs and cannot promote internalization of agonist-activated GPCRs, still is able to potentiate the activity of nuclear retinoid receptors (Piu et al., 2006). The reverse is also true: mutation in the NES of b-arrestin2 did not affect the kinetics of endocytosis of plasma membrane GPCR (i.e. thyrotropin-releasing hormone receptor-1; Scott

Concluding remarks and future questions
The nuclear functions of endocytic proteins appear to be quite distinct from their roles played in membrane trafficking. Examples for nucleocytoplasmic shuttling and involvement in gene transcription of the evolutionary conserved endocytic proteins are present in

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et al., 2002). These observations indicate that at least in particular cases there are separate pools of proteins found on CCVs/endosomes and in the nucleus. The mechanisms, which diversify these functionally distinct pools, are not clear. Whether the same molecule of a given protein can function interchangeably in the nucleus and in the cytosol during its lifetime remains also an open question. It is likely that the endocytic and nuclear functions are mutually exclusive, and that the binding to endocytic complexes prevents translocation to the nucleus. Only upon solubilization and detachment from endocytic compartments, the adaptors are free to function in the nucleus. This has been proposed for HIP1, as the mutation impairing binding to lipids and CCPs enhances its coregulatory effect on AR-dependent transcription (Mills et al., 2005). In addition to regulation of transcription, adaptor proteins that have nuclear functions are commonly found to affect cell proliferation and/or apoptosis, and have a role as tumor suppressor (e.g. TSG101, Dab2, HIP1). Genes encoding endocytic proteins are involved in genetic translocations that are found in leukemias, such as HIP1/PDGFb receptor (Ross et al., 1998), CALM/AF10 (Dreyling et al., 1996) and MLL/CALM (Wechsler et al., 2003). Even though the tumorigenic properties of such fusions may be due to impaired endocytic functions, it is possible that the nuclear activities are also altered. The endocytic adaptor proteins are known for their self-assembling properties, due to the multiple protein– protein interaction domains. They were proposed to act as scaffold platforms not only in endocytic compartments, but also recruiting transcription factors and cofactors. For the proteins that display both nuclear and cytoplasmic distribution, it is conceivable that they exhibit different preferences for binding partners and/or various post-translational modifications in distinct subcellular compartments. Given the fact that ESCRTII subunits have dual functions acting as an intact complex in MVBs and in the nucleus, it would be interesting to find other mechanistic similarities responsible for the cargo sorting and transcriptional regulation by endocytic adaptors. The scaffolding function of endocytic proteins in the nucleus is reminiscent of specific nuclear functions of the actin meshwork (Miralles and Visa, 2006; Rando et al., 2000). Actin has an NES and accumulates in the nucleus upon treatment with LMB (Wada et al., 1998). Involvement of actin in gene transcription occurs at several levels: binding RNA polymerases, controlling nucleocytoplasmic distribution of transcription factors, and participation in chromatin-remodeling complexes. Intriguingly, very recently nuclear functions have been described for clathrin itself. A small fraction of clathrin heavy chain (CHC) is present in the nucleus, where it specifically enhances p53-dependent transactivation (Enari et al.,

2006). CHC binds to the p53-responsive promoter and stabilizes interaction between p53 and p300 histone acetyltransferase. Binding with p53 competes with clathrin light chain. Moreover, complete clathrin triskelia are important for kinetochore fiber stability during mitosis (Royle and Lagnado, 2006). A number of open questions will be likely addressed in future research, including a possible functional interdependence between the nuclear and endocytic roles of the same proteins. It will be interesting to determine whether other classes of proteins regulating membrane transport also possess specific roles in the nucleus or whether endocytic adaptors preferentially developed nuclear functions. Studies on the mechanisms underlying partitioning of endocytic proteins to the cytoplasmic and nuclear pools will contribute to our understanding of the inter-compartmental trafficking between different organelles. Undoubtedly, we should expect more surprises as the endocytic proteins continue to reveal their double life.

Acknowledgments
We thank the group members for critical reading of the manuscript. Research in M. Miaczynska’s laboratory is supported by an International Research Scholar grant from the Howard Hughes Medical Institute, a Senior Research Fellowship from the Wellcome Trust (076469/Z/05/Z), by the European Union LSHG-CT2006-019050 (EndoTrack), Polish Ministry of Science and Higher Education (2P04A03828), and Max Planck Society (Partner Group programme).

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