PERSPECTIVES OPINION
DEP domains: structurally similar but functionally different Sarah V. Consonni, Madelon M. Maurice and Johannes L. Bos
Abstract | The Dishevelled, EGL‑10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation. This article is dedicated to the memory of Tony Pawson, who taught us the importance of protein domains in signal transduction. Cell surface receptors transduce extracellu‑ lar signals by assembling protein complexes at the inner side of the plasma membrane. The targeting of signalling complex compo‑ nents to the membrane not only increases the possibility of complex formation but also increases the strength of the transduced signal1. Moreover, due to the modular nature of signalling molecules, different types of complexes can easily form in time and space2. Each protein domain recognizes specific protein sequences or lipids, and the binding is frequently regulated by a distinc‑ tive signalling event. One example is the Dishevelled, EGL‑10 and pleckstrin (DEP) domain: Dishevelled (dsh; also known as DVL in higher eukaryotes) was first identi‑ fied in Drosophila melanogaster, whereas egg-laying defective protein 10 (EGL‑10) and pleckstrin were first identified in Caenorhabditis elegans and mammalian cells, respectively 3–5. In mammals, the DEP domain is present in a limited number of protein families (FIG. 1) that have diverse functions in signal transduction. DEP domains have also been identified in bacte‑ ria (for further information see the SMART (simple modular architecture research tool) database), which indicates that the DEP domain did arise early during evolution.
Some DEP domain-containing proteins, for example the regulator of G protein signal‑ ling (RGS) family of proteins, are present in yeast, whereas others such as DVL appeared later in evolution and are only found in higher eukaryotes6,7. RGS proteins contain both a DEP domain and an RGS domain; the RGS domain confers GTPase-activating protein (GAP) activity 8. Interestingly, the RGS domain–DEP domain module that is present in yeast and mammalian RGS pro‑ teins seems to be conserved in the mamma‑ lian DVL signalosome, in which the scaffold protein Axin contains the RGS domain9,10. A predominant function of DEP domains is plasma membrane anchoring, but DEP domains carry out several addi‑ tional functions by using different binding interfaces (TABLE 1). In this respect, DEP domains seem to differ from many other protein domains with a more restricted mechanism of action. However, the use of multiple binding surfaces may be more common than originally anticipated. For instance, SRC homology 2 (SH2) domains were long considered to have a single binding pocket for phosphotyrosine moieties, but more recently it was shown that SH2 domains can interact with kinase domains that use a different binding inter‑ face11,12. DEP domains can be considered a paradigm for the growing group of protein domains that use different interfaces for different functions.
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In the past few years, analyses on the structure and function of DEP domains have shown that this protein module is one of the prime examples of a regulated domain in signal transduction. In this Opinion article we focus on the DEP domain-containing proteins DVL, RGS, exchange protein directly activated by cAMP 1 (EPAC1; also known as RAPGEF3), pleckstrin and PtdIns(3,4,5)P3-dependent RAC exchanger (PREX). We examine the various mechanisms of DEP domainmediated interaction with proteins and lipids, and discuss how DEP domains function in the spatiotemporal control of diverse signalling events. Structure of DEP domains DEP domains consist of approximately 100 amino acids and they all share a high degree of sequence and structural similari‑ ties. They have a characteristic α/β fold, and they are comprised of a conserved helical core and a protruding β‑hairpin arm, which is located between the core helices α1 and α2 (REFS 4,13) (FIG. 2). The β‑hairpin module, which often bears residues that are impor‑ tant for mediating molecular interactions, is less conserved than the helical core14. In some cases, such as for DVL and EPAC, polybasic residues in the β‑hairpin are required for the recruitment to the plasma membrane14–16 (FIG. 2a,b). Furthermore, studies revealed that DEP domains encompass distinct positive and negative patches that influence their plasma membrane binding abilities. The DEP domain of RGS9 (FIG. 2c) is oriented in such a way that a cluster of positively charged residues faces the membrane17. Conversely, the DEP domain of pleckstrin (FIG. 2d) has mainly a negatively charged cluster on its exposed surface, whereas the DEP domains of EPAC and DVL combine both positive and negative patches. In some instances, the DEP domain requires an additional fold or motif to interact with membrane anchors. This is the case for the DEP domain of RGS9, which needs an adjacent DEP helical extension (DHEX) fold for membrane binding 17,18. Likewise, the DEP domain of DVL2 requires a neighbouring Tyr-based YHEL motif for membrane anchoring VOLUME 15 | MAY 2014 | 357
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PERSPECTIVES DVL
DIX
PDZ
EGL-10, RGS EPAC Pleckstrin FYVE PREX1, PREX2 GPR155
DH
DEP DEP
GGL
RGS
CNB
DEP
CNB
REM
PH
DEP
PH
FYVE
DEP
CPN
PH
DEP
DEP
PDZ
MTD
DEP DEP
PDZ
DEPTOR
DEP
RA
CDC25HD
PtdIns(5)K PDZ
IP4P
Figure 1 | Domain architecture of mammalian DEP domain-containing proteins. The Dishevelled, EGL‑10 and pleckstrin (DEP) domain is shown in pink and other domains that |are present Cell in more than Nature Reviews Molecular Biology one protein are represented in an identical manner. Protein modules that are adjacent to the DEP domain often aid the DEP domain in its membrane binding capabilities. This is the case for the DVL (Dishevelled) DEP domain that also requires the flanking carboxy-terminal region to bind to Frizzled receptor and for the RGS (regulator of G protein signalling) DEP domain that uses the GGL (Gγ subunitlike) domain to bind to Gβ5. In the case of EPAC (exchange protein directly activated by cAMP), pleckstrin and PREX (PtdIns(3,4,5)P3-dependent RAC exchanger), conformational changes that relieve the protein from an autoinhibited state or intramolecular interactions are needed for facilitating mem‑ brane localization of the protein. For other proteins the function of the DEP domain is still elusive. CDC25HD, CDC25 homology domain; CNB, cyclic nucleotide-binding; CPN, chaperonin; DEPTOR, DEP domain-containing mTOR-interacting protein; DH, DBL homology; DIX, Dishevelled and Axin; FYVE, FAB 1, YOTB, VAC 1 and EEA1; GPR155, G protein-coupled receptor 155; IP4P, inositol polyphos‑ phate 4‑phosphatase; MTD, membrane transport domain; PDZ, post-synaptic density 95, discs large and zonula occludens 1; PH, Pleckstrin homology; PtdIns (5)K, phosphatidylinositol‑5‑kinase; RA, RAS-association; REM, RAS exchange motif.
during non-canonical signalling, and DVL1 contains a DEP domain together with a flanking carboxy terminal region, to bind to a discontinuous motif on the seven-span receptor Frizzled (FZ) during canonical signalling 19,20. Thus, despite the structural similarity in their core regions, small differences between DEP domains account for their differential mode of regulation and for their ability to coordinate diverse signalling events in space and time. DVL DEP domain in signal regulation DVL is a cytoplasmic phosphoprotein involved in the regulation of WNT signalling that was first identified in D. melanogaster as a regulator of early embryo polarity 21. It is composed of an amino‑terminal DVL and Axin (DIX) autointeraction domain that is responsible for the formation of multimeric complexes, a central post-synaptic density 95, discs large and zonula occludens 1 (PDZ) domain that is required for binding to FZ receptor and a C‑terminal DEP domain that is important for specific membrane localization14,22–24 (FIG. 1).
DVL DEP domain in membrane targeting. Activation of FZ receptors by WNT ligands activates several downstream signalling pathways, most notably the canonical WNT pathway that stabilizes β‑catenin signalling to control cell fate and the non-canonical planar cell polarity (PCP) signalling path‑ way for cytoskeletal remodelling 25,26. In the canonical pathway, WNT binding to FZ and the low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 (LRP5/6) receptor results in the formation of a DVL-dependent signalosome, which includes the receptors FZ, LRP5/6, DVL, the adaptor protein Axin and additional proteins (FIG. 3a). This interaction is mediated in part by a weak interaction of FZ with both the PDZ and the DEP domains of DVL. The PDZ domain binds to a short linear sequence in the cytosolic tail of FZ, whereas the DEP domain together with the C termi‑ nus interact with three discontinuous regions that are located in the third intracellular loop and C-terminal tail of FZ20. Each of these interactions is required for membrane recruitment and the proper function of DVL in this pathway. In the context of the noncanonical PCP signalling in D. melanogaster,
358 | MAY 2014 | VOLUME 15
the DEP domain of Dsh interacts with nega‑ tively charged membrane phospholipids and, aided by the Na+/H+ hydrogen exchanger 2 (Nhe2), which keeps an alkaline pH suited for charge-dependent interactions, it interacts with Fz16. Hence, the DEP–phospholipid interaction may contribute to the forma‑ tion of a robust FZ–DVL complex at the plasma membrane (FIG. 3b). Moreover, the DEP domain of another of the three mam‑ malian DVL isoforms, DVL2, aided by a YHEL motif at its C terminus, can interact with μ2 adaptor complexes at the membrane to facilitate clathrin-mediated endocytosis of FZ4 (REF. 19). This is required to regulate surface expression of FZ and therefore for desensitization of signalling 27. DVL DEP domain in signalling complex formation. A number of important WNTinduced post-translational modifications of WNT pathway proteins depend on DVL DEP domain activity. A recent study showed that RIP kinase 4 (RIPK4) binds to DVL and mediates phosphorylation of the PDZ and DEP domains28. The combined phosphorylation events promote the assem‑ bly of DVL2 into large signalling complexes, which leads to maximal β‑catenin stabiliza‑ tion and transcription of WNT-responsive target genes. How DEP domain-mediated intermolecular and intramolecular interac‑ tions are regulated through phosphorylation remains unresolved. In addition, the DVL DEP domain itself controls post-translational modification of other DVL domains and protein partners. Increased Lys63‑linked polyubiquitylation of the DVL DIX domain drives WNT–β‑catenin signalling in cells that lack the deubiquitylation enzyme CYLD; this phenomenon depends on an intact DEP domain29,30. In addition, Xenopus laevis fz3 is phosphorylated after association with dvl. This phosphorylation requires the DEP domain of dvl and is needed for dvl-mediated fz3 desensitization and thus for regulation of PCP signalling 29,30. Hence, the interactions of the DVL DEP domain with both proteins and phospholipids mediate the formation and modification of essential protein complexes in the regulation of WNT signalling (FIG. 3). RGS DEP domain in signal termination Members of the RGS family are GAPs of heterometric G proteins. They use their RGS domain to promote GTP hydrolysis of the Giα subunit thereby switching off G proteincoupled receptor (GPCR) signalling (FIG. 3c). Currently, the best characterized of the RGS family of proteins is the R7 subfamily, which includes RGS6, RGS7, RGS9 and RGS11. www.nature.com/reviews/molcellbio
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PERSPECTIVES Table 1 | Properties of DEP domains DEP protein
DEP binding partner
DEP domain length (amino acids)
Additional requirements for function
Function
Refs
Dishevelled
PA, FZ and μ2 adaptor
~87
DEP with carboxy-terminal flanking region and YHEL motif
Membrane targeting
16,19,20
EGL‑10 and RGS
Gβ5, R9AP and R7BP
~107
DHEX fold
Membrane targeting
17,33–35
EPAC
PA
120 or 121
cAMP-induced conformational change
Membrane targeting
15
Pleckstrin
Intramolecular interaction with PH domain
~116
PKC-induced phosphorylation
Autoinhibition
FYVE
Unknown
81
Unknown
Unknown
4,49
50
PREX1 and PREX2 Intramolecular ~80 interaction with PDZ and IP4P domains, and mTOR
PtdIns(3,4,5)P3‑induced conformational Protein function change
45,48
GPR155
Unknown
~82
Unknown
Unknown
51
DEPTOR
Unknown
81
Unknown
Unknown
52
cAMP, cyclic AMP; DEPTOR, DEP domain-containing mTOR-interacting protein; DHEX, DEP helical extension; EGL‑10, egg-laying defective protein 10; EPAC, exchange protein directly activated by cAMP; FYVE, FAB 1, YOTB, VAC 1 and EEA1; FZ, Frizzled; Gβ5, G protein β subunit subtype 5; GPR155, G protein-coupled receptor 155; IP4P, inositol polyphosphate 4‑phosphatase; PA, phosphatidic acid; PDZ, post-synaptic density 95, Discs large and zonula occludens 1; PH, pleckstrin homology; PKC, protein kinase C; PREX, PtdIns(3,4,5)P3-dependent RAC exchanger; PtdIns(3,4,5)P3, phosphatidylinositol‑3,4,5‑trisphosphate; R7BP, R7 familybinding protein; R9AP, RGS9‑anchoring protein; RGS, regulator of G protein signalling.
RGS proteins of the R7 subfamily are made up of three domains: a DEP domain for membrane targeting at its N terminus, a Gγ subunit-like (GGL) domain for binding Gβ subunits and a C‑terminal RGS domain that is required for its signal termination31,32 (FIG. 1). Structural analysis revealed that the DEP domain of RGS9 has a C‑terminal DHEX that is not found in other DEP domains. The DHEX domain, together with the DEP and GGL domains, interacts with Gβ5 (REF. 17). In addition to binding to Gβ5, RGS proteins of the R7 subfamily a DVL DEP
b EPAC DEP
α1
α3
contributes to the stabilization of the complex and subsequent regulation of the G proteinmediated signalling cascade18. This highlights the multifaceted nature of DEP domainmediated complex assembly at the membrane. The DEP domains that are found in Sst2, the yeast homologue of the RGS family, medi‑ ate binding to the GPCR fungal pheromone mating factor (Ste2 in MATa and Ste3 in MATα cells). Interestingly, Sst2 DEP domains can only bind to the GPCR in its nonphosphorylated state, which suggests that this interaction is regulated35.
c RGS9 DEP
Hairpin
Hairpin
α2
interact with the membrane targeting proteins RGS9‑anchoring protein (R9AP; also known as RGS9BP) and R7 family-binding protein (R7BP; also known as RGS7BP). The DEP domain, despite being needed for this interac‑ tion, is not sufficient for the binding 33. Recent protein–protein interaction mapping revealed that the DHEX domain, together with the DEP domain, is also involved in the inter action with R9AP and R7BP18,34. Site-directed mutagenesis studies suggest that R7BP inter‑ acts with the DEP domain at the surface that also interacts with Gβ5, and R7BP therefore
d Pleckstrin DEP
Hairpin
α1
Hairpin
α1
α1
α2
α2 α3
Figure 2 | DEP domain structures. Cartoon structures of Dishevelled, EGL‑10 and pleckstrin (DEP) domains that show their characteristic α/β fold. DEP domains comprise a conserved helical core, which consists of three heli‑ ces and a protruding β‑hairpin arm between the helices α1 and α2. This helical core is highly conserved in all DEP domains. Only in the case of pleckstrin, there is an additional helix located at the end of the DEP domain, which is
α2 α3
α3
thought to be important for proteinNature mobility.Reviews Arrows |show the location of the Molecular Cell Biology residues in the β‑hairpin that are required for membrane binding. a | Image is based on Protein Data Bank (PDB) entry 1fsh of Dishevelled (DVL)13. b | Image is based on PDB entry 2byv of exchange protein directly activated by cAMP (EPAC)40. c | Image is based on PDB entry 2pbi of regulator of G protein signalling 9 (RGS9)17. d | Image is based on PDB entry 1w4m of pleckstrin4.
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VOLUME 15 | MAY 2014 | 359 © 2014 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES a
b
Frizzled WNT
LRP5, LRP6
DVL
c
Frizzled
WNT
Plasma membrane
GPCR
PA DVL
Axin
R7BP–R9AP β α γ RGS GDP
DAAM
GTP hydrolysis β-catenin
β-catenin
RHO
β-catenin
ROCK Nucleus
d
β-catenin TCF
β-adrenergic receptor PA EPAC1 RAP1 cAMP AF6 RIAM ARAP3 RASIP1 Others
• Polarity • Endothelial junction formation • Cell adhesion • Cell spreading
• Polarity • Actin polymerization
Signal termination
e
f GPCR
β α γ G protein
PtdIns(3,4,5)P3
X PI3K
PRE
RAC
PtdIns(4,5)P2
PKC
Pleckstrin P
Phosphorylation
PAK WAVE MEK1/2 Others Phosphoinositide hydrolysis • Proliferation • Migration • Invasion
• Platelet aggregation • Platelet secretion
Figure 3 | Diversity of pathways that are regulated by DEP domain-containing proteins. Nature Reviews | Molecular Cell in Biology Dishevelled, EGL‑10 and pleckstrin (DEP) domain-containing proteins are shown pink. a | Dishevelled (DVL) during canonical WNT signalling. The DEP domain of DVL binds to the G proteincoupled receptor (GPCR) Frizzled and enables formation of a signalling complex that results in β‑catenin translocation to the nucleus and subsequent transcription of WNT target genes. b | DVL during non-canonical planar cell polarity (PCP)–WNT signalling. By interacting with PA (phosphatidic acid) at the membrane (shown in yellow), the DEP domain of DVL mediates cytoskeletal changes and polarity through DAAM (DVL associated activator of morphogenesis) and subsequent downstream activation of RHO–ROCK (RHO-associated kinase) signalling. c | Regulator of G protein signalling (RGS). To promote GPCR signal termination, RGS proteins associate with their cognate receptor, which is aided by their DEP domain and promotes GTP hydrolysis of the Gα subunit. d | Exchange protein directly activated by cAMP 1 (EPAC1). In response to cyclic AMP (cAMP), EPAC1 translocates to the plasma membrane, where it binds to PA through its DEP domain. This results in activation of plasma membrane-located repressor/activator protein 1 (RAP1), downstream effectors and of RAP-mediated cellular processes, such as cell polarity, cell adhesion, cell spreading and cell–cell junction formation. e | PtdIns(3,4,5)P3‑dependent RAC exchanger (PREX). After activation by phosphatidylinositol‑3,4,5‑ trisphosphate (PtdIns(3,4,5)P3), PREX translocates to the plasma membrane where it binds to the Gβγ subunits and PtdIns(3,4,5)P3 (depicted in red). This results in RAC activation and subsequent cell migra‑ tion, proliferation and invasion. f | Pleckstrin phosphorylation (P) by protein kinase C (PKC) is required for its localization to the plasma membrane, where it binds to PtdIns‑4,5‑bisphosphate (PtdIns(4,5)P2; depicted in red). Downstream signalling then results in the induction of platelet aggregation, and secretion and inhibition of phosphoinositide hydrolysis. AF6, ALL1‑fused gene from chromosome 6 (also known as Afadin); ARAP3, ARF-GAP with RHO-GAP domain, ANK repeat and PH domaincontaining protein 3; LRP, low-density lipoprotein receptor-related protein; MEK, MAPK/ERK kinase (also known as MAPKK); PAK, p21‑activated kinase; R7BP, R7 family-binding protein; R9AP, RGS9‑anchoring protein; RASIP1, RAS-interacting protein 1; RIAM, RAP1‑GTP-interacting adapter molecule (also known as APBB1IP); TCF, transcription factor; WAVE, WASP-family verprolin homology domain-containing. Dashed arrows indicate the end result of the signalling pathway.
360 | MAY 2014 | VOLUME 15
The DEP domain of RGS7 is required for binding to and inhibition of muscarinic acetylcholine receptor M3 but not for other GPCRs36. This clearly shows that DEP domains are not only simply involved in membrane anchoring but also in conferring specificity by selectively interacting with cognate receptors. EPAC DEP domain in phospholipid binding EPAC proteins are guanine nucleotide exchange factors (GEFs) for the small G protein RAP that is involved, among others, in the control of cell adhesion and actin cytoskeletal rearrangements37,38. The two EPAC proteins, EPAC1 (also known as RAPGEF3) and EPAC2 (also known as RAPGEF4), are differentially expressed — EPAC1 is more abundant in the central nervous system, ovaries and uterus, whereas EPAC2 is most prevalent in the pancreas and adrenal gland in mammals39. Both are multi‑ domain proteins that contain an N‑terminal regulatory region, which comprises one (in the case of EPAC1) or two (in the case of EPAC2) cyclic nucleotide-binding (CNB) domains and a DEP domain. The catalytic region at their C terminus harbours the CDC25 homology domain (CDC25HD) for exchange activity, a RAS exchange motif (REM) and a RAS-association (RA) domain (FIG. 1). Both proteins assume a closed auto‑ inhibitory conformation, which is relieved after cyclic AMP (cAMP) binding 40. In the case of EPAC1, the allosteric activation by cAMP is accompanied by a very rapid relocalization of the protein from the cytosol to the plasma membrane. This transloca‑ tion is mediated by the DEP domain, which directly interacts with phosphatidic acid at the membrane (FIG. 3d). A polybasic stretch that encompasses residue Arg82 in the DEP domain is responsible for this binding 15,41 (FIG. 2). This interaction is required for the proper function of EPAC1, as deletion of the DEP domain or alteration of Arg82 to a nonpolar amino acid inhibits cAMP-induced and EPAC1‑mediated cell adhesion41. Amide hydrogen and deuterium mass spectrometry studies on EPAC2 elucidated the possible regulatory mechanisms of its cAMP-induced and DEP domain-mediated tethering to the plasma membrane. After cAMP sig‑ nalling, some regions of the DEP domain become more solvent exposed, resulting in reorientation of the β‑hairpin that contains the essential Arg82 residue and in this way enabling plasma membrane anchoring 15,42. Interestingly, the cAMP-binding domain is located at the same position as the C‑terminal helical extension of the DEP www.nature.com/reviews/molcellbio
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PERSPECTIVES domain (which is important for its func‑ tion) of the R7 subfamily of RGS proteins and DVL. So far, no additional interacting proteins have been identified for EPAC DEP domains. It should be noted that, currently, the function of the DEP domain of EPAC2 is elusive as the DEP domain does not seem to be required for its localization to the plasma membrane15,43. PREX DEP domain in intradomain binding PREX proteins act as GEFs for the small GTPase RAC. They both contain a catalytic DBL homology (DH) domain at their N‑terminal adjacent to a pleckstrin homol‑ ogy (PH) domain that is required for binding to phosphatidylinositol‑3,4,5‑trisphosphate (PtdIns(3,4,5)P3), which is followed by two tandem DEP domains and two tandem PDZ domains. At their C terminus they have an inositol polyphosphate 4‑phosphatase (IP4P) domain that is needed for binding to protein phosphatase 1α (PP1α)44,45 (FIG. 1). PREX is likely to be in an autoinhibited state in the cytosol and translocates to the membrane, where it binds to Gβγ subunits and PtdIns(3,4,5)P3 through its DH and PH domains after activation by PtdIns(3,4,5)P3 (REF. 45) (FIG. 3e). PREX has recently attracted much attention, as it was found to have a role in metastatic cancers. Indeed, PREX1 was found to increase cell proliferation in breast cancer cells and to induce migration and invasion of prostate cancer cells by activat‑ ing RAC46,47. Interestingly, the DEP domain mediates intramolecular interactions with the PDZ and the IP4P domains. Such bind‑ ing is needed for association with Gβγ and for Gβγ-induced GEF activity 48. Pleckstrin DEP domain in autoinhibition Pleckstrin is another DEP domaincontaining protein family. It is one of the main substrates for protein kinase C (PKC) in platelets and comprises two PH domains for protein or lipid interactions and a cen‑ tral DEP domain3 (FIG. 1). The pleckstrin DEP domain structure is more divergent than that of DEP domains in other DEP domain-containing proteins, as it bears an additional β‑strand that is positioned after the β‑hairpin and an additional helix, which is located towards the end of the DEP domain; the helix is thought to be important for increased protein mobility and to facili‑ tate protein–protein interactions4,49. The DEP domain of pleckstrin does not directly mediate membrane anchoring of the pro‑ tein, but it interacts intramolecularly with the N‑terminal PH domain. Such interac‑ tion is only relieved after phosphorylation
by PKC, which enables activation and membrane localization of the protein4 (FIG. 3f). This finding suggests a function for DEP domains in autoinhibitory domain interactions. Concluding remarks and future directions DEP domains are globular protein domains that, despite having diverse mechanisms of action, generally assist in the transloca‑ tion of the cognate protein to the plasma membrane. Often the presence of a specific sequence or exposure of a particular amino acid is pivotal for such accurate spatial regulation of proteins, thus highlighting the importance of sequence context and specificity 14,41. The presence of additional domains or folds that are adjacent to the DEP domain seems to be important for signalling speci‑ ficity. The DVL DEP domain requires a YHEL motif for membrane anchoring during non-canonical signalling, and both the PDZ domain and a C‑terminal region cooperate with DEP to bind to FZ during canonical signalling 19,20. Similarly, the DEP domain of RGS9 needs a DHEX helical fold for membrane localization17,18. Conformational changes also play an important part in the regulation of DEP domain-mediated signalling. This is the case for EPAC and possibly for PREX, which require a conformational change that is triggered by cAMP and PtdIns(3,4,5)P3, respectively, to become active and to local‑ ize to the plasma membrane40,45. Additional levels of regulation might be involved, such as post-translational modifications and domain–domain interactions4,48. This illustrates the diversity of DEP domain-mediated signalling regulation and also emphasizes that the DEP domain is not simply a localization device, but that it also affects protein function by being itself subject to regulation. As our understanding of how DEP domains function and are regulated increases, it is becoming clear that they use many ways to carry out their task in various cellular events (FIG. 3). Further identification of all the factors and regulatory mechanisms that are involved in such regulation will help us to understand how DEP domains achieve such signalling specificity, despite their structural similarities. Importantly, the ability of protein domains to have different functional properties by providing diverse binding interfaces is being more and more appreciated as a recurrent theme in the control of signalling in space and time.
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Sarah V. Consonni and Johannes L. Bos are at the Molecular Cancer Research and Cancer Genomics Netherlands, Center for Molecular Medicine, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, Utrecht, The Netherlands. Madelon M. Maurice is at the Department of Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands. Correspondence to J.L.B. e-mail: [email protected] doi:10.1038/nrm3791 Published online 16 April 2014 Kholodenko, B. N., Hoek, J. B. & Westerhoff, H. V. Why cytoplasmic signalling proteins should be recruited to cell membranes. Trends Cell Biol. 10, 173–178 (2000). 2. Scott, J. D. & Pawson, T. Cell signaling in space and time: where proteins come together and when they’re apart. Science 326, 1220–1224 (2009). 3. Kharrat, A. et al. Conformational stability studies of the pleckstrin DEP domain: definition of the domain boundaries. Biochim. Biophys. Acta 1385, 157–164 (1998). 4. Civera, C., Simon, B., Stier, G., Sattler, M. & Macias, M. J. Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily. Proteins 58, 354–366 (2005). 5. Ponting, C. P. & Bork, P. Pleckstrin’s repeat performance: a novel domain in G-protein signaling? Trends Biochem. Sci. 21, 245–246 (1996). 6. Sierra, D. A. et al. Evolution of the regulators of G-protein signaling multigene family in mouse and human. Genomics 79, 177–185 (2002). 7. Dillman, A. R., Minor, P. J. & Sternberg, P. W. Origin and evolution of dishevelled. G3 (Bethesda) 3, 251–262 (2013). 8. Ross, E. M. & Wilkie, T. M. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827 (2000). 9. Schwarz-Romond, T., Metcalfe, C. & Bienz, M. Dynamic recruitment of axin by Dishevelled protein assemblies. J. Cell Sci. 120, 2402–2412 (2007). 10. Julius, M. A. et al. Domains of axin and disheveled required for interaction and function in wnt signaling. Biochem. Biophys. Res. Commun. 276, 1162–1169 (2000). 11. Pawson, T., Gish, G. D. & Nash, P. SH2 domains, interaction modules and cellular wiring. Trends Cell Biol. 11, 504–511 (2001). 12. Grebien, F. et al. Targeting the SH2‑kinase interface in Bcr–Abl inhibits leukemogenesis. Cell 147, 306–319 (2011). 13. Wong, H. C. et al. Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway. Nature Struct. Biol. 7, 1178–1184 (2000). 14. Axelrod, J. D., Miller, J. R., Shulman, J. M., Moon, R. T. & Perrimon, N. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev. 12, 2610–2622 (1998). 15. Consonni, S. V., Gloerich, M., Spanjaard, E. & Bos, J. L. cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane. Proc. Natl Acad. Sci. USA 109, 3814–3819 (2012). 16. Simons, M. et al. Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization. Nature Cell Biol. 11, 286–294 (2009). 17. Cheever, M. L. et al. Crystal structure of the multifunctional Gβ5–RGS9 complex. Nature Struct. Mol. Biol. 15, 155–162 (2008). 18. Masuho, I., Wakasugi-Masuho, H., Posokhova, E. N., Patton, J. R. & Martemyanov, K. A. Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors. J. Biol. Chem. 286, 21806–21813 (2011). 1.
VOLUME 15 | MAY 2014 | 361 © 2014 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES 19. Yu, A. et al. Association of Dishevelled with the clathrin AP‑2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling. Dev. Cell 12, 129–141 (2007). 20. Tauriello, D. V. et al. Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled. Proc. Natl Acad. Sci. USA 109, E812–E820 (2012). 21. Perrimon, N. & Mahowald, A. P. Multiple functions of segment polarity genes in Drosophila. Dev. Biol. 119, 587–600 (1987). 22. Pan, W. J. et al. Characterization of function of three domains in dishevelled‑1: DEP domain is responsible for membrane translocation of dishevelled‑1. Cell Res. 14, 324–330 (2004). 23. Schwarz-Romond, T. et al. The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization. Nature Struct. Mol. Biol. 14, 484–492 (2007). 24. Wong, H. C. et al. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled. Mol. Cell 12, 1251–1260 (2003). 25. Clevers, H. & Nusse, R. Wnt/β-catenin signaling and disease. Cell 149, 1192–1205 (2012). 26. Gao, B. Wnt regulation of planar cell polarity (PCP). Curr. Top. Dev. Biol. 101, 263–295 (2012). 27. Schulte, G. & Bryja, V. The Frizzled family of unconventional G-protein-coupled receptors. Trends Pharmacol. Sci. 28, 518–525 (2007). 28. Huang, X. et al. Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling. Science 339, 1441–1445 (2013). 29. Yanfeng, W. A., Tan, C., Fagan, R. J. & Klein, P. S. Phosphorylation of frizzled‑3. J. Biol. Chem. 281, 11603–11609 (2006). 30. Mlodzik, M. Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet. 18, 564–571 (2002). 31. Chasse, S. A. & Dohlman, H. G. RGS proteins: G protein-coupled receptors meet their match. Assay Drug Dev. Technol. 1, 357–364 (2003).
32. Narayanan, V. et al. Intramolecular interaction between the DEP domain of RGS7 and the Gβ5 subunit. Biochemistry 46, 6859–6870 (2007). 33. Martemyanov, K. A. et al. The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo. J. Neurosci. 23, 10175–10181 (2003). 34. Anderson, G. R., Posokhova, E. & Martemyanov, K. A. The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling. Cell Biochem. Biophys. 54, 33–46 (2009). 35. Ballon, D. R. et al. DEP-domain-mediated regulation of GPCR signaling responses. Cell 126, 1079–1093 (2006). 36. Sandiford, S. L. & Slepak, V. Z. The Gβ5-RGS7 complex selectively inhibits muscarinic M3 receptor signaling via the interaction between the third intracellular loop of the receptor and the DEP domain of RGS7. Biochemistry 48, 2282–2289 (2009). 37. de Rooij, J. et al. Epac is a Rap1 guanine-nucleotideexchange factor directly activated by cyclic AMP. Nature 396, 474–477 (1998). 38. Gloerich, M. et al. Spatial regulation of cyclic AMP‑Epac1 signaling in cell adhesion by ERM proteins. Mol. Cell. Biol. 30, 5421–5431 (2010). 39. Gloerich, M. & Bos, J. L. Epac: defining a new mechanism for cAMP action. Annu. Rev. Pharmacol. Toxicol. 50, 355–375 (2010). 40. Rehmann, H., Das, J., Knipscheer, P., Wittinghofer, A. & Bos, J. L. Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state. Nature 439, 625–628 (2006). 41. Ponsioen, B. et al. Direct spatial control of Epac1 by cyclic AMP. Mol. Cell. Biol. 29, 2521–2531 (2009). 42. Li, S. et al. Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS). J. Biol. Chem. 286, 17889–17897 (2011). 43. Li, Y. et al. The RAP1 guanine nucleotide exchange factor Epac2 couples cyclic AMP and Ras signals at the plasma membrane. J. Biol. Chem. 281, 2506–2514 (2006).
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44. Hill, K. et al. Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gβγ subunits. J. Biol. Chem. 280, 4166–4173 (2005). 45. Pandiella, A. & Montero, J. C. Molecular pathways: p-rex in cancer. Clin. Cancer Res. 19, 4564–4569 (2013). 46. Sosa, M. S. et al. Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer. Mol. Cell 40, 877–892 (2010). 47. Qin, J. et al. Upregulation of PIP3‑dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis. Oncogene 28, 1853–1863 (2009). 48. Urano, D., Nakata, A., Mizuno, N., Tago, K. & Itoh, H. Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein betagamma subunits and PKA. Cell Signal 20, 1545–1554 (2008). 49. Ma, A. D., Brass, L. F. & Abrams, C. S. Pleckstrin associates with plasma membranes and induces the formation of membrane projections: requirements for phosphorylation and the NH2‑terminal PH domain. J. Cell Biol. 136, 1071–1079 (1997). 50. Stenmark, H. & Aasland, R. FYVE-finger proteins-effectors of an inositol lipid. J. Cell Sci. 112, 4175–4183 (1999). 51. Vassilatis, D. K. et al. The G protein-coupled receptor repertoires of human and mouse. Proc. Natl Acad. Sci. USA 100, 4903–4908 (2003). 52. Peterson, T. R. et al. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137, 873–886 (2009).
Acknowledgements
The authors thank H. Rehmann, University Medical Center Utrecht, The Netherlands, for providing the ribbon diagrams of DVL, EPAC, RGS9 and pleckstrin DEP.
Competing interests statement
The authors declare no competing interests.
FURTHER INFORMATION SMART database: http://smart.embl-heidelberg.de ALL LINKS ARE ACTIVE IN THE ONLINE PDF
www.nature.com/reviews/molcellbio © 2014 Macmillan Publishers Limited. All rights reserved
DEP domains: structurally similar but functionally different Sarah V. Consonni, Madelon M. Maurice and Johannes L. Bos
Abstract | The Dishevelled, EGL‑10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation. This article is dedicated to the memory of Tony Pawson, who taught us the importance of protein domains in signal transduction. Cell surface receptors transduce extracellu‑ lar signals by assembling protein complexes at the inner side of the plasma membrane. The targeting of signalling complex compo‑ nents to the membrane not only increases the possibility of complex formation but also increases the strength of the transduced signal1. Moreover, due to the modular nature of signalling molecules, different types of complexes can easily form in time and space2. Each protein domain recognizes specific protein sequences or lipids, and the binding is frequently regulated by a distinc‑ tive signalling event. One example is the Dishevelled, EGL‑10 and pleckstrin (DEP) domain: Dishevelled (dsh; also known as DVL in higher eukaryotes) was first identi‑ fied in Drosophila melanogaster, whereas egg-laying defective protein 10 (EGL‑10) and pleckstrin were first identified in Caenorhabditis elegans and mammalian cells, respectively 3–5. In mammals, the DEP domain is present in a limited number of protein families (FIG. 1) that have diverse functions in signal transduction. DEP domains have also been identified in bacte‑ ria (for further information see the SMART (simple modular architecture research tool) database), which indicates that the DEP domain did arise early during evolution.
Some DEP domain-containing proteins, for example the regulator of G protein signal‑ ling (RGS) family of proteins, are present in yeast, whereas others such as DVL appeared later in evolution and are only found in higher eukaryotes6,7. RGS proteins contain both a DEP domain and an RGS domain; the RGS domain confers GTPase-activating protein (GAP) activity 8. Interestingly, the RGS domain–DEP domain module that is present in yeast and mammalian RGS pro‑ teins seems to be conserved in the mamma‑ lian DVL signalosome, in which the scaffold protein Axin contains the RGS domain9,10. A predominant function of DEP domains is plasma membrane anchoring, but DEP domains carry out several addi‑ tional functions by using different binding interfaces (TABLE 1). In this respect, DEP domains seem to differ from many other protein domains with a more restricted mechanism of action. However, the use of multiple binding surfaces may be more common than originally anticipated. For instance, SRC homology 2 (SH2) domains were long considered to have a single binding pocket for phosphotyrosine moieties, but more recently it was shown that SH2 domains can interact with kinase domains that use a different binding inter‑ face11,12. DEP domains can be considered a paradigm for the growing group of protein domains that use different interfaces for different functions.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
In the past few years, analyses on the structure and function of DEP domains have shown that this protein module is one of the prime examples of a regulated domain in signal transduction. In this Opinion article we focus on the DEP domain-containing proteins DVL, RGS, exchange protein directly activated by cAMP 1 (EPAC1; also known as RAPGEF3), pleckstrin and PtdIns(3,4,5)P3-dependent RAC exchanger (PREX). We examine the various mechanisms of DEP domainmediated interaction with proteins and lipids, and discuss how DEP domains function in the spatiotemporal control of diverse signalling events. Structure of DEP domains DEP domains consist of approximately 100 amino acids and they all share a high degree of sequence and structural similari‑ ties. They have a characteristic α/β fold, and they are comprised of a conserved helical core and a protruding β‑hairpin arm, which is located between the core helices α1 and α2 (REFS 4,13) (FIG. 2). The β‑hairpin module, which often bears residues that are impor‑ tant for mediating molecular interactions, is less conserved than the helical core14. In some cases, such as for DVL and EPAC, polybasic residues in the β‑hairpin are required for the recruitment to the plasma membrane14–16 (FIG. 2a,b). Furthermore, studies revealed that DEP domains encompass distinct positive and negative patches that influence their plasma membrane binding abilities. The DEP domain of RGS9 (FIG. 2c) is oriented in such a way that a cluster of positively charged residues faces the membrane17. Conversely, the DEP domain of pleckstrin (FIG. 2d) has mainly a negatively charged cluster on its exposed surface, whereas the DEP domains of EPAC and DVL combine both positive and negative patches. In some instances, the DEP domain requires an additional fold or motif to interact with membrane anchors. This is the case for the DEP domain of RGS9, which needs an adjacent DEP helical extension (DHEX) fold for membrane binding 17,18. Likewise, the DEP domain of DVL2 requires a neighbouring Tyr-based YHEL motif for membrane anchoring VOLUME 15 | MAY 2014 | 357
© 2014 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES DVL
DIX
PDZ
EGL-10, RGS EPAC Pleckstrin FYVE PREX1, PREX2 GPR155
DH
DEP DEP
GGL
RGS
CNB
DEP
CNB
REM
PH
DEP
PH
FYVE
DEP
CPN
PH
DEP
DEP
PDZ
MTD
DEP DEP
PDZ
DEPTOR
DEP
RA
CDC25HD
PtdIns(5)K PDZ
IP4P
Figure 1 | Domain architecture of mammalian DEP domain-containing proteins. The Dishevelled, EGL‑10 and pleckstrin (DEP) domain is shown in pink and other domains that |are present Cell in more than Nature Reviews Molecular Biology one protein are represented in an identical manner. Protein modules that are adjacent to the DEP domain often aid the DEP domain in its membrane binding capabilities. This is the case for the DVL (Dishevelled) DEP domain that also requires the flanking carboxy-terminal region to bind to Frizzled receptor and for the RGS (regulator of G protein signalling) DEP domain that uses the GGL (Gγ subunitlike) domain to bind to Gβ5. In the case of EPAC (exchange protein directly activated by cAMP), pleckstrin and PREX (PtdIns(3,4,5)P3-dependent RAC exchanger), conformational changes that relieve the protein from an autoinhibited state or intramolecular interactions are needed for facilitating mem‑ brane localization of the protein. For other proteins the function of the DEP domain is still elusive. CDC25HD, CDC25 homology domain; CNB, cyclic nucleotide-binding; CPN, chaperonin; DEPTOR, DEP domain-containing mTOR-interacting protein; DH, DBL homology; DIX, Dishevelled and Axin; FYVE, FAB 1, YOTB, VAC 1 and EEA1; GPR155, G protein-coupled receptor 155; IP4P, inositol polyphos‑ phate 4‑phosphatase; MTD, membrane transport domain; PDZ, post-synaptic density 95, discs large and zonula occludens 1; PH, Pleckstrin homology; PtdIns (5)K, phosphatidylinositol‑5‑kinase; RA, RAS-association; REM, RAS exchange motif.
during non-canonical signalling, and DVL1 contains a DEP domain together with a flanking carboxy terminal region, to bind to a discontinuous motif on the seven-span receptor Frizzled (FZ) during canonical signalling 19,20. Thus, despite the structural similarity in their core regions, small differences between DEP domains account for their differential mode of regulation and for their ability to coordinate diverse signalling events in space and time. DVL DEP domain in signal regulation DVL is a cytoplasmic phosphoprotein involved in the regulation of WNT signalling that was first identified in D. melanogaster as a regulator of early embryo polarity 21. It is composed of an amino‑terminal DVL and Axin (DIX) autointeraction domain that is responsible for the formation of multimeric complexes, a central post-synaptic density 95, discs large and zonula occludens 1 (PDZ) domain that is required for binding to FZ receptor and a C‑terminal DEP domain that is important for specific membrane localization14,22–24 (FIG. 1).
DVL DEP domain in membrane targeting. Activation of FZ receptors by WNT ligands activates several downstream signalling pathways, most notably the canonical WNT pathway that stabilizes β‑catenin signalling to control cell fate and the non-canonical planar cell polarity (PCP) signalling path‑ way for cytoskeletal remodelling 25,26. In the canonical pathway, WNT binding to FZ and the low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 (LRP5/6) receptor results in the formation of a DVL-dependent signalosome, which includes the receptors FZ, LRP5/6, DVL, the adaptor protein Axin and additional proteins (FIG. 3a). This interaction is mediated in part by a weak interaction of FZ with both the PDZ and the DEP domains of DVL. The PDZ domain binds to a short linear sequence in the cytosolic tail of FZ, whereas the DEP domain together with the C termi‑ nus interact with three discontinuous regions that are located in the third intracellular loop and C-terminal tail of FZ20. Each of these interactions is required for membrane recruitment and the proper function of DVL in this pathway. In the context of the noncanonical PCP signalling in D. melanogaster,
358 | MAY 2014 | VOLUME 15
the DEP domain of Dsh interacts with nega‑ tively charged membrane phospholipids and, aided by the Na+/H+ hydrogen exchanger 2 (Nhe2), which keeps an alkaline pH suited for charge-dependent interactions, it interacts with Fz16. Hence, the DEP–phospholipid interaction may contribute to the forma‑ tion of a robust FZ–DVL complex at the plasma membrane (FIG. 3b). Moreover, the DEP domain of another of the three mam‑ malian DVL isoforms, DVL2, aided by a YHEL motif at its C terminus, can interact with μ2 adaptor complexes at the membrane to facilitate clathrin-mediated endocytosis of FZ4 (REF. 19). This is required to regulate surface expression of FZ and therefore for desensitization of signalling 27. DVL DEP domain in signalling complex formation. A number of important WNTinduced post-translational modifications of WNT pathway proteins depend on DVL DEP domain activity. A recent study showed that RIP kinase 4 (RIPK4) binds to DVL and mediates phosphorylation of the PDZ and DEP domains28. The combined phosphorylation events promote the assem‑ bly of DVL2 into large signalling complexes, which leads to maximal β‑catenin stabiliza‑ tion and transcription of WNT-responsive target genes. How DEP domain-mediated intermolecular and intramolecular interac‑ tions are regulated through phosphorylation remains unresolved. In addition, the DVL DEP domain itself controls post-translational modification of other DVL domains and protein partners. Increased Lys63‑linked polyubiquitylation of the DVL DIX domain drives WNT–β‑catenin signalling in cells that lack the deubiquitylation enzyme CYLD; this phenomenon depends on an intact DEP domain29,30. In addition, Xenopus laevis fz3 is phosphorylated after association with dvl. This phosphorylation requires the DEP domain of dvl and is needed for dvl-mediated fz3 desensitization and thus for regulation of PCP signalling 29,30. Hence, the interactions of the DVL DEP domain with both proteins and phospholipids mediate the formation and modification of essential protein complexes in the regulation of WNT signalling (FIG. 3). RGS DEP domain in signal termination Members of the RGS family are GAPs of heterometric G proteins. They use their RGS domain to promote GTP hydrolysis of the Giα subunit thereby switching off G proteincoupled receptor (GPCR) signalling (FIG. 3c). Currently, the best characterized of the RGS family of proteins is the R7 subfamily, which includes RGS6, RGS7, RGS9 and RGS11. www.nature.com/reviews/molcellbio
© 2014 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES Table 1 | Properties of DEP domains DEP protein
DEP binding partner
DEP domain length (amino acids)
Additional requirements for function
Function
Refs
Dishevelled
PA, FZ and μ2 adaptor
~87
DEP with carboxy-terminal flanking region and YHEL motif
Membrane targeting
16,19,20
EGL‑10 and RGS
Gβ5, R9AP and R7BP
~107
DHEX fold
Membrane targeting
17,33–35
EPAC
PA
120 or 121
cAMP-induced conformational change
Membrane targeting
15
Pleckstrin
Intramolecular interaction with PH domain
~116
PKC-induced phosphorylation
Autoinhibition
FYVE
Unknown
81
Unknown
Unknown
4,49
50
PREX1 and PREX2 Intramolecular ~80 interaction with PDZ and IP4P domains, and mTOR
PtdIns(3,4,5)P3‑induced conformational Protein function change
45,48
GPR155
Unknown
~82
Unknown
Unknown
51
DEPTOR
Unknown
81
Unknown
Unknown
52
cAMP, cyclic AMP; DEPTOR, DEP domain-containing mTOR-interacting protein; DHEX, DEP helical extension; EGL‑10, egg-laying defective protein 10; EPAC, exchange protein directly activated by cAMP; FYVE, FAB 1, YOTB, VAC 1 and EEA1; FZ, Frizzled; Gβ5, G protein β subunit subtype 5; GPR155, G protein-coupled receptor 155; IP4P, inositol polyphosphate 4‑phosphatase; PA, phosphatidic acid; PDZ, post-synaptic density 95, Discs large and zonula occludens 1; PH, pleckstrin homology; PKC, protein kinase C; PREX, PtdIns(3,4,5)P3-dependent RAC exchanger; PtdIns(3,4,5)P3, phosphatidylinositol‑3,4,5‑trisphosphate; R7BP, R7 familybinding protein; R9AP, RGS9‑anchoring protein; RGS, regulator of G protein signalling.
RGS proteins of the R7 subfamily are made up of three domains: a DEP domain for membrane targeting at its N terminus, a Gγ subunit-like (GGL) domain for binding Gβ subunits and a C‑terminal RGS domain that is required for its signal termination31,32 (FIG. 1). Structural analysis revealed that the DEP domain of RGS9 has a C‑terminal DHEX that is not found in other DEP domains. The DHEX domain, together with the DEP and GGL domains, interacts with Gβ5 (REF. 17). In addition to binding to Gβ5, RGS proteins of the R7 subfamily a DVL DEP
b EPAC DEP
α1
α3
contributes to the stabilization of the complex and subsequent regulation of the G proteinmediated signalling cascade18. This highlights the multifaceted nature of DEP domainmediated complex assembly at the membrane. The DEP domains that are found in Sst2, the yeast homologue of the RGS family, medi‑ ate binding to the GPCR fungal pheromone mating factor (Ste2 in MATa and Ste3 in MATα cells). Interestingly, Sst2 DEP domains can only bind to the GPCR in its nonphosphorylated state, which suggests that this interaction is regulated35.
c RGS9 DEP
Hairpin
Hairpin
α2
interact with the membrane targeting proteins RGS9‑anchoring protein (R9AP; also known as RGS9BP) and R7 family-binding protein (R7BP; also known as RGS7BP). The DEP domain, despite being needed for this interac‑ tion, is not sufficient for the binding 33. Recent protein–protein interaction mapping revealed that the DHEX domain, together with the DEP domain, is also involved in the inter action with R9AP and R7BP18,34. Site-directed mutagenesis studies suggest that R7BP inter‑ acts with the DEP domain at the surface that also interacts with Gβ5, and R7BP therefore
d Pleckstrin DEP
Hairpin
α1
Hairpin
α1
α1
α2
α2 α3
Figure 2 | DEP domain structures. Cartoon structures of Dishevelled, EGL‑10 and pleckstrin (DEP) domains that show their characteristic α/β fold. DEP domains comprise a conserved helical core, which consists of three heli‑ ces and a protruding β‑hairpin arm between the helices α1 and α2. This helical core is highly conserved in all DEP domains. Only in the case of pleckstrin, there is an additional helix located at the end of the DEP domain, which is
α2 α3
α3
thought to be important for proteinNature mobility.Reviews Arrows |show the location of the Molecular Cell Biology residues in the β‑hairpin that are required for membrane binding. a | Image is based on Protein Data Bank (PDB) entry 1fsh of Dishevelled (DVL)13. b | Image is based on PDB entry 2byv of exchange protein directly activated by cAMP (EPAC)40. c | Image is based on PDB entry 2pbi of regulator of G protein signalling 9 (RGS9)17. d | Image is based on PDB entry 1w4m of pleckstrin4.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 15 | MAY 2014 | 359 © 2014 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES a
b
Frizzled WNT
LRP5, LRP6
DVL
c
Frizzled
WNT
Plasma membrane
GPCR
PA DVL
Axin
R7BP–R9AP β α γ RGS GDP
DAAM
GTP hydrolysis β-catenin
β-catenin
RHO
β-catenin
ROCK Nucleus
d
β-catenin TCF
β-adrenergic receptor PA EPAC1 RAP1 cAMP AF6 RIAM ARAP3 RASIP1 Others
• Polarity • Endothelial junction formation • Cell adhesion • Cell spreading
• Polarity • Actin polymerization
Signal termination
e
f GPCR
β α γ G protein
PtdIns(3,4,5)P3
X PI3K
PRE
RAC
PtdIns(4,5)P2
PKC
Pleckstrin P
Phosphorylation
PAK WAVE MEK1/2 Others Phosphoinositide hydrolysis • Proliferation • Migration • Invasion
• Platelet aggregation • Platelet secretion
Figure 3 | Diversity of pathways that are regulated by DEP domain-containing proteins. Nature Reviews | Molecular Cell in Biology Dishevelled, EGL‑10 and pleckstrin (DEP) domain-containing proteins are shown pink. a | Dishevelled (DVL) during canonical WNT signalling. The DEP domain of DVL binds to the G proteincoupled receptor (GPCR) Frizzled and enables formation of a signalling complex that results in β‑catenin translocation to the nucleus and subsequent transcription of WNT target genes. b | DVL during non-canonical planar cell polarity (PCP)–WNT signalling. By interacting with PA (phosphatidic acid) at the membrane (shown in yellow), the DEP domain of DVL mediates cytoskeletal changes and polarity through DAAM (DVL associated activator of morphogenesis) and subsequent downstream activation of RHO–ROCK (RHO-associated kinase) signalling. c | Regulator of G protein signalling (RGS). To promote GPCR signal termination, RGS proteins associate with their cognate receptor, which is aided by their DEP domain and promotes GTP hydrolysis of the Gα subunit. d | Exchange protein directly activated by cAMP 1 (EPAC1). In response to cyclic AMP (cAMP), EPAC1 translocates to the plasma membrane, where it binds to PA through its DEP domain. This results in activation of plasma membrane-located repressor/activator protein 1 (RAP1), downstream effectors and of RAP-mediated cellular processes, such as cell polarity, cell adhesion, cell spreading and cell–cell junction formation. e | PtdIns(3,4,5)P3‑dependent RAC exchanger (PREX). After activation by phosphatidylinositol‑3,4,5‑ trisphosphate (PtdIns(3,4,5)P3), PREX translocates to the plasma membrane where it binds to the Gβγ subunits and PtdIns(3,4,5)P3 (depicted in red). This results in RAC activation and subsequent cell migra‑ tion, proliferation and invasion. f | Pleckstrin phosphorylation (P) by protein kinase C (PKC) is required for its localization to the plasma membrane, where it binds to PtdIns‑4,5‑bisphosphate (PtdIns(4,5)P2; depicted in red). Downstream signalling then results in the induction of platelet aggregation, and secretion and inhibition of phosphoinositide hydrolysis. AF6, ALL1‑fused gene from chromosome 6 (also known as Afadin); ARAP3, ARF-GAP with RHO-GAP domain, ANK repeat and PH domaincontaining protein 3; LRP, low-density lipoprotein receptor-related protein; MEK, MAPK/ERK kinase (also known as MAPKK); PAK, p21‑activated kinase; R7BP, R7 family-binding protein; R9AP, RGS9‑anchoring protein; RASIP1, RAS-interacting protein 1; RIAM, RAP1‑GTP-interacting adapter molecule (also known as APBB1IP); TCF, transcription factor; WAVE, WASP-family verprolin homology domain-containing. Dashed arrows indicate the end result of the signalling pathway.
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The DEP domain of RGS7 is required for binding to and inhibition of muscarinic acetylcholine receptor M3 but not for other GPCRs36. This clearly shows that DEP domains are not only simply involved in membrane anchoring but also in conferring specificity by selectively interacting with cognate receptors. EPAC DEP domain in phospholipid binding EPAC proteins are guanine nucleotide exchange factors (GEFs) for the small G protein RAP that is involved, among others, in the control of cell adhesion and actin cytoskeletal rearrangements37,38. The two EPAC proteins, EPAC1 (also known as RAPGEF3) and EPAC2 (also known as RAPGEF4), are differentially expressed — EPAC1 is more abundant in the central nervous system, ovaries and uterus, whereas EPAC2 is most prevalent in the pancreas and adrenal gland in mammals39. Both are multi‑ domain proteins that contain an N‑terminal regulatory region, which comprises one (in the case of EPAC1) or two (in the case of EPAC2) cyclic nucleotide-binding (CNB) domains and a DEP domain. The catalytic region at their C terminus harbours the CDC25 homology domain (CDC25HD) for exchange activity, a RAS exchange motif (REM) and a RAS-association (RA) domain (FIG. 1). Both proteins assume a closed auto‑ inhibitory conformation, which is relieved after cyclic AMP (cAMP) binding 40. In the case of EPAC1, the allosteric activation by cAMP is accompanied by a very rapid relocalization of the protein from the cytosol to the plasma membrane. This transloca‑ tion is mediated by the DEP domain, which directly interacts with phosphatidic acid at the membrane (FIG. 3d). A polybasic stretch that encompasses residue Arg82 in the DEP domain is responsible for this binding 15,41 (FIG. 2). This interaction is required for the proper function of EPAC1, as deletion of the DEP domain or alteration of Arg82 to a nonpolar amino acid inhibits cAMP-induced and EPAC1‑mediated cell adhesion41. Amide hydrogen and deuterium mass spectrometry studies on EPAC2 elucidated the possible regulatory mechanisms of its cAMP-induced and DEP domain-mediated tethering to the plasma membrane. After cAMP sig‑ nalling, some regions of the DEP domain become more solvent exposed, resulting in reorientation of the β‑hairpin that contains the essential Arg82 residue and in this way enabling plasma membrane anchoring 15,42. Interestingly, the cAMP-binding domain is located at the same position as the C‑terminal helical extension of the DEP www.nature.com/reviews/molcellbio
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PERSPECTIVES domain (which is important for its func‑ tion) of the R7 subfamily of RGS proteins and DVL. So far, no additional interacting proteins have been identified for EPAC DEP domains. It should be noted that, currently, the function of the DEP domain of EPAC2 is elusive as the DEP domain does not seem to be required for its localization to the plasma membrane15,43. PREX DEP domain in intradomain binding PREX proteins act as GEFs for the small GTPase RAC. They both contain a catalytic DBL homology (DH) domain at their N‑terminal adjacent to a pleckstrin homol‑ ogy (PH) domain that is required for binding to phosphatidylinositol‑3,4,5‑trisphosphate (PtdIns(3,4,5)P3), which is followed by two tandem DEP domains and two tandem PDZ domains. At their C terminus they have an inositol polyphosphate 4‑phosphatase (IP4P) domain that is needed for binding to protein phosphatase 1α (PP1α)44,45 (FIG. 1). PREX is likely to be in an autoinhibited state in the cytosol and translocates to the membrane, where it binds to Gβγ subunits and PtdIns(3,4,5)P3 through its DH and PH domains after activation by PtdIns(3,4,5)P3 (REF. 45) (FIG. 3e). PREX has recently attracted much attention, as it was found to have a role in metastatic cancers. Indeed, PREX1 was found to increase cell proliferation in breast cancer cells and to induce migration and invasion of prostate cancer cells by activat‑ ing RAC46,47. Interestingly, the DEP domain mediates intramolecular interactions with the PDZ and the IP4P domains. Such bind‑ ing is needed for association with Gβγ and for Gβγ-induced GEF activity 48. Pleckstrin DEP domain in autoinhibition Pleckstrin is another DEP domaincontaining protein family. It is one of the main substrates for protein kinase C (PKC) in platelets and comprises two PH domains for protein or lipid interactions and a cen‑ tral DEP domain3 (FIG. 1). The pleckstrin DEP domain structure is more divergent than that of DEP domains in other DEP domain-containing proteins, as it bears an additional β‑strand that is positioned after the β‑hairpin and an additional helix, which is located towards the end of the DEP domain; the helix is thought to be important for increased protein mobility and to facili‑ tate protein–protein interactions4,49. The DEP domain of pleckstrin does not directly mediate membrane anchoring of the pro‑ tein, but it interacts intramolecularly with the N‑terminal PH domain. Such interac‑ tion is only relieved after phosphorylation
by PKC, which enables activation and membrane localization of the protein4 (FIG. 3f). This finding suggests a function for DEP domains in autoinhibitory domain interactions. Concluding remarks and future directions DEP domains are globular protein domains that, despite having diverse mechanisms of action, generally assist in the transloca‑ tion of the cognate protein to the plasma membrane. Often the presence of a specific sequence or exposure of a particular amino acid is pivotal for such accurate spatial regulation of proteins, thus highlighting the importance of sequence context and specificity 14,41. The presence of additional domains or folds that are adjacent to the DEP domain seems to be important for signalling speci‑ ficity. The DVL DEP domain requires a YHEL motif for membrane anchoring during non-canonical signalling, and both the PDZ domain and a C‑terminal region cooperate with DEP to bind to FZ during canonical signalling 19,20. Similarly, the DEP domain of RGS9 needs a DHEX helical fold for membrane localization17,18. Conformational changes also play an important part in the regulation of DEP domain-mediated signalling. This is the case for EPAC and possibly for PREX, which require a conformational change that is triggered by cAMP and PtdIns(3,4,5)P3, respectively, to become active and to local‑ ize to the plasma membrane40,45. Additional levels of regulation might be involved, such as post-translational modifications and domain–domain interactions4,48. This illustrates the diversity of DEP domain-mediated signalling regulation and also emphasizes that the DEP domain is not simply a localization device, but that it also affects protein function by being itself subject to regulation. As our understanding of how DEP domains function and are regulated increases, it is becoming clear that they use many ways to carry out their task in various cellular events (FIG. 3). Further identification of all the factors and regulatory mechanisms that are involved in such regulation will help us to understand how DEP domains achieve such signalling specificity, despite their structural similarities. Importantly, the ability of protein domains to have different functional properties by providing diverse binding interfaces is being more and more appreciated as a recurrent theme in the control of signalling in space and time.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
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Acknowledgements
The authors thank H. Rehmann, University Medical Center Utrecht, The Netherlands, for providing the ribbon diagrams of DVL, EPAC, RGS9 and pleckstrin DEP.
Competing interests statement
The authors declare no competing interests.
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