Signaling by Small Gtpases at Cell–Cell Junctions: Protein Interactions Building Control and Networks

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Signaling by Small GTPases at Cell–Cell Junctions: Protein Interactions Building Control and Networks

Vania Braga

Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom Correspondence: [email protected]

A number of interesting reports highlight the intricate network of signaling proteins that coordinate formation and maintenance of cell–cell contacts. We have much yet to learn about how the in vitro binding data is translated into protein association inside the cells and whether such interaction modulates the signaling properties of the protein. What emerges from recent studies is the importance to carefully consider small GTPase activation in the context of where its activation occurs, which upstream regulators are involved in the activation/inactivation cycle and the GTPase interacting partners that determine the intracellular niche and extent of signaling. Data discussed here unravel unparalleled cooperation and coordination of functions among GTPases and their regulators in supporting strong adhesion between cells.

he presence of tight cell–cell attachment is a In different cell types, cell–cell junctions are Thallmark of different cell types such as epi- “hot spots” for the localization of signaling mol- thelial, endothelial, cardiac, or smooth muscle ecules such as small GTPases, their regulators cells, which require strong cohesion for their and effectors, kinases, phosphatases, and others. specialized functions and to sustain mechanical In the past decade, exciting studies have mapped stress. In these cell types, different members of the molecular complexes that support and the cadherin family of cell–cell adhesion recep- strengthen attachment of cadherin receptors be- tors drive and orchestrate the assembly of addi- tween neighboring cells (Zaidel-Bar 2013; Citi tional adhesion complexes, thereby providing et al. 2014; Zaidel-Bar et al. 2015), the regulation spatial signals to organize the cytoskeleton and of receptor interaction with the underlying cy- signaling components at junctions. The rele- toskeleton and how cytoskeletal remodeling is vance of cadherin-dependent adhesion to tissue driven by the presence of cell–cell contacts (Hu- morphogenesis and function is highlighted in veneers and de Rooij 2013; Ladoux et al. 2015; the strong defects in tissue organization and pa- Strale et al. 2015). In parallel with these advance- thologies associated with disruption of junction ments, a number of regulators of junction as- structure (Macara et al. 2014). sembly or maintenance have been identiﬁed

Editors: Carien M. Niessen and Alpha S. Yap Additional Perspectives on Cell–Cell Junctions available at www.cshperspectives.org Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a028746 Cite this article as Cold Spring Harb Perspect Biol 2018;10:a028746

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V. Braga

among cytoskeletal or signaling proteins (Mc- SIGNALING ACTIVATION: WHERE Cormack et al. 2013; van Buul et al. 2014; Sluys- AND HOW mans et al. 2017). However, this extensive knowledge requires The majority of Rho family of small GTPases (or now a systematic approach to integrate the var- Rho GTPases, for short) are found in an inactive, ious regulators in the context of their intracellu- GDP-bound form and most likely associated lar compartmentalization, cycles of activation/ with RhoGDI in the cytoplasm (Cook et al. inactivation, interaction with selected effectors, 2014). The classical view of activation of Rho and precise positioning at cytoskeletal structures GTPases at adhesive sites is that it occurs (Fig. and junctional adhesive complexes. From the 1A) via stimulation of specific guanine nucleo- large number of regulators of cell–cell contacts tide exchange factors (GEF) or by inactivation of identified so far, research is moving on to dissect a GTPase activating protein (GAP) (Cook et al. the interplay among key signaling pathways, 2014; Schaefer et al. 2014; Hodge and Ridley scaffolding proteins, and the modulation of cel- 2016). Small GTPase activation may occur local- lular processes driven by junctions. This per- ly at the adhesion site and membrane regions, spective discusses recent insights in our under- where its function is required and then interact standing of the integration of signaling at with its effectors and modulators (Fig. 1B). In- cell–cell junctions in epithelial cells. Excellent active GTPases may be sequestered in the cyto- reviews on Rho GTPase regulators in other cell plasm via interaction with RhoGDI (Fig. 1A). types (Ngok and Anastasiadis 2013; van Buul In any case, at steady state, GEFs and GAPs et al. 2014) and their specific interaction polarity must avoid random interaction with substrate complexes (Citi et al. 2014; Ngok et al. 2014) can GTPases and indeed they have evolved different be found elsewhere. mechanisms to do so (Schaefer et al. 2014; Rai-

A GTP GEF GDP

GDP GTP

RhoA RhoA Pi RhoGDI GAP

B Protein Direct binding Outcome GTP Effector Signal transduction

GDP Binding, sequestering Partner localization, retention

GTP GDP Potential regulation? Modulator localization, retention

Figure 1. (A) Classical regulation of Rho GTPases. Rho small GTPases are mostly found in an inactive, GDP- bound form, and is transiently activated when bound to GTP. This cycle is tightly modulated by guanine nucleotide exchange factors (GEF), which facilitates replacement of GDP for GTP. GTPase inactivation is facilitated by guanine nucleotide activating protein (GAP), as the latter increases the intrinsic GTP hydrolysis of small GTPases. Inactivated GTPases may be removed from membranes and maintained in the cytoplasm by interaction with RhoGDI. (B) Different proteins are able to interact directly with active or inactive GTPases or both forms. On interaction, different functions and cellular outcomes may occur (see text for more details).

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Signaling by Small GTPases at Cell–Cell Junctions

mondi et al. 2015; Hodge and Ridley 2016). Cur- tivation-independent interaction may serve to rent data reinforce a potential key function of either localize the GTPase or to retain the interacting partners and additional domains of GTPase at junctions (Nola et al. 2011; Reyes et GEFs and GAPs to drive the specificity and lo- al. 2014). Examples are the F-Bar protein calization of GTPase regulation. For example, PACSIN (Lam and Hordijk 2013) and the actin the catalytic domain of GAPs are found to be bundling and LIM-domain protein Ajuba (see nonselective in vitro and additional domains below) (JJ McCormack, S Bruche, ABD Ouadda, may govern specificity toward GTPase sub- et al. in press; Nola et al. 2011). strates (Amin et al. 2016). These results help to explain the different substrate specificity of in WHERE TO BE, WHERE TO FUNCTION vitro (with purified catalytic domains) and in vivo GAP assays following a specific stimulus. A signaling molecule can be found in various It becomes clear that the classical regulation intracellular and cortical structures and dis- of Rho GTPases cannot fully account for a much persed in the cytoplasm. Subcellular localization more complex and wired regulation of GTPase is a recognized mechanism to compartmentalize function. Among additional regulatory events of signaling pathways to control specific cellular GTPase signaling, there are (Hodge and Ridley processes. The current dogma in interpreting 2016): (1) posttranslation modifications such as the function of a junction regulator is that it ubiquitination and sumoylation to potentially must be detected at cell–cell adhesive sites. In regulate GTPase protein levels; (2) phosphory- one hand, this rational is clearly applicable to lation, which can modulate binding affinity; and cytoskeletal proteins that support scaffolding (3) interaction with cytoskeletal proteins (Tables structures at junctions or cytoskeletal proteins 1 and 2; Fig. 1B). Evidently, these additional that are transiently recruited to junctions by dif- forms of GTPase regulation may complement ferent stimuli, such as mechanical stress, growth and be coordinated with the classical activation factor stimulation, etc. (Ladoux et al. 2015). On and inactivation cycles by GEFs and GAPs. the other hand, additional points should be con- Conceptually, these distinct regulatory events sidered for enzymatic regulators such as GTPase also pose different questions and potential signaling components such as the control of mechanisms of how a given signaling pathway localization and activation/inactivation status. operates at cell–cell contacts. Below I discuss some considerations on these This review focus on the potential participa- topics and summarize current evidence. tion of cytoskeletal proteins to fine-tune the ac- It is highly likely that the amount of a partic- tion and localization of Rho GTPases signaling. ular GTPase or regulator detected at junctions by A number of cytoskeletal proteins are known standard microscopy represents an inactive pool. effectors of GTPases, that is, they interact with First, rapid activation and inactivation is predict- cytoskeletal filaments and the GTP-bound ac- ed to be in place to ensure tight GTPase regula- tive form of GTPases to transduce signaling to- tion temporally and to avoid over-activation that ward a specific cellular outcome. Other proteins can be deleteriousto junctions (Cooket al. 2014). interact with the inactive form of GTPases, and Second, a specific treatment drives a surge of are thought to behave as sequesters, similar to stimulation; however, only a very small fraction RhoGDI. A classic example is p120CTN, protein of the GTPase is activated transiently. At steady- that interacts with the cadherin tail: p120CTN state, the fraction of active GTPase or its regula- cytoplasmic pool can interact with RhoA and tor(s) at junctions may be even smaller, consid- inactivate this pathway in different cells (Kour- ering the heterogeneity of stimulus at different tidis et al. 2013; Peglion and Etienne-Manneville regions of cell–cell contacts (different receptors, 2013). Finally, other cytoskeletal proteins may trafficking, mechanical stress, and others). bind a GTPase independently of its GTP or GDP The lack of correlation between localization loading (Lam and Hordijk 2013), and are thus and steady-state pathway activation is supported not considered effectors or sequesters. Such ac- by three sets of data. First, constitutively active

Cite this article as Cold Spring Harb Perspect Biol 2018;10:a028746 3 Downloaded from http://cshperspectives.cshlp.org/ .Braga V. 4 Table 1. Selected list of exchange factors (GEF) shown to interact with lipids and cytoskeletal proteins found at epithelial junctions GEF Names GTPase Partnera Effect interactionb Functional outcome Additional observations References Trio ARHGEF23 Rac1 Tara Inhibition Regulates E-cadherin Increases Trio binding to E- Seipel et al. 2001; transcriptional levels cadherin tail; Tara binds Yano et al. 2011 directly and stabilize F-actin Filamin A Recruitment Reduces RhoA activation Association with cadherin Tu and You, 2014 induced by junction complexes increase with assembly junction formation Tiam-1 — Rac1 Paracingulin Recruitment Paragingulin depletion Depletion paracingulin Guillemot et al. inactivates Rac1 during decreases Tiam1, but not 2008, 2014 onSeptember25,2021-PublishedbyColdSpringHarborLaboratoryPress junction formation Ect2, at contacts Par3 (PDZ2) ND Regulates TJ assembly Par3 depleted cells, Rac1 is Chen and Macara, constitutively activated 2005; Mertens et al. 2005 ieti ril as article this Cite Par3 ND Inactivates and promotes a Mack et al. 2012 gradient of Rac1 activity along cell–cell contacts β2-syntrophin activation Restrict Rac1 activation to Regulates TJ assembly Mack et al. 2012 (PDZ domain) TJ

odSrn abPrpc Biol Perspect Harb Spring Cold GEF-H1 ARHGEF2 RhoA Cingulin (amino Inhibition Mechanism inhibition Aijaz et al. 2005 acids 782–1025) unknown Paracingulin Recruitment RhoA activity increases on Guillemot et al. depletion of 2008 paracingulin PDZ ARHGEF11 RhoA ZO-1 ND Regulates myosin Localizes to primordial Itoh et al. 2012 RhoGEF activation at junctions contacts and then tight junctions ND ND Celsr1 necessary for Restricts RhoA activation Sai et al. 2014 recrutiment apically to induce

2018;10:a028746 constriction Continued Downloaded from http://cshperspectives.cshlp.org/ ieti ril as article this Cite Table 1. Continued GEF Names GTPase Partnera Effect interactionb Functional outcome Additional observations References p114 ARHGEF18 RhoA LKB1 (amino acids Activation Maturation of apical Independent of LKB1 activity Xu et al. 2013 RhoGEF 155–433) junctions Cingulin ND Regulates junctional RhoA Cell-type speciﬁc interaction Terry et al. 2012 odSrn abPrpc Biol Perspect Harb Spring Cold and myosin activation at cell–cell contacts Lulu2 Activation Regulation of Interaction modulated by Nakajima and circumferential actin Lulu2 phosphorylation Tanoue, 2011

ring onSeptember25,2021-PublishedbyColdSpringHarborLaboratoryPress Tuba ARHGEF36 Cdc42 Tricellulin Activation Tension to produce Recruitment to adherens Oda et al. 2014; straight junctions junctions Otani et al. 2006 ZO-1 ND Recruitment to tight junctions N-WASP ND Cooperate in nascent N-WASP polyproline domain Kovacs et al. 2006 junctions in interacts with Tuba carboxy- 2018;10:a028746 luminogenesis terminal TEM4 ARHGEF17 RhoA Cadherin complex ND Knockdown generates Association independent of F- Ngok et al. 2013 elongated cells and actin interaction; curved junctions coprecipitates with a number Cell at GTPases Small by Signaling of cytoskeletal proteins Ect2 ARHGEF31 RhoA Anillin (772–940) Stabilizes central spindle Interaction with the PH Frenette et al. 2012 microtubules domain Ect2 Centralspindlin Localization Localizes ECT2 at Stabilizes E-cadherin at Ratheesh et al. 2012 complex adherens junctions, contact-sites microtubule- dependent α-catenin Retention Coprecipitation is microtubule- Ratheesh et al. 2012 dependent Asef ARHGEF4 RhoA PI(3,4,5)P Recruitment Overexpression Asef Muroya et al. 2007 increases E-cadherin at –

junctions Junctions Cell Continued 5 Downloaded from http://cshperspectives.cshlp.org/ .Braga V. 6

Table 1. Continued GEF Names GTPase Partnera Effect interactionb Functional outcome Additional observations References

Vav2 — ND p120CTN Recrutiment to Vav2 depletion reduces Binding to E-cadherin tail in a Erasmus et al. 2016 onSeptember25,2021-PublishedbyColdSpringHarborLaboratoryPress cadherin tail cadherin levels at p120-dependent manner junctions Rac1,Cdc42 p120CTN ND ND Complex does not contain E- Noren et al. 2000 cadherin ieti ril as article this Cite Rac1 p120CTN ND ND Binding cytosolic pools of Espejo et al. 2014; p120CTN , binding stimulated Valls et al. 2013 by Wnt 3A treatment β-PIX ARHGEF7 Cdc42 P-cadherin Recruitment Activation of Cdc42 to Also interacts with N- and E- Plutoni et al. 2016 promote collective cadherin in C2C12 cells migration odSrn abPrpc Biol Perspect Harb Spring Cold Solo ARHGEF40 RhoA Keratin 18 May mediate junction- Endothelial cells Abiko et al. 2015; dependent responses to Fujiwara et al. mechanical stretch 2016 Listed are the exchange factor two different names, small GTPase showed to be modulated in the particular example, interacting partner, what is the effect of the interaction (if known), and the functional outcome for junctions or other cellular processes. Also shown are additional observations and the references reporting the results. aPartners listed are those that interact biochemically with the GEF (directly or indirectly); TJ, tight junctions. bTwo major effects caused by the interaction are reported: modulation of the localization of the GEF (recruitment, retention) at junctions or regulation of the activity status either directly (in vitro via the binding to key domains) or indirectly (via measurement of GTPase activity levels in cellulo). ND, not determined. 2018;10:a028746 Downloaded from http://cshperspectives.cshlp.org/ ieti ril as article this Cite Table 2. Selected list of GTPase activating proteins (GAP) shown to interact with lipids and cytoskeletal proteins found at epithelial junctions Additional GAP Names GTPase Partnera Effect interactionb Effect on junctions observations Reference FILGAP ARHGAP24 Rac1 ND Activated by ROCK-1 Stimulates accumulation Nakahara et al. odSrn abPrpc Biol Perspect Harb Spring Cold phosphorylation to of E-cadherin at 2015 inactivate Rac1 junctions; GAP activity required Rich1 ARHGAP17 Rac1, Cdc42 Amot (coil-coiled Inhibition of Rich1 Recruitment to TJ Wells et al. 2006;

domain) activity; release by Yi et al. 2011 onSeptember25,2021-PublishedbyColdSpringHarborLaboratoryPress competitive binding with Merlin DLC1 ARHGAP7 RhoA α-catenin (aa 117– Accumulation of α- Reduces active RhoA Tripathi et al. 161) catenin and DLC1 at levels at membrane; 2012 membranes and depletion destabilizes 2018;10:a028746 cytosol E-cadherin at junctions DLC2 ARHGAP37 Cdc42 KIF1B (forkhead- ND-complex ND. DLC2 depletion Elbediwy et al. associated coprecipitates with leads to a mild junction 2012; Vitiello domain) p120CTN defect. et al. 2014 Cell at GTPases Small by Signaling DLC3 ARHGAP38 RhoA Scribble (PDZ3) Recruitment Inactivates RhoA at Via its PDZ ligand Hendrick et al. junctions motif 2016; Holeiter et al. 2012 p190RhoGAP-A ARHGAP35 RhoA p120CTN (amino Subcellular targeting ND To endothelial Zebda et al. 2013 acids 820–843) junctions p120CTN PDGFR-induced N-cadherin contacts Wildenberg et al. transient translocation regulated by GAP 2006 p190RhoGAP-B ARHGAP5 RhoA p120CTN ND Depletion GAP increases Association regulated Ponik et al. 2013 RhoA activity at by matrix junctions compliance RhoA Active Rac1 Recruitment to junctions Control levels of active Recruitment Ratheesh et al. –

via interaction with RhoA at cadherin dependent on 2012 Junctions Cell active Rac1 levels contacts McgRacGAP myosin IXa — RhoA F-actin ND Regulate assembly F-actin Binding via the motor Omelchenko and to native junctions domain; Hall, 2012 Continued 7 Downloaded from http://cshperspectives.cshlp.org/ .Braga V. 8

Table 2. Continued Additional GAP Names GTPase Partnera Effect interactionb Effect on junctions observations Reference onSeptember25,2021-PublishedbyColdSpringHarborLaboratoryPress MgcRacGAP RACGAP1, Rac1 Cingulin, Recruitment, ND Inhibits Rac1 at newly Direct binding Guillemot et al. Cyk4 paracingulin formed tight junctions 2014 Rac1, RhoA ND ND Perturbs AJ, inhibits Xenopus cells Breznau et al. RhoA and Rac1 activity 2015 ieti ril as article this Cite at junctions Rac1 α-catenin (1–507) Retention of McgGAP at Coprecipitation is Ratheesh et al. junctions microtubule- 2012 dependent β2 chimerin ARHGAP3 Rac1 DAG Recruitment to apical Suppresses Rac1 Depletion increases Yagi et al. 2012a,b

odSrn abPrpc Biol Perspect Harb Spring Cold domain of epithelial activation at the apical number of cells cysts domain inside lumen Listed are the GAP two different names, small GTPase showed to be modulated in the particular report (rather than known in vitro substrates), interacting partner, what is the effect of the interaction (if known), and the functional outcome for junctions or other cellular processes. Also shown are additional observations and the references reporting the results. aPartners listed are those that interact directly or indirectly with the GEF; TJ, tight junctions. bTwo major effects caused by the interaction are reported: modulation of the localization of the GAP (recruitment, retention) at junctions or regulation of the activity status either directly (in vitro via the binding to key domains) or indirectly (via measurement of GTPase activity levels in cellulo). ND, not determined. 2018;10:a028746 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Signaling by Small GTPases at Cell–Cell Junctions

or dominant forms of GTPases when expressed held at the junctions primarily in an inactivated in epithelial cells localize at junctions (Takaishi conformation, such as the GEFs Trio and GEF- et al. 1997; Braga et al. 2000; Ehrlich et al. 2002; H1 or the GAPs Rich1, β2 chimerin, and Yamada and Nelson 2007). Second, constitutive CdGAP (Tables 1 and 2). activation of GTPases (by locking on a GTP- For upstream regulators such as GEFs and bound status) or their regulators (truncations or GAPs, a number of mechanisms are known to point mutations) have pathological and dire con- mediate their specific distribution or activation sequences for normal cellular function (Alan and (the reader is referred to excellent reviews on Lundquist 2013; Porter et al. 2016). Although these topics, e.g., McCormack et al. 2013; Citi Rac1 is essential for junction stability, activated et al. 2014; van Buul et al. 2014; Sluysmans et al. Rac1 expression strongly disrupt cell–cell con- 2017). In terms of their intracellular localization, tacts (Marei and Malliri 2016). In addition, fast the low expression levels of GEFs and GAPs in cycling mutations of small GTPases have been different cell types hinders the investigation of identified in tumor samples (Alan and Lundquist endogenous proteins. Unfortunately, the lack of 2013; Porter et al. 2016). They behave as consti- suitable biosensors for GEF or GAP activation tutively active mutants, as they undergo faster further complicates the precise mapping of cycles of activation/inactivation and have thus where their dynamic regulation occurs. Of note enhanced kinetics of GTP hydrolysis. is that higher levels of expression of a regulator in Finally, the distribution of the active GTPase a given cell do not necessarily correlate with its pool detected by biosensors (selectively recog- importance on junction stabilization, as argued nize the activated status) forms an essential tool for endothelial cells (van Buul et al. 2014) and to understand the dynamic behavior of GTPases highlighted in Tables 1 and 2. Indeed, such argu- in space and time and have expanded exponen- ment is in line with the prediction that a very tially our understanding of the mechanisms at small pool of an upstream regulator is activated play. There are a variety of biosensors for differ- by a specific stimulus at any given time and place, ent GTPases (fluorescently tagged forexpression similar to what has been shown for GTPases. in cells or as FRET/FLIM probes) (Schaefer Altogether, dynamic studies of upstream regula- et al. 2014). Differential localization of reporters tors lag behind our understanding of how Rho (fluorescently tagged) and activated GTPases GTPases themselves operate. (biosensor FRET/FLIM signal) has been shown Nevertheless, the same principles would op- at cell–cell contacts (e.g., Priya et al. 2016) and erate for both classes of enzymes: steady-state during cellmigration (e.g., Machaceket al. 2009). localization of endogenous/expressed proteins Collectively, these data indicate that a con- may reflect an inactive pool. When required, centration of predominantly active species of cycles of activation/inactivation results in a tran- GTPases in a particular subcellular space is not sient activation of small GTPases to perform desirable for the maintenance of homeostasis. specific tasks. For example, GEF-H1, a RhoA There should be extensive controls to minimize and Rac1 GEF, is localized in an inactivated sta- inappropriate activation of Rho GTPase signal- tus at cell–cell contacts and at microtubule fila- ing, by ensuring a strong spatial and temporal ments (Krendel et al. 2002; Aijaz et al. 2005). It is modulation of GTPase cycling. feasible that GEF-H1 may be transiently activat- Building from past research of junction sta- ed to control vesicular trafficking locally (Pathak bilization, it is clear that the correlation between et al. 2012), and could thus participate in some localization of a regulator GAP or GEF and its aspects of adhesive receptor regulation. The positive role in contact stabilization does not Cdc42/Rac1 CdGAP is not essential for junction hold true for all examples. Tables 1 and 2 list formation and is found inactivated at cell–cell selected examples of interactions of GTPase reg- contacts (McCormack et al., in press). Dysregu- ulators with cytoskeletal proteins and lipids that lation of the mechanisms that maintain CdGAP underpin their localization or function at junc- inactive severely disrupts cell –cell contacts and tions. Of note is that some GTPase regulators are causes cell retraction.

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V. Braga

HOW LOCALIZED ACTIVATION TAKES junctions as inactive complexes, which may or PLACE? may not contain a given GTPase, its activator, and negative regulator in close proximity. A po- Two models underlying transient GTPase acti- tential advantage of the latter mechanism is a vation have been proposed so far (Fig. 2). The better flow of information, as activation/inacti- first model involves the transient recruitment to vation cycles are faster and more efficient, with- junctions of a GTPase and its regulator(s) to out the need to coordinate independent and become activated. A variation of this model timely recruitment of different molecules to would have the GTPases already localized in cell–cell contacts (see sections below). an inactive status and the upstream regulator The two described models are clearly chal- recruited to activate the Rho protein in situ. lenging to dissect (Fig. 2). Examples of de novo Such model would imply a spatial segregation recruitment of specific GTPases or regulators to between a GTPase and its regulators at different junctions following a stimulus have been report- intracellular regions to prevent unwelcomed and ed, whereas the presence of inactive pools of inappropriate activation. The second model GTPase or regulators is also well documented proposes a transient stimulation of a pool of (Tables 1 and 2). Furthermore, different mech- regulators/GTPases already present at cell–cell anisms to maintain regulators at junctions may contacts. This second model predicts that regu- generate distinct outcomes in different cell latory signaling units may already be in place at types. Tiam1, p114RhoGEF, or Tuba are able

Model 1 Model 2 GEF Partner Partner 2 RhoA GAP GTP Partner 3 GEF or GDP Effector

RhoA GAP Stimuli Pi Effector GDP

RhoA

GTP

RhoA Nucleus Nucleus

Figure 2. Potential models via which GTPase signaling is modulated at cell–cell contacts. In model 1, Rho GTPases are maintained at cell–cell contacts via association with binding proteins in an inactive, GDP-bound form. On stimulation, the regulator of GTPases (GEF) is translocated to junctions to activate the small GTPase, enabling interaction with effector proteins. The process is reversed via recruitment of a GAP, which help with the hydrolysis of GTP into GDP, releasing phosphate (Pi). In model 2, it is predicted that inactivated Rho proteins are maintained at junctions in a complex with cytoskeletal proteins that may or may not interact with selected GEF or GAP (see also Fig. 3). Alternatively, GEF/GAP may interact with different partners at the membrane and be kept at close proximity to the Rho GTPase. On stimuli, transient activation/inactivation is achieved coordinately in a speedy manner. In both models, Rho GTPase activation may also occur by blocking locally the function of a GAP, enabling a shift to GTP-loaded GTPase (not shown for simplicity). See text and Tables 1 and 2 for more details and references. Diagrams are not drawn to scale.

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Signaling by Small GTPases at Cell–Cell Junctions

to associate with multiple cytoskeletal proteins isolated migrating cells (Plutoni et al. 2016). Col- at junctions (Table 1), which restricts their po- lectively, the data indicate that the precise activa- sitioning at distinct adhesive sites (i.e., apico- tion of individual GTPases in specific cellular basolateral axis, adherens junctions versus tight events is determined by the ability of associated junctions) by controlling their recruitment and/ partnersandstimulitorecruitdistinctregulators. or retention at cell–cell contacts. The presence of a regulatory signaling unit Importantly,such interactionscan alsomod- (GTPase, GAP, and GEF) at junctions is predict- ulate the GEF activation status depending on the ed by the second model (Fig. 2). Unsuspected associated protein. Cingulin, a tight junction direct interactions among GEFs and GAPs with protein, has been shown to interact with GEF- overlapping specificity for the same GTPase H1 and keep it inhibited at junctions (Fig. 3) could provide fast, localized activation and inac- (Aijaz et al. 2005). Cingulin also binds to tivation. The existence of such complexes have p114RhoGEF in a separate complex (Terry been shown biochemically; for example, Inter- et al. 2012), but it is not clear whether this GEF sectin (a GEF for Cdc42) and CdGAP (ARH- is also inhibited by cingulin interaction. Instead, GAP31, a GAP for Rac1 and Cdc42), although association of p114RhoGEF with Lulu2, a their potential function at cell–cell adhesion has FERM-containing actin binding protein, leads not been addressed (Jenna et al. 2002; Primeau to GEF activation and modulation of tension at et al. 2011). perijunctional actin ring (Nakajima and Tanoue Alternatively, interactions among distinct 2011). Another example is the association of P- regulators may coordinate activities of different cadherin with the GEF β-PIX, which recruits a GTPases in the local control of a cellular process pool of β-PIX to sites of cell–cell contacts in important for junction stabilization (Fig. 2). C2C12 cells. Expression of P-cadherin increases There are numerous examples of such cross- the activation of Cdc42 and Rac1 specifically in talk during cytokinesis (Chircop 2014; Zuo migrating cells, rather than in confluent cells or et al. 2014), single cell wounding (Vaughan et

p120CTN α-catenin Cingulin Ajuba junctions Partners at

Vav2 Ect2 GEF-H1 p114RhoGEF

p190 RhoGAP-A/B DLC1 MgcRacGAP CdGAP Regulators

GTP GDP GTP GDP GDP GTP GTP GDP

GTPases Rac1 RhoA RhoA RhoA Rac1 RhoA Rac1 Rac1

Localization at junctions (recruitment, retention) Regulators GEFs GAPs

Figure 3. Examples of multiple interactions converging on specific proteins resident at junctions. Selected examples of the ability of a cytoskeletal protein or junctional protein to associate with different regulators is depicted here. Such association modulates the activation and localization of regulators (GEF and GAP) by controlling their recruitment or retention at junctions. Each regulator can then modulate the activity of a specific GTPase; shown here is the GTPase that is modified specifically at junctions on modulation of the partner. Ternary complexes among partners and more than one regulator at junctions has not yet been formally shown. Of note is that, in some examples, interactions between partners and regulators can also be found in the cytoplasm in addition to junctions. For more details and references, see text and Tables 1 and 2. Diagrams are not drawn to scale.

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V. Braga

al. 2011), or at attachment to extracellular ma- GTP-bound Rac1, and when phosphorylated, trices (Devreotes and Horwitz 2015). Similar has higher affinity for the activated, GTP-bound regulatory complexes are being identified at Rac1, retaining this form at cell–cell contacts junctions (Tables 1,2). Paracingulin, a tight (Nola et al. 2011). Depletion of Ajuba interferes junction protein, interacts with GEF-H1 and with junction-dependent Rac1 activation (Nola Tiam1 (a Rac1 GEF) and mediates their locali- et al. 2011). Pulsatile behavior of RhoA activa- zation at junctions (Guillemot et al. 2008). The tion has been shown at Xenopus cell–cell con- catenins may also play a prominent role in the tacts (Reyes et al. 2014). Depletion of anillin re- coordination of such activities (Fig. 3; Tables 1 sults in a higher frequency of active RhoA flares, and 2): α-catenin associates with DLC1 (a RhoA but with shorter duration. As anillin interacts GAP), Ect2 (RhoA GEF), and MgcRacGAP (a preferentially with active RhoA (Piekny and GAP for Rac1), whereas p120CTN binds to Glotzer 2008), the data is interpreted as a func- p190RhoGAP and Vav2 (a Rac1 GEF) (Ponik tion for anillin to retain RhoA.GTP at cell–cell et al. 2013; Erasmus et al. 2016). However, ter- contacts. However, as shown during cytokinesis, nary complexes among these proteins have not anillin is also able to interact and co-localize with yet been characterized in the same cellular set- Rho regulators such as Ect2 (Piekny and Glotzer ting. Thus, the relevance of these interactions as 2008; Frenette et al. 2012), p190RhoGAP-A or a signaling hub still remains to be shown. Fur- MgcRacGAP (D’Avino et al. 2008; Gregory et al. ther investigation should seek to clarify key ele- 2008; Manukyan et al. 2015). It will be interesting ments controlling recruitment or retention of to address whether such complexes exist also at each regulator at epithelial contacts, the site junctions and may contribute to the changes in and mechanism of activation and how interact- RhoA activation observed on anillin depletion. ing partners help to modulate these processes. What becomes clear is that an interaction The wealth of information on the cytoskeletal with cytoskeletal filaments may strongly influ- and signaling molecules found at junctions ence the activation status of different GEFs and (Bertocchi et al. 2012; Zaidel-Bar 2013; Zaidel- GAPs. A large number of GEFs has been shown Bar et al. 2015) provides an excellent platform to to interact directly with myosin II assembled spring board such studies. onto F-actin filaments in different cell types (Lee et al. 2010). Such association appears to be a general property of GEFs: it occurs via their CYTOSKELETAL STRUCTURES AS A catalytic domain (dbl domain) and suppresses PLATFORM FOR SIGNALING COMPLEXES GEF activation. In fibroblasts, a variety of GEFs The organization of the cytoskeleton at different localize to stress fibers and on myosin II phos- adhesive sites and intracellular regions has been phorylation and activation, their association is long recognized as an important structure to transiently decreased (Lee et al. 2010). Among harbor signaling complexes. Binding of GTPase the GEF shown to interact with myosin on con- regulators to cytoskeletal proteins/structures tractile F-actin filaments, there are known regu- provides localization and retention cues at junc- lators of cell–cell contacts such as Trio, Tiam1, tions but also a mechanism to control their ac- GEF-H1, or β-PIX (Table 1). It will be interest- tivation. The latter may be direct (preferential ing to determine whether similar association is binding to activated forms) or indirect by cou- found at perijunctional contractile filaments in pling distinct regulators in a complex. Below, I epithelial cells. discuss examples of modulation of Rho GTPases Furthermore, pharmacological disruption of and regulators by interfering with their associa- actin filaments releases GEFs, which are predict- tion with specific cytoskeletal proteins or fila- ed to become activated and increase active levels mentous structures. of Rho GTPases. Indeed, treatment with blebbi- Modulation of levels of interacting partners statin or cytochalasin D leads to higher levels of indeed alter GTPase activation levels. For exam- active RhoA, Cdc42, or Rac1 (Lee et al. 2010). ple, Ajuba interacts directly with both GDP- and Similarly, mechanical strain or myosin II-driven

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Signaling by Small GTPases at Cell–Cell Junctions

forces has been shown to release FILGAP, a GAP GTPases. To cite a few examples, inhibition of for Rac1, from filaminA, a cross-linking actin epidermal growth factor receptor (EGFR) func- binding protein (Ehrlicher et al. 2011). In this tion (pharmacologically or via RNAi) can acti- case, FILGAP attachment to filaminA leads to vate Rac1 and perturb cell–cell contacts (Betson FILGAP activation and suppression of Rac1 ac- et al. 2002; Erasmus et al. 2015). In contrast, tivity, thereby enabling cellular responses to me- inhibition of mammalian target of rapamycin chanical tension (Shifrin et al. 2009). complex (mTORC)1 and mTORC2 with rapa- The microtubule cytoskeleton has been mycin decreasesthe activation of both RhoA and shown to be a repository for ARHGAP21 (Bar- Rac1, promotes cell–cell adhesion and reduces cellos et al. 2013), DLC2 (Vitiello et al. 2014), cell migration (Gulhati et al. 2011). Treatment and Rho GEFs such as GEF-H1 (Krendel et al. with platelet-derived growth factor (PDGF) in- 2002) or Ect2 (Piekny and Glotzer 2008; Su et al. duces atransient dissociation of β-PIX from con- 2011). Whether microtubule binding to ARH- tractile actin filaments and concomitant activa- GAP21, DLC2, or Ect2 modulates their catalytic tion of Rac1 (Lee et al. 2010). Such examples activity is not clear. Disruption of microtubules reinforce the complex interplay between struc- pharmacologically releases and activates GEF- tural components and the GEF/GAP signaling H1 (Chang et al. 2008; Pathak et al. 2012). In machinery and the need to understand GTPase contrast, in lung endothelium, stimulation with signaling in a broader cellular architecture. hepatocyte growth factor (HGF) increases the activation of the GEFs Asef and Tiam1, and the former specifically shows higher levels of CONCLUDING REMARKS interaction with peripheral microtubules (Hig- GEFs, GAPs, and Rho small GTPases are local- ginbotham et al. 2014). KIF17 is a microtubule ized to different intracellular spaces and struc- motor that interacts with the plus-end microtu- tures via association with lipids, interaction with bule capture machinery at the cell cortex. Ex- cytoskeletal proteins, microtubules and micro- pression of KIF17 increases active RhoA and filaments. Although the key role of these inter- junctional actin levels, thereby stabilizing cell– actions for localization of signaling has long cell contacts (Acharya et al. 2016). Although the been recognized, it now emerges that cytoskele- GEF responsible for these effects is not known, tal partners provide networking capabilities to these results suggest that modification of micro- concatenate temporal enrolment of distinct sig- tubule dynamics in more subtle ways also have naling molecules in a particular place. Further- an impact on RhoA signaling. more, direct interaction with cytoskeletal struc- Our understanding on the modulation of tures and proteins can influence the availability, keratin filaments and desmosomes by Rho extent and frequency of activation/inactivation GTPase signaling is emerging. Potential interac- cycles, thereby providing signaling fine-tuning. tion of GTPase regulators with keratin filaments The intricate relationship between cytoskeletal is poorly understood in epithelia (Todorovic structures and signaling at junctions provides an et al. 2010; Dubash et al. 2013, 2014). An exam- exciting platform to build our knowledge on ple has been reported in endothelial cells, where how to integrate cell–cell adhesion with cellular ARHGEF40 (SOLO), a RhoA GEF stimulated by function. mechanical stress, interacts with keratin8/18 filaments and its overexpression induces keratin bundling (Abiko et al. 2015; Fujiwara et al. REFERENCES 2016). It is likely that further studies will unravel Abiko H, Fujiwara A, Ohashi K, Hiatari R, Mashiko T, similar interactions and regulatory events asso- Sakamoto N, Sato M, Mizuno K. 2015. Rho guanine ciated with keratin dynamics in epithelial cells. nucleotide exchange factors involved in cyclic-stretch-induced reorientation of vascular endothelial cells. J Cell Sci Finally, treatment with different drugs that 128: – fi 1683 1695. do not speci cally target the cytoskeleton can Acharya BR, Espenel C, Libanje F, Raingeaud J, Morgan J, unexpectedly alter the basal activity status of Jaulin F, Kreitzer G. 2016. KIF17 regulates RhoA-depen-

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Signaling by Small GTPases at Cell–Cell Junctions

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Signaling by Small GTPases at Cell−Cell Junctions: Protein Interactions Building Control and Networks

Vania Braga

Cold Spring Harb Perspect Biol 2018; doi: 10.1101/cshperspect.a028746 originally published online September 11, 2017

Subject Collection Cell-Cell Junctions

Vascular Endothelial (VE)-Cadherin, Endothelial Signaling by Small GTPases at Cell−Cell Adherens Junctions, and Vascular Disease Junctions: Protein Interactions Building Control Maria Grazia Lampugnani, Elisabetta Dejana and and Networks Costanza Giampietro Vania Braga Adherens Junctions and Desmosomes Making Connections: Guidance Cues and Coordinate Mechanics and Signaling to Receptors at Nonneural Cell−Cell Junctions Orchestrate Tissue Morphogenesis and Function: Ian V. Beamish, Lindsay Hinck and Timothy E. An Evolutionary Perspective Kennedy Matthias Rübsam, Joshua A. Broussard, Sara A. Wickström, et al. Cell−Cell Contact and Receptor Tyrosine Kinase The Cadherin Superfamily in Neural Circuit Signaling Assembly Christine Chiasson-MacKenzie and Andrea I. James D. Jontes McClatchey Hold Me, but Not Too Tight−−Endothelial Cell−Cell Mechanosensing and Mechanotransduction at Junctions in Angiogenesis Cell−Cell Junctions Anna Szymborska and Holger Gerhardt Alpha S. Yap, Kinga Duszyc and Virgile Viasnoff Connexins and Disease Beyond Cell−Cell Adhesion: Sensational Mario Delmar, Dale W. Laird, Christian C. Naus, et Cadherins for Hearing and Balance al. Avinash Jaiganesh, Yoshie Narui, Raul Araya-Secchi, et al. Cell Junctions in Hippo Signaling Cell−Cell Junctions Organize Structural and Ruchan Karaman and Georg Halder Signaling Networks Miguel A. Garcia, W. James Nelson and Natalie Chavez

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Loss of E-Cadherin-Dependent Cell−Cell Adhesion Cell Biology of Tight Junction Barrier Regulation and the Development and Progression of Cancer and Mucosal Disease Heather C. Bruner and Patrick W.B. Derksen Aaron Buckley and Jerrold R. Turner Desmosomes and Intermediate Filaments: Their Integration of Cadherin Adhesion and Consequences for Tissue Mechanics Cytoskeleton at Adherens Junctions Mechthild Hatzfeld, René Keil and Thomas M. René Marc Mège and Noboru Ishiyama Magin

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