WNT-3A Modulates Platelet Function by Regulating Small Gtpase Activity ⇑ Brian M

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FEBS Letters 586 (2012) 2267–2272

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WNT-3a modulates platelet function by regulating small GTPase activity ⇑ Brian M. Steele a, , Matthew T. Harper b, Albert P. Smolenski a, Naheda Alkazemi a, Alastair W. Poole b, ⇑ Desmond J. Fitzgerald a, Patricia B. Maguire a,

a UCD Conway Institute, University College Dublin, Belﬁeld, Dublin D4, Ireland b Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom

article info abstract

Article history: Here we provide evidence that WNT-3a modulates platelet function by regulating the activity of four Received 20 March 2012 key GTPase proteins: Rap1, Cdc42, Rac1 and RhoA. We observe WNT-3a to differentially regulate Revised 4 May 2012 small GTPase activity in platelets, promoting the GDP-bound form of Rap1b to inhibit integrin-aIIbb3 Accepted 23 May 2012 adhesion, while concomitantly increasing Cdc42 and Rac1-GTP levels thereby disrupting normal Available online 13 June 2012 platelet spreading. We demonstrate that Daam-1 interacts with Dishevelled upon platelet activation, Edited by Lukas Huber which correlates with increased RhoA-GTP levels. Upon pre-treatment with WNT-3a, this complex disassociates, concurrent with a reduction in RhoA-GTP. Together these data implicate WNT-3a as a novel upstream regulator of small GTPase activity in platelets. Keywords: Daam-1 GTPase Structured summary of protein interactions: Platelets DVL physically interacts with DAAM-1 by anti bait co-immunoprecipitation (View interaction) WNT signalling DVL and DAAM-1 colocalise by ﬂuorescence microscopy (View interaction)

1. Introduction traditionally thought to be a canonical ligand, emerging evidence suggests that WNT-3a may also activate non-canonical pathways Anucleate platelets are the principal effectors of haemostasis to regulate small GTPase activity [7–10]. In particular, it has been and are found circulating in a non-adhesive, quiescent state. Small shown that WNT-3a regulates RhoA-GTP levels by engaging Fzd GTPase proteins such as Rho, Rac and Rap play an important signal- receptors to regulate the interaction between the cytoplasmic ling role during platelet activation where they regulate a variety of WNT signalling protein Dishevelled (Dvl) and the formin protein intracellular functions including integrin activation, stress fibre Daam-1 [11–13]. formation and cytoskeletal reorganisation [1–3]. GTPases cycle be- In platelets, a number of formin-related proteins are known to tween an active and inactive state, regulated by Guanine nucleo- be present, including mDia1, mDia2, Fhod1 and Daam-1 [14]. tide Exchange Factors (GEFs) and GTPase Activating Proteins Although Daam-1 is known to promote actin assembly through (GAPs). In resting platelets, small GTPase proteins exist mainly in the small GTPase RhoA [15], the upstream signalling processes that a GDP-bound state, with GAPs promoting the hydrolysis of GTP regulate Daam-1 activity in platelets are currently unknown. In to GDP. Upon platelet stimulation, GEFs then promote their activa- other cell types, the formation of a Dvl/Daam-1 complex is critical tion by increasing the binding of GTP. This allows the GTPase pro- for RhoA activation, since Daam-1 does not have the ability to acti- tein to bind its effector and so initiate a signalling response [4,5]. vate RhoA on its own. Instead Daam-1 is thought to act as a scaf- We have previously shown the canonical WNT signalling path- fold protein, first binding to Dvl to allow ‘unfolding’ of the way to be present and active in platelets and the WNT-3a ligand to protein, before recruiting Rho GEF proteins to enhance GTPase acti- inhibit platelet adhesion, secretion and shape change during the vation [12]. This activates the downstream effector Rho-associated platelet activation response [6]. The Frizzled-6 (Fzd6) receptor kinase (ROCK), which mediates cytoskeletal rearrangement by pro- was shown in part to mediate this inhibitory response; however moting myosin light chain phosphorylation [5,16]. As we previ- the underlying signalling effect and the exact downstream WNT ously identified Dvl in human platelets and found WNT-3a to signalling effectors involved are currently unknown. Although reduce platelet RhoA-GTP levels [6], we hypothesised a link between WNT signalling in platelets and small GTPase function. ⇑ Here we provide evidence that WNT-3a is differentially modu- Corresponding authors. Address: UCD Conway Institute, School of Biomolecular lating the activity of four key platelet GTPase proteins: Rap1, & Biomedical Science, University College Dublin, D4, Ireland. Fax: +353 1 7166700. E-mail addresses: [email protected] (B.M. Steele), [email protected] Cdc42, Rac1 and RhoA. Furthermore, we demonstrate that (P.B. Maguire). WNT-3a regulates a Dvl/Daam-1 signalling complex in platelets

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.05.060 2268 B.M. Steele et al. / FEBS Letters 586 (2012) 2267–2272 to control downstream RhoA-GTP activity. Regulation of small adhesion and spreading was monitored by differential interference GTPase activity by WNT-3a can have a major impact on the platelet contrast (DIC) microscopy with a wide-ﬁeld microscope DM IRB at- signalling response and represents a possible mechanism through tached to an ORCA ER camera (Leica, Wetzlar, Germany). Images which WNT signalling negatively regulates platelet function. were processed with Volocity OpenLab 4.0.3 (Improvision, Wal- tham, MA) to produce Quicktime videos of platelet spreading. 2. Methods Adherence and spread platelet size was calculated using Image J 1.42q software (http://rsb.info.nih.gov/ij/) at 720 s with any plate- 2.1. Platelet isolation and co-immunoprecipitation let producing lamellipodia deemed ‘spread’.

Platelets were isolated by centrifugation and recombinant 3. Results WNT-3a (R&D Systems, Abingdon, UK) added 30 s before agonist loading as previously described [6]. Platelet activation was for 3.1. WNT-3a inhibits Rap1b activation by promoting Rap1GAP2 30 s unless otherwise stated and co-immunoprecipitation experi- activity ments carried out on the lysed platelets (1 Â 109/ml) as before [17] using an antibody speciﬁc for anti-Dvl (sc-25534, Santa Cruz, Rap1b is a key intracellular signalling protein in platelets

Heidelberg, Germany). Recombinant WNT-3a vehicle controls known to control integrin aIIbb3-receptor activation and platelet were tested in a wide range of platelet assays, with no significant aggregation [3]. Since small GTPase activation occurs rapidly fol- effects observed. lowing agonist stimulation, and platelet aggregation has been previously shown to induce the inactivation of Rap1b [20], samples 2.2. Measurement of small GTPase activation were lysed after only 30 s in the absence of aggregation. We observed no Rap1-GTP in resting platelets but found levels to increase Washed platelets (1 Â 109/ml) were activated with 1.5 lM after 30 s of 1.5 lM TRAP activation (p = 0.0004) (Fig. 1A). Pre- TRAP for 30 s before lysis. 20 ll lysate was kept for analysis of total treating platelets with 32 or 50 nM WNT-3a prior to TRAP activa- protein expression and the remaining lysate used to precipitate tion inhibited Rap1-GTP levels (p = 0.1446 and p = 0.0081, respec- GTP-bound protein using agarose beads conjugated to either tively), while WNT-3a on its own had no effect. Since total Rap1 GST-Rhotekin-RBD for RhoA (Cytoskeleton, CO, USA), GST-PAK- levels were found to remain stable, it is likely that WNT-3a is reg- PBD for Rac1 (Cytoskeleton) or GST-RalGDS-RBD for Rap1b as pre- ulating the conversion of Rap1-GTP into its inactive GDP-bound viously described [18]. Total and precipitated GTPase protein was state. measured by immunoblot analysis as per the manufacturer’s As the only Rap1 GAP protein identified in platelets to date, instructions. GTP-bound Cdc42 protein was measured using a G- Rap1GAP2 plays a pivotal role in platelet Rap1b GTPase regulation LISA assay (Cytoskeleton). by converting it into an inactive GDP-bound form [18]. Phosphor- ylation of Rap1GAP2 at Ser7, a residue present only in the platelet 2.3. SDS–PAGE and immunoblotting splice variants of Rap1GAP2, is a known marker of GAP activity [18]. Using a phospho-specific Ser7 antibody we observed only Equal amounts of protein were analysed by Western blotting background levels of Ser7 phosphorylation in resting and 1.5 lM and visualised as described [6]. Antibodies used were as follows: TRAP-activated platelets (Fig. 1B). However, when platelets were Abcam (Cambridge, UK): GAPDH (ab9485); Santa Cruz (Heidelberg, pre-treated with 32 or 50 nM WNT-3a (p = 0.0510 and p = 0.0222, Germany): a-actinin (sc-17829), b-actin (sc-130300), Dvl (sc- respectively) prior to TRAP activation, we observed an increase in 25534), RhoA (sc-418) and Rap1 (sc-166556); Cytoskeleton: Ser7 phosphorylation suggesting that WNT-3a may be promoting Cdc42 (ACD03) and Rac1 (ARC03). Anti-Daam-1 was a kind gift Rap1GAP2 activity during platelet activation. This in turn may ex- from Prof. Raymond Habas (Temple University, PA, USA.), while plain the reduction in Rap1b-GTP levels observed in Fig. 1A. phospho-Rap1GAP2 (Ser7) antibody was made as described [19]. Densitometry analysis was carried out using Image J 1.42q software 3.2. Increased Cdc42 and Rac1-GTP levels correlate with faster platelet (http://rsb.info.nih.gov/ij/), normalising to the control, and is rep- spreading over a fibrinogen extracellular matrix resentative of three independent experiments. Statistical analysis was then performed on the resulting data using unpaired two- Small GTPase activation is also critical for platelet spreading tailed t-tests (GraphPad Prism v5.0). with Cdc42 implicated during filopodia formation [21] and Rac1 essential for lamellipodia production [22]. In washed platelets, 2.4. Confocal microscopy Cdc42-GTP levels peaked after 25 s in response to 1.5 lM TRAP activation (solid line), before returning to basal levels (Fig. 2A). Platelets (2 Â 104/lL) were activated for 30 s with 1.5 lM TRAP, Pre-treating platelets with 50 nM WNT-3a alone increased in the absence and presence of 50 nM WNT-3a, prior to fixing in Cdc42-GTP levels slightly (data not shown), while incubation for 3.7% paraformaldehyde. The fixed platelets were adhered to un- 30 s prior to TRAP stimulation (dotted line) was found to elevate coated glass slides for 30 min. Slides were permeabilised in 0.1% Cdc42-GTP levels across all time points, including resting platelets Triton-X100 before incubating with primary antibodies for anti- (0 s), however this was statistically significant only at 25 s Daam-1 (a kind gift from Prof. Raymond Habas) and anti-Dvl (sc- (p = 0.0281). We also observed Racl-GTP levels to increase during 25534, Santa Cruz), followed by an appropriate fluorescent second- WNT-3a treatment (Fig. 2B). Similar to the other small GTPases, ary antibody. All images were acquired using an Olympus FV1000 immunoblot analysis showed little Rac1 activation in resting plate- confocal microscope (Olympus Europa Germany). lets. However, Rac1-GTP levels increased upon 1.5 lM TRAP activation (p = 0.0090) and were further increased in the presence of 2.5. Platelet spreading WNT-3a (p = 0.0256). Next we used a qualitative in vitro platelet spreading assay to Glass coverslips were coated overnight with 100 lg/ml fibrino- test the ability of washed platelets to adhere and spread on immo- gen (Sigma, Arklow, Ireland) before blocking with BSA and mount- bilised fibrinogen (Fb) (Fig. 2C) (videos in Supplementary informa- ing in a live-cell microscopy chamber. Platelet (2 Â 108/ml) tion). In the absence of WNT-3a, platelets adhere to Fb and produce B.M. Steele et al. / FEBS Letters 586 (2012) 2267–2272 2269

A B

1.5µM TRAP - +++ - - 1.5µM TRAP - +++ - - 32nM WNT-3a --- + + - 32nM WNT-3a --- + + - 50nM WNT-3a - --+ - + 50nM WNT-3a - --+ - +

Rap1b-GTP 27kDa pRap1GAP2 (Ser7) 90kDa

Total Rap1 27kDa α-actinin 100kDa

Fig. 1. WNT-3a inhibits Rap1 activation by promoting Rap1GAP2 activity. Resting and 1.5 lM TRAP-activated platelets were prepared in the absence and presence of WNT-3a before (A) Incubating with GST-RalGDS-RBD agarose beads to pull-down the active form of Rap1, or (B) immunoblotting for phosphorylated Rap1GAP2 (Ser7) levels. Forskolin-treated platelets (10 lM) were used as the positive control (data not shown) and total Rap1 and a-actinin immunoblots show equal loading. Densitometry analysis is from three independent experiments ± S.E.M. and represents the GTP-bound or phosphorylated protein level normalised for total Rap1 or a-actinin protein expression (⁄, p < 0.05).

filopodia and lamellipodia over a 300 s time point, until the plate- interaction detected between these proteins when platelets were let is fully spread and remains unchanged after 720 s (Fig. 2C(i)). in a resting state (0 s). However, Dvl co-immunoprecipitated with Following WNT-3a treatment, the total number of platelets adher- Daam-1 within 30–60 s of TRAP activation (1.5 lM) and this inter- ing to Fb was significantly reduced (Fig. 2D; column 2), while the action was inhibited when platelets were pre-treated with 50 nM remaining adherent platelets showed faster spreading in compari- WNT-3a. Together these data propose that the Dvl/Daam-1 com- son to the control samples. These platelets displayed only transient plex plays a role during early platelet activation and shape change. filopodia production, and had extensive lamellipodial formation While the Dvl/Daam-1 complex is known to bind and activate visible after only 180 s (Fig. 2C(iii) (180 s)). Analysis of the mean Rho-GTP in other cell types [12], it is unknown whether this occurs surface area (Fig. 2D; column 3) revealed that platelets treated in platelets. In the absence of WNT-3a, we found platelet RhoA-GTP with 32 or 50 nM WNT-3a were significantly larger than the con- levels to increase following 30 s of TRAP (1.5 lM) stimulation be- trol platelets (p = 0.038 and p = 0.010, respectively). Furthermore, fore returning to basal levels at 60 s (Fig. 3C). This coincided with instead of the centrally located granules seen under basal condi- a dramatic increase in Daam-1 binding to RhoA-GTP at 30 s and tions (Fig. 2C(i) (720 s)), WNT-3a treated platelets displayed an to a lesser extent 60 s. These results are consistent with the Dvl/ abnormal granule distribution with a concave centre in the fully Daam-1 complex forming 30 s after TRAP-activation (Fig. 3A and spread platelet (Fig. 2C(iii) (720 s)). This did not change for the B) suggesting that the Dvl/Daam-1 complex is a novel upstream remainder of the 30 min observation period. As microtubule rear- promoter of Rho-GTP activity in platelets. WNT-3a (50 nM) abol- rangement and actin assembly are essential for platelet shape ished the effect of TRAP on both RhoA-GTP levels and Daam-1 change it suggests a possible role for WNT-3a in modulating the binding to Rho-GTP. These findings are consistent with prior re- platelet cytoskeleton. ports showing that the WNT pathway controls Dvl/Daam1/Rho- signalling in other cell types [12]. Total RhoA levels were found 3.3. WNT-3a regulates RhoA-GTP activity via a Dvl/Daam-1 signalling to remain constant, suggesting that the WNT-mediated reduction complex in RhoA-GTP is due to WNT-3a converting Rho-GTP into its inactive GDP-bound state. In other cells, transmission of the WNT-3a signal via the Fzd receptor regulates the interaction between Dvl Daam-1 [12].We 4. Discussion have previously reported that Fzd4, Fzd6 and Dvl are all present in human platelets. As we also found WNT-3a to reduce platelet In this study, we provide strong evidence that WNT-3a influ- RhoA-GTP levels [6], we hypothesised a possible interaction be- ences platelet activation by regulating the activity of four key small tween Dvl and Daam-1 in platelets. Using confocal microscopy, GTPase proteins; Rap1b, Cdc42, Rac1 and RhoA. Previously we we provide evidence for the interaction between Dvl and Daam-1 established that WNT-3a inhibited activation of the integrin aIIbb3 following 30 s of 1.5 lM TRAP stimulation (Fig. 3A). In the absence surface receptor, resulting in decreased platelet adhesion to Fb and of WNT-3a Dvl and Daam-1 co-localised in response to TRAP. How- aggregation [6]. While aIIbb3 requires intracellular calcium signal- ever upon WNT-3a pre-treatment, while both proteins still reside ling for activation [23], we previously demonstrated that WNT- at the outer edge of the platelet, they do not appear to co-localise 3a inhibits platelets independent of altered Ca2+ mobilisation [6]. to the same extent. Similarly, WNT-3a treatment alone was also Our observation that WNT-3a inhibits Rap1b therefore provides a found to have no effect on Dvl/Daam-1 association (data not viable alternative explanation for WNT-3a mediated inhibition of shown). Next, we blotted Dvl immunoprecipitates with a Daam- inside-out activation of this major integrin on platelets. Since 1-specific antibody (Fig. 3B). Similar to microscopy, there was no Rap1b is carefully regulated by GAPs and GEFs, one could presume 2270 B.M. Steele et al. / FEBS Letters 586 (2012) 2267–2272

A B *

1.5µM TRAP - ++- 50nM WNT-3a - - + +

Rac1-GTP 22kDa

Total Rac1 22kDa

C Time (seconds)

(i) Untreated

(ii) 32nM WNT-3a

(iii) 50nM WNT-3a

D Platelet % Spread Mean Platelet Treatment Platelets Surface Area (μm2)

None 54 ± 3.7 42.05 ± 0.89 32nM WNT-3a 22 ± 2.0* 46.82 ± 1.95* 50nM WNT-3a 18 ± 2.8* 48.57 ± 1.73*

Fig. 2. Increased Cdc42 and Rac1-GTP levels correlates with faster platelet spreading over a fibrinogen extracellular matrix. Resting and 1.5 lM TRAP-activated platelets were used to measure (A) Cdc42-GTP levels by G-LISA in the absence (solid line) and presence (dotted line) of 50 nM WNT-3a or (B) Rac1-GTP levels using GST-PAK-PBD agarose beads in the absence and presence of WNT-3a. Densitometry analysis is from three independent experiments and total Rac1 protein expression was used to normalise Rac1- GTP levels. Results are presented as mean values ± S.E.M. (C) Resting platelets were allowed to spread over Fb in the absence and presence of WNT-3a, using differential interference contrast microscopy. (D) Adherence and spread platelet size was calculated using Image J software. (⁄, p < 0.05). that WNT signalling may either be promoting GAP activity to spreading. However, recent studies suggest that filopodia forma- switch off GTPase function or inhibiting the GEFs that promote tion can also occur independantly of Cdc42 [26]. In addition, acti- small GTPase activity. Indeed, WNT signalling has been shown to vation of Rac1 is known to be essential for correct lamellipodia regulate GAP and GEF activity in other cells, with WNT-3a activat- formation [22]. Therefore, increased activation may explain the ing the Rac1 GEF Tiam-1 to increase Rac1-GTP levels in chondro- faster, more extended platelet spreading observed with WNT-3a. cytes [24]. In platelets, we observed Ser7 phosphorylation of However, while WNT signalling would possibly reduce the number Rap1GAP2 to increase during WNT signalling, although it remains of platelets adhering at sites of vascular damage, it remains unclear unclear why phosphorylation occurs only in the presence of TRAP why the rate and extent of platelet spreading would be increased activation. Since phospho-Rap1GAP2 (Ser7) has previously been for the remaining adherent population and further work will be re- linked to increased GAP activity [25], it suggests that WNT-3a quired to specifically understand this. Indeed a similar phenotype may be increasing Rap1 GAP activity during platelet activation has been reported in WNT-3a treated chondrocytes, where en- and offers a likely explanation for the lower Rap1b-GTP levels hanced expression of the Rac1 GEF, Tiam-1, increased cell spread- and inhibition of integrin aIIbb3 activation and platelet aggregation ing size [24]. A comparable phenotype has also been reported in as described previously [6]. However, further work will be required melanoma A7 cells lacking the FilGAP1 protein [27], which is a to directly prove that this is the case. Rac GAP protein activated downstream of RhoA. RhoA-GTP acti- We also found WNT-3a to increase Cdc42 and Rac1-GTP levels vates FilGAP1 which converts Rac1 into its GDP-bound form and during platelet activation. Since Cdc42 is thought to be required thus switches Rac1 activity off. Therefore, as WNT-3a also inhibits for filopodia growth [21], this may offer an explanation for the RhoA activation, it may also reduce FilGAP1 levels or increase rapid, transient filopodia formation observed during platelet Tiam-1 expression, either of which would increase Rac1 activation B.M. Steele et al. / FEBS Letters 586 (2012) 2267–2272 2271

AB DIC Daam-1 Dvl merged IP: Dvl Time (seconds)

50nM WNT-3a - - - - - + + + - - - - - 50nM 20μm 1.5μM TRAP + + + + WNT-3a PL -- IgG 0 30 60 0 30 60

Daam-1 122kDa 2.5μm Dvl 90kDa

β-actin 40kDa + 50nM 20μm WNT-3a

2.5μm

C Pull Down: Rho-GTP Time (seconds) 50nM WNT-3a - - - + + + 1.5μM TRAP - + + - + + GTPγS 0 30 60 0 30 60 RhoA-GTP 24kDa

Daam-1 122kDa

Total RhoA 24kDa

Fig. 3. WNT-3a regulates RhoA-GTP activity via a Dvl/Daam-1 signalling complex. Resting and 1.5 lM TRAP-activated platelets were prepared in the absence and presence of 50 nM WNT-3a before (A) Using confocal microscopy to investigate the cellular location of Daam-1 and Dvl, (B) Immunoprecipitating with Dvl antibody prior to blotting for Daam-1 (Platelet Lysate (PL) was used as the positive control and anti-Dvl and anti-b-actin immunoblots show equal protein loading) or (C) incubating with GST-rhotekin- RBD agarose beads to pull-down the active form of RhoA. GTPcS-treated platelet lysate was used as the positive control and total RhoA immunoblots show equal protein loading across all sample lanes. All blots are representative of three independent experiments. and thus promote lamellipodia formation and faster platelet Thus, WNT effectors can assemble to provide diverse and cell-spe- spreading. This inverse Rho/Rac relationship has been noted previ- cific signalling responses. This is especially relevant in the anucle- ously during platelet activation [28] and both RhoA and Rac1 play ate platelet as any WNT-3a effect is independent of changes in key roles during the WNT signalling response [29,30]. However, gene expression. since platelets cannot respond to changes in expression in the tra- Taken together, our results demonstrate that WNT-3a differen- ditional sense (i.e., transcription–translation), further work will be tially modulates the downstream signalling activity of four key required to establish exactly how WNT signalling is producing such GTPase proteins in platelets. We hypothesise that WNT-3a is most effects on platelets as WNT-3a may affect post-translational mod- likely regulating key GAPs and GEFs during platelet activation to ification of these signalling proteins or their compartmentalisation either suppress (Rho, Rap1b) or promote (Rac1, Cdc42) small within the platelet. GTPase function. In conclusion, our data implicates WNT and down- In this study, we also demonstrate that TRAP promotes the stream WNT signalling effectors as novel upstream regulators of binding of the WNT signalling protein Dvl to Daam-1 to control small GTPase activity in platelets and represents a possible mecha- downstream RhoA GTPase activity. The involvement of Dvl in the nism through which WNT-3a signalling regulates platelet function. platelet signalling response has not been previously demonstrated however, Dvl has been shown to interact specifically with Ga13 Acknowledgements during PAR1 activation in HEK cells [31], suggesting that this also could be occurring in platelets. Further investigation will be re- We thank Dr. Gerard Cagney for his helpful comments upon quired to establish whether this specific interaction is present in reading the manuscript and Prof. Raymond Habas for the kind gift the anucleate platelet and whether other platelet agonists are of Daam-1 antibody. We would like to acknowledge funding by capable of eliciting a similar Dvl/Daam-1 signalling response. Science Foundation Ireland (PBM: 10/IN.1/B3012 and APS: 08/ While we found WNT-3a to inhibit Dvl/Daam-1 binding during IN.1/B1855), The Biochemical Society (PBM, KG), Programme for platelet activation, in other cells WNT has an opposing effect, pro- Research in Third-Level Institutions (PRTLI), Ireland (DJF, PBM), moting Dvl/Daam-1 binding to increase RhoA activation [12,29]. the Ministry of Kuwait (NA) and support from the British Heart Such opposing cell-specific responses to WNT-3a have already Foundation (AWP and MTH; RG/10/006/28299). been demonstrated [32]. For example, WNT-3a activates gene transcription in HUVEC cells [7], while in monocytes and THP-1 cells it Appendix A. Supplementary data has no effect on TCF-promoter activity or the expression of classi- cal WNT target genes [33,34]. Likewise, Daam-1 is not required for Supplementary data associated with this article can be found, in WNT signalling in Drosophila [35], yet in Xenopus embryos Daam-1 the online version, at http://dx.doi.org/10.1016/j.febslet.2012.05. is essential for WNT/Fzd/Dvl-dependent RhoA activation [12]. 060. 2272 B.M. Steele et al. / FEBS Letters 586 (2012) 2267–2272

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