© 2021. Published by The Company of Biologists Ltd | Journal of Cell Science (2021) 134, jcs251140. doi:10.1242/jcs.251140 RESEARCH ARTICLE Daam2 couples translocation and clustering of Wnt receptor signalosomes through Rac1 Carlo D. Cristobal1,QiYe2, Juyeon Jo2, Xiaoyun Ding3, Chih-Yen Wang2, Diego Cortes2, Zheng Chen4 and Hyun Kyoung Lee1,3,5,* ABSTRACT Dynamic polymerization of the Dishevelled proteins functions at Wnt signaling plays a critical role in development across species and the core of the Wnt signalosome by interacting with both the is dysregulated in a host of human diseases. A key step in signal Frizzled Wnt receptors and low-density lipoprotein receptor-related transduction is the formation of Wnt receptor signalosomes, during protein 5/6 (LRP5/6), leading to recruitment of Axin proteins β which a large number of components translocate to the membrane, from the -catenin destruction complex (MacDonald et al., 2009; cluster together and amplify downstream signaling. However, the Schwarz-Romond et al., 2007). However, the exact composition molecular processes that coordinate these events remain poorly and mechanisms of signalosome assembly at the plasma membrane defined. Here, we show that Daam2 regulates canonical Wnt remain unclear. signaling via the PIP –PIP5K axis through its association with Rac1. The hallmark of canonical Wnt signaling is the accumulation and 2 β Clustering of Daam2-mediated Wnt receptor complexes requires both translocation of -catenin into the nucleus for gene transcription, β Rac1 and PIP5K, and PIP5K promotes membrane localization of these whereas non-canonical Wnt signaling is -catenin independent and complexes in a Rac1-dependent manner. Importantly, the localization involves assembly/disassembly of the actin cytoskeleton, polarized of Daam2 complexes and Daam2-mediated canonical Wnt signaling is cell shape changes and cell migration (Niehrs, 2012; Schlessinger dependent upon actin polymerization. These studies – in chick spinal et al., 2009). Rho family GTPase proteins play important roles in cord and human and monkey cell lines – highlight novel roles for Rac1 actin/microtubule organization such as cell adhesion and migration and the actin cytoskeleton in the regulation of canonical Wnt signaling in all eukaryotic cells (Hodge and Ridley, 2016). Although Rho and define Daam2 as a key scaffolding hub that coordinates membrane GTPases are known to be mainly involved in the non-canonical translocation and signalosome clustering. Wnt/planar cell polarity signaling pathways (Niehrs, 2012; Schlessinger et al., 2009), evidence indicates a potential link KEY WORDS: Daam2, Rac1, PIP5K, Wnt signalosome, Chick spinal between Rho GTPases and canonical Wnt signaling, with Rac1 cord, Dorsal patterning emerging as a possible major player (Schlessinger et al., 2009). Several studies in different systems have reported that Rac1 INTRODUCTION promotes nuclear import of β-catenin (Wu et al., 2008), enhances Wnt signaling is a highly conserved signaling pathway required for β-catenin–LEF-1 complex assembly (Jamieson et al., 2015) cell fate determination during development and homeostasis in adult and interacts directly with Dishevelled proteins (Dvls) (Cajanek tissues (Logan and Nusse, 2004; Nusse and Clevers, 2017), and et al., 2013; Soh and Trejo, 2011), resulting in the activation of emerging evidence has implicated Wnt dysregulation in both genetic canonical Wnt signaling. Moreover, emerging studies reported the and non-genetic neurological disorders (Fancy et al., 2014). Wnt association between Rac1 and phosphatidylinositol 4-phosphate-5 signaling effectors have relatively low affinities for each other, kinase (PIP5K) proteins, which mediate phosphatidylinositol 4,5- preventing unregulated interactions at their physiological levels and bisphosphate (PIP2) production for signalosome formation (Pan et al., allowing fine-tuning of proper signaling activation at the appropriate 2008; van den Bout and Divecha, 2009; Weernink et al., 2004). This time (Kishida et al., 2001; Niehrs, 2012; Schwarz-Romond et al., finding raises an important question regarding a direct functional 2007). Therefore, a key regulatory step for Wnt signaling activation is relationship between Rho GTPases and proximal events of canonical the strengthening of modest interactions between Wnt effectors when Wnt receptor complex signaling, which remains to be addressed. the Wnt ligand binds the receptor. As a result, transmembrane Previously, we found that dishevelled associated activator of receptors and signal transducers assemble to form the Wnt morphogenesis 2 (Daam2) is required for the clustering of Wnt signalosome (Gammons et al., 2016; MacDonald et al., 2009). receptor complexes and canonical Wnt signaling through its association with PIP5K and, consequently, PIP2 production (Lee and Deneen, 2012; Lee et al., 2015). The presence of a GTPase- 1Program in Integrative Molecular and Biomedical Sciences, Baylor College of binding domain in Daam2 and previous studies implicating Rho Medicine, Houston, TX 77030, USA. 2Department of Pediatric, Baylor College of GTPase Rac1 in PIP5K activation suggested a possible role for Rac1 Medicine, Houston, TX 77030, USA. 3Program in Developmental Biology, Baylor – College of Medicine, Houston, TX 77030, USA. 4Department of Biochemistry and in Daam2 PIP5K-mediated canonical Wnt signaling. Here, we Molecular Biology, The University of Texas Health Science Center at Houston, show that the association of Daam2, Rac1 and PIP5K is critical for Houston, TX 77030, USA. 5Jan and Dan Duncan Neurological Research Institute, Wnt receptor complex clustering, membrane translocation and Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030, USA. subsequent activation of canonical Wnt signaling during cell fate *Author for correspondence ([email protected]) determination. Moreover, pharmacological manipulation of actin dynamics regulates Daam2-mediated Wnt signal transduction H.K.L., 0000-0001-5793-4398 during central nervous system development. Taken together, our in vivo Handling Editor: Daniel Billadeau study provides and biochemical evidence that Rac1 is Received 3 July 2020; Accepted 7 December 2020 required for canonical Wnt signaling, and that its function in actin Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2021) 134, jcs251140. doi:10.1242/jcs.251140 polymerization is crucial for Daam2–PIP5K-mediated complex absence of Daam2. We performed functional epistatic experiments translocation at the membrane. utilizing a Daam2-targeted short hairpin RNA interference (shRNAi) (or mutant shRNAi control) system we previously RESULTS employed (Lee and Deneen, 2012), together with expression of Rho Daam2 regulates Wnt signaling through Rac1 and PIP5K GTPases and canonical Wnt reporter TOP–nRFP in the chick spinal To further dissect the direct interaction of Daam2 and PIP5K (Lee cord at embryonic day 4 (E4). Effective knockdown of endogenous et al., 2015), we performed co-immunoprecipitation (co-IP) assays Daam2 was verified by in situ hybridization (Fig. 1F,J,N), and exploring the nature of their association. Our results revealed that overexpression of GFP-tagged Rho GTPases was verified by GFP PIP5K associates with the GTPase-binding domain (GBD) of Daam2, fluorescence (Fig. 1K,O). We found that Rac1 overexpression an essential domain for canonical Wnt signaling (Fig. 1A; Fig. S1A, partially restores canonical Wnt signaling (TOP–nRFP) and dorsal B). These data, together with our previous studies (Lee et al., 2015), progenitor marker expression (Pax7) in the absence of Daam2 suggest that the Daam2 GBD functions through PIP5K to regulate during chick spinal cord development (Fig. 1J–M,R–S), while canonical Wnt signaling. The GBDs of Daam family proteins have leaving ventral marker expression (Nkx2.2) unaffected. However, been shown to interact with Rho GTPases (Habas et al., 2001), and overexpression of another Rho-GTPase, Cdc42, did not rescue Rac1 binds to activate PIP5K (Weernink et al., 2004; Wei et al., 2002), canonical Wnt activity or dorsal marker expression in the absence suggesting that Rac1 or other Rho GTPases may contribute to Daam2 of Daam2 (Fig. 1N–S), indicating that this function is specific to function in this context. To examine the link between Daam2 and Rho Rac1. GTPases, we performed co-IP assays in human embryonic kidney Previously, we showed that Daam2 is required for canonical Wnt (HEK)293T cells and found, through western blot analysis, that signaling through PIP2 production in developing spinal cord (Lee Daam2 associates with Rac1 and Cdc42 and that these associations et al., 2015). To examine whether Rac1 regulates Daam2 to induce require the GBD domain of Daam2 (Fig. 1A; Fig. S1C,D). PIP2 production, we performed PIP2 enzyme-linked immunosorbent Next, we examined the ability of Rac1 or Cdc42 to rescue dorsal assays (ELISAs) with these chick spinal cords. Indeed, we found that patterning and canonical Wnt signaling in the spinal cord in the Rac1 restored PIP2 production in the absence of Daam2 (Fig. S1E). Fig. 1. Daam2 regulates Wnt signaling through Rac1. (A) Immunoblots of HEK293T cell extracts expressing tagged versions of Daam2, Rac1 and PIP5K. Immunoprecipitation shows that Daam2 and PIP5K associate with Rac1. (B–Q) Overexpression of Rac1 rescues canonical Wnt signaling and dorsal patterning in the absence of Daam2 in chick spinal cord. (P–Q) Overexpression of Cdc42 does not rescue canonical Wnt signaling nor dorsal patterning in the absence