Mechanism of activation of the Formin protein Daam1 Wei Liu*, Akira Sato*, Deepak Khadka*, Ritu Bharti*, Hector Diaz*, Loren W. Runnels†, and Raymond Habas*‡§ Departments of *Biochemistry and †Pharmacology and ‡Cancer Institute of New Jersey, Robert Wood Johnson School of Medicine, Piscataway, NJ 08854 Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved November 15, 2007 (received for review August 2, 2007) The Formin proteins are central players in mediating cytoskeletal activation allows for Rho-GTP to bind to the GBD and release this reorganization and are epistatically positioned in a pathway down- molecule from autoinhibition. The FH1 and FH2 can then bind to stream of Rho activation. These proteins exist in the cytoplasm in effectors to mediate effects on the cytoskeleton. Intriguingly, the an autoinhibited state, which is mediated by intramolecular inter- FH2 domain has recently been shown to be capable of nucleating actions between the amino-terminal GTPase binding domain (GBD) actin filaments by itself in vitro, suggesting a complex interplay that encompasses the diaphanous inhibitory domain (DID) and the between the FH1 and FH2 domains along with their effectors for carboxyl-terminal diaphanous autoregulatory domain (DAD). It has actin polymerization (12). However, it remains unclear how the been proposed that the binding of Rho within the GBD releases this Formin proteins are activated in vivo, and one molecule shown to molecule from autoinhibition by disrupting the DID/DAD inter- bind the Formin proteins is Profilin1, which functions in actin actions. Here we report that Daam1 is not significantly activated by polymerization (12, 14). Rho binding but rather by its interaction with Dishevelled (Dvl). Here we report on the mechanisms of activation of Daam1. We Removal of the DAD domain disrupts interactions between Dvl and show that Daam1 exists in an autoinhibited state via intramolecular Daam1, and the binding of Dvl to Daam1 disrupts the interaction interactions between its amino-terminal GBD and carboxyl- between the GBD and DAD that mediates Daam1 autoinhibition. terminal DAD. Daam1 is not significantly activated by binding of Mutations within or removal of the DAD converts Daam1 into an Rho but rather by binding of Dvl, which interacts with the DAD and active protein that can induce Rho activation. We further demon- relieves autoinhibition of Daam1. In the Xenopus embryo we show strate that Dvl synergizes with Daam1 to regulate gastrulation that coexpression of Dvl with Daam1 leads to activation of Daam1 during Xenopus embryogenesis and that expression of activated as monitored by hyperactivation of the noncanonical Wnt pathway. Daam1 can rescue impaired convergent extension movements Deletion or mutation of conserved amino acid residues of the DAD resulting from deregulated noncanonical Wnt signaling. Our stud- further converts Daam1 into an activated protein that can rescue ies together define the importance of a carboxyl-terminal binding defects in gastrulation induced by noncanonical Wnt signaling partner, Dvl, that leads to the activation of Daam1. components. Our studies together uncover the role of Dvl as an important factor that mediates Daam1’s activation. ͉ ͉ Dishevelled Wnt Rho Results Daam1 Is Autoinhibited by Interactions Between the GBD and DAD. irectional cell migration is required for the development of We had previously shown that the full-length Daam1 protein Dan organism with proper polarity including dorsoventral, appeared to be autoinhibited because it was incapable of inducing anterior–posterior, and left–right symmetry. Examples of these Rho activation and cytoskeletal changes whereas a fragment of cell movements include those of gastrulation and neural fold Daam1 termed C-Daam, harboring the FH1, FH2, and DAD, can closure. These cell movements are tightly regulated by secreted induce Rho activation and cytoskeletal changes (7) [supporting ligands (1, 2). One of these signaling pathways required for cell information (SI) Fig. 6A]. We explored whether Daam1 was movements is the noncanonical Wnt pathway (3–5). autoinhibited by interactions between its GBD and DAD similar to Noncanonical Wnt signaling, also termed the planar cell polarity other Formin proteins (12). To test for interactions we performed pathway, regulates cell movements through modification of the GST pull-down assays. We generated GST fusion proteins that actin cytoskeleton (1, 3, 4, 6). A number of molecular components encompass the PDZ domain of Dvl (GST-PDZ) (7) and another for this pathway have been identified including Wnt11, Fz, Dvl, containing the amino-terminal domain of Daam1 encompassing Daam1, Rho, Rac, JNK, Strabismus, and Prickle (reviewed in ref. the GBD (GST-N-Daam) and used in vitro translated HA-T-Daam, 5). For noncanonical Wnt signaling, the binding of Wnt to the which contains the DAD of Daam1. These pull-down assays Frizzled (Fz) receptor stimulates a signal that is transduced to the demonstrated a direct and specific interaction between DAD and cytoplasmic phosphoprotein Dishevelled (Dvl). At the level of Dvl, the PDZ domain of Dvl or the GBD domain of Daam1, because the two independent and parallel pathways lead to the activation of the DAD fragment did not bind to GST protein alone (Fig. 1A). small GTPases Rho and Rac. The first pathway signaling to the To further delineate interactions between the GBD and DAD small GTPase Rho occurs through the molecule Daam1 (7). This of Daam1, we examined interactions by coimmunoprecipitation Rho pathway leads to the activation of the Rho-associated kinase using epitope-tagged proteins expressed in mammalian HEK393T Rock and mediates cytoskeletal reorganization (5, 8). The second cells (SI Fig. 6A). We find that N-Daam can bind to T-Daam and activates another small GTPase of the Rho-family, Rac, which in C-Daam, both of which contain the DAD, but its binding to turns stimulates JNK activity (9–11). Daam1 is a Formin protein family and has been shown to regulate gastrulation; however, how Daam1 is activated for its function remains unknown. Author contributions: W.L., A.S., and D.K. contributed equally to this work; W.L., A.S., D.K., The Formin proteins are central players in regulating cytoskel- L.W.R., and R.H. designed research; W.L., A.S., D.K., R.B., H.D., L.W.R., and R.H. performed research; W.L., A.S., D.K., R.B., L.W.R., and R.H. analyzed data; and L.W.R. and R.H. wrote etal reorganization in mammalian cells (12). The Formin proteins the paper. contain three major domains termed the GTPase binding domain The authors declare no conflict of interest. (GBD), Formin homology 1 (FH1) domain, and Formin homology This article is a PNAS Direct Submission. 2 (FH2) domain (13). These proteins are proposed to exist in the §To whom correspondence should be addressed at: Department of Biochemistry, Robert cytoplasm in an autoinhibited state, which is mediated by a domain Wood Johnson School of Medicine, Research Tower, Room 629, 675 Hoes Lane, Piscat- termed the diaphanous autoinhibitory domain (DAD) (12). This away, NJ 08854. E-mail: [email protected]. DAD found in the carboxyl terminus mediates interaction with the This article contains supporting information online at www.pnas.org/cgi/content/full/ amino terminus of the protein and serves to ‘‘lock’’ the protein in 0707277105/DC1. a folded or closed conformation (12). It is proposed that Rho © 2007 by The National Academy of Sciences of the USA 210–215 ͉ PNAS ͉ January 8, 2008 ͉ vol. 105 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707277105 Downloaded by guest on September 27, 2021 C + Myc-N-Daam1 A + Flag-Dvl B + HA-T-Daam1 1 1 am Daam C-Daam1 A 1 - l Da D HA- HA-C-Daam1 - T am o A t T r - - Control HA-C-Daam1 pu T S S a t A-C C ∆D GST GST-PDZ GST GST-PDZ S G Z G -D on HA- α-HA nI G PD N C H Pull- α-HA α down HA -Myc IP: Flag α-Flag P : M y c I P : Input α -HA α-Flag Coomassie Coomassie α-HA IP: HA α-HA IgG HA-C-Daam1 + Flag-Dvl 1 I α-Myc C D -N-Daam1 m Myc-N-Daam1 a 1 1 a B m m --- -Daa -D -Daa Flag-Dvl Control -N c -T HA-T c My My HA + Myc-N-Daam1 α-HA D + Control HA-C-Daam1HA-C-Daam1A2HA-C-Daam1A12 2 1 A IgG α 1 1 am1A α-HA α IP Myc, -HA am aam -Myc N-Daam l a a o -D C-D -D IP: Myc tr α -C - -C -Flag n IP: Flag o HA A A IP HA,α -Myc C H2H α-HA α-Flag α H A -Myc α-Myc α IP: HA α : -HA Lysate -Myc Lysate P I α-HA -Flag α Lysate -HA y c α-HA Flag-Dvl M IgG I P : α-Myc E Flag-Dvl HA-Daam1 Flag-Dvl HA-Daam1 + HA-Daam1 + GST + GST + GST-Rho + GST-Rho + GST-Rho Fig. 1. Autoinhibition of Daam1 is mediated by interactions between its Input Input GDP GTP GDP GTP GDP GTP GDP GTP GDP GTP amino-terminal and carboxyl-terminal regions. (A) GST pull-down assays re- Flag-Dvl veal that T-Daam binds to the PDZ domain of Dvl and to N-Daam. Precipitated HA-Daam1 T-Daam was detected with Western blotting, and input of GST proteins was visualized by using Coomassie staining. (B–D) Coimmunoprecipitation assays. F Plasmids encoding tagged Daam1 fragments were cotransfected into am1 am1 -Da Da t C m -C-Daam1 2 P P HEK293T cells, and cell lysates were immunoprecipitated (IP) with indicated F FP-Daam1 A2 FP GF GFP-Daam1G GFP G GFP-Daam1 A12G GFP- GFP-C FH- Abs.