DOI 10.1515/hsz-2012-0246 Biol. Chem. 2013; 394(3): 421–432 Sarah L. Risse, Belen Vaz, Matthew F. Burton, Pontus Aspenström, Roland P. Piekorz, Luc Brunsveld and Mohammad R. Ahmadian* SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins Abstract: Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-ter- minus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor- bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low- affinity Wrch1-binding partners. Interestingly, under cell- based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indi- cating that high avidity of the adaptor proteins can com- pensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties. Keywords: proline rich; PxxP motif; Rho family; SH3 domain; unique GTPases. *Corresponding author: Mohammad R. Ahmadian, Department of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, D-40225 Düsseldorf, Germany, e-mail: reza.ahmadian@uni-duesseldorf.de Sarah L. Risse and Roland P. Piekorz: Department of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, D-40225 Düsseldorf, Germany Belen Vaz, Matthew F. Burton and Luc Brunsveld: Laboratory of Chemical Biology, Department of Biomedical Engineering, Technical University Eindhoven, NL-5612 AZ Eindhoven, The Netherlands Pontus Aspenström: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, S-171 77 Stockholm, Sweden Introduction RhoU/Wrch1 (Wnt-responsive Cdc42 homolog 1), an atypi- cal member of the Rho GTPase family, was first identified to be upregulated by Wnt1 signaling in mouse mammary epithelial cells (Tao et al., 2001) contributing to the Wnt phenotype (Taneyhill and Pennica, 2004). In addition to the Wnt1 pathway, Wrch1 is also a transcriptional target of the RANKL, gp130/STAT3, EGFR, and Notch1 signaling pathways (Tao et al., 2001; Brazier et al., 2006; Schiavone et al., 2009; Zhang et al., 2011; Bhavsar et al., 2012). Like Cdc42, Wrch1 induces formation of filopodia (Saras et al., 2004; Ruusala and Aspenstrom, 2008), which is associa- ted with disassembly of stress fibers and focal adhesions and, thus, mediates cell motility (Tao et al., 2001; Saras et al., 2004; Shutes et al., 2004; Chuang et al., 2007; Ory et al., 2007). In epithelial cells, Wrch1 interacts with Par6, which modulates cell polarity (Brady et al., 2009). On the effector level, Wrch1 has been reported to interact with different downstream kinases including PAK1, JNK, and Pyk2 (Chuang et al., 2007; Ruusala and Aspenstrom, 2008). In cells, Wrch1 is found mostly in a GTP-bound as it exhibits a high intrinsic nucleotide exchange rate (Shutes et al., 2004; Aspenstrom et al., 2007). In contrast to conventional members of the Rho family, Wrch1 contains N- and C-terminal extensions. Membrane targeting of Wrch1 is distinct to the classi- cal CaaX motif-containing Rho GTPases and has been reported to be achieved by palmitoylation of a CFV (C: Cysteine, F: Phenylalanine, V: Valine) motif in the C-ter- minus of Wrch1 (Berzat et al., 2005). It has been suggested that the 46-amino acid N-terminal extension of Wrch1 (nWrch1) exhibits an autoinhibitory function via binding to the G-domain of Wrch1 (Shutes et al., 2004). nWrch1 contains three putative PxxP motifs (P: Proline, x: any amino acid, mostly hydrophobic), which may be responsi- ble for its interaction with the Src homology region 3 (SH3) domains of noncatalytic region of tyrosine kinase adaptor protein 2 (Nck2), growth factor receptor-bound protein 2 (Grb2), CRKL, CRKL-II, LCK, CIN85, p85 α, Abi-1, and Abi-2 (Tao et al., 2001; Saras et al., 2004; Shutes et al., 2004; Liu et al., 2006; Zhang et al., 2011). Here, elucidation of the Brought to you by | Rice University Authenticated | 128.42.202.150 Download Date | 6/2/13 4:27 AM