Investigation of the TOCA1-Cdc42 interaction

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.JRW is supported by a Herchel Smith studentship. JLG is supported by a Wellcome Trust Research Career Development Fellowship (WT095829AIA), European Research Council Starting Grant (281971) and Gurdon Institute funding provided by the Wellcome Trust (092096) and CRUK (C6946/A14492). HMF is supported by a Wellcome Trust PhD Studentship (WT099740Z12Z). We would like to thank Dr A Walrant for help with the pyrene actin assays and liposome preparation. We are also grateful to Dr J.R. Peterson (Fox Chase Cancer Center) for human TOCA1 clones.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M116.72429