The RhoA GEF Syx Is a Target of Rnd3 and Regulated via a Raf1-Like Ubiquitin-Related Domain |
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Authors: | Liuh Ling Goh Ed Manser |
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Institution: | 1. Rho GTPases in Stem Cells (RGS) Group, Institute of Medical Biology (IMB), Singapore, Singapore.; 2. Small G-Protein Signaling and Kinases (sGSK) Group, Institute of Molecular and Cell Biology (IMCB), Neuroscience Research Partnership, Singapore, Singapore.;University of Chicago, United States of America |
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Abstract: | BackgroundRnd3 (RhoE) protein belongs to the unique branch of Rho family GTPases that has low intrinsic GTPase activity and consequently remains constitutively active 1], 2]. The current consensus is that Rnd1 and Rnd3 function as important antagonists of RhoA signaling primarily by activating the ubiquitous p190 RhoGAP 3], but not by inhibiting the ROCK family kinases.Methodology/Principal FindingsRnd3 is abundant in mouse embryonic stem (mES) cells and in an unbiased two-step affinity purification screen we identified a new Rnd3 target, termed synectin-binding RhoA exchange factor (Syx), by mass spectrometry. The Syx interaction with Rnd3 does not occur through the Syx DH domain but utilizes a region similar to the classic Raf1 Ras-binding domain (RBD), and most closely related to those in RGS12 and RGS14. We show that Syx behaves as a genuine effector of Rnd3 (and perhaps Rnd1), with binding characteristics similar to p190-RhoGAP. Morpholino-oligonucleotide knockdown of Syx in zebrafish at the one cell stage resulted in embryos with shortened anterior-posterior body axis: this phenotype was effectively rescued by introducing mouse Syx1b mRNA. A Rnd3-binding defective mutant of Syx1b mutated in the RBD (E164A/R165D) was more potent in rescuing the embryonic defects than wild-type Syx1b, showing that Rnd3 negatively regulates Syx activity in vivo.Conclusions/SignificanceThis study uncovers a well defined Rnd3 effector Syx which is widely expressed and directly impacts RhoA activation. Experiments conducted in vivo indicate that Rnd3 negatively regulates Syx, and that as a RhoA-GEF it plays a key role in early embryonic cell shape changes. Thus a connection to signaling via the planar cell polarity pathway is suggested. |
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