title: The RhoA GEF Syx is a target of Rnd3 and regulated via a Raf1-like ubiquitin-related domain.
abstract: BACKGROUND
  Rnd3 (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.
  
  
  RESULTS
  Rnd3 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
  This 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.
authors.affiliation: Rho GTPases in Stem Cells Group, Institute of Medical Biology, Singapore.
authors.forename: Liuh Ling
authors.initials: LL
authors.lastname: Goh
authors.forename: Ed
authors.initials: E
authors.lastname: Manser
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D054262:Gastrulation; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry*; D015027:Zebrafish; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C511269:Syx protein, mouse; D025801:Ubiquitin; D019908:Proto-Oncogene Proteins c-raf; C513341:Rnd3 protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
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