The RhoA GEF Syx is a target of Rnd3 and regulated via a Raf1-like ubiquitin-related domain.
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.
Goh
Liuh Ling
LL
Rho GTPases in Stem Cells Group, Institute of Medical Biology, Singapore.
Manser
Ed
E
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*
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
PLoS One. 2008 Aug 20;3(8):e3001
18714354
Prog Mol Subcell Biol. 1999;22:51-83
10081065
Genes Dev. 2002 Jul 1;16(13):1587-609
12101119
EMBO Rep. 2005 May;6(5):426-31
15815683
Circ Res. 2008 Sep 26;103(7):710-6
18757825
Biochem J. 2009 Oct 23;424(1):153-61
19723022
Curr Biol. 2003 Jul 1;13(13):1106-15
12842009
Biochemistry. 2004 Feb 3;43(4):837-42
14744125
J Neurosci Res. 2008 Feb 1;86(2):270-80
17828770
J Mol Biol. 2007 Jul 27;370(5):1020-32
17544445
Mol Cell Biol. 1998 Aug;18(8):4761-71
9671486
Oncogene. 2001 Nov 1;20(50):7307-17
11704860
EMBO J. 2005 Mar 23;24(6):1170-80
15775972
Mol Cell Biol. 1996 Jun;16(6):2689-99
8649376
Mol Cell Biol. 2003 Jun;23(12):4219-29
12773565
EMBO J. 2008 Feb 20;27(4):606-17
18256687
J Neurochem. 2010 Feb;112(4):1074-87
19968760
PLoS Genet. 2007 May 25;3(5):e78
17530925
Nat Rev Mol Cell Biol. 2006 Jan;7(1):54-62
16493413
Nature. 1995 Jun 15;375(6532):554-60
7791872
Development. 1999 Dec;126(23):5339-51
10556059
Cell Death Differ. 2008 Aug;15(8):1221-31
18369372
Nat Cell Biol. 2008 Feb;10(2):127-37
18204440
Science. 1998 Jan 23;279(5350):560-3
9438849
Cell. 2001 Dec 28;107(7):843-54
11779461
Exp Cell Res. 2005 Apr 15;305(1):74-82
15777789
Mol Cell Biol. 2002 May;22(9):2952-64
11940653
J Neurochem. 2005 Feb;92(4):850-8
15686487
Nat Rev Mol Cell Biol. 2005 Feb;6(2):167-80
15688002
Mol Biol Cell. 2006 Apr;17(4):1880-7
16467373
Dev Biol. 2003 Jul 15;259(2):318-35
12871704
J Cell Biol. 1998 Apr 6;141(1):187-97
9531558
Blood. 2009 Jan 1;113(1):244-53
18824598
Mol Biol Cell. 2008 Feb;19(2):498-508
18045987
Nat Biotechnol. 2007 Jun;25(6):681-6
17529971
Nat Cell Biol. 2002 Aug;4(8):621-5
12134164
Curr Biol. 2002 Jun 4;12(11):876-84
12062050
Curr Biol. 2006 Dec 19;16(24):2466-72
17174923
Mol Cell Biol. 2000 Dec;20(24):9364-75
11094087
EMBO J. 2008 Dec 3;27(23):3175-85
18946488
Cell Signal. 2006 Mar;18(3):359-72
16019189
J Biol Chem. 2009 Dec 18;284(51):35962-72
19843518