Raymond K, Bergeret E, Dagher M C, Breton R, Griffin-Shea R, Fauvarque M O
Département de Biologie Moléculaire et Structurale, CEA-CNRS-UJF, UMR 5092, 17 rue des Martyrs, Grenoble 38054, France.
J Biol Chem. 2001 Sep 21;276(38):35909-16. doi: 10.1074/jbc.M105779200. Epub 2001 Jul 23.
RhoGTPases are negatively regulated by GTPase-activating proteins (GAPs). Here we demonstrate that Drosophila RotundRacGAP is active in vitro on Drac1 and Dcdc42 but not Drho1. Similarly, in yeast, RotundRacGAP interacts specifically with Drac1 and Dcdc42, as well as with their activated V12 forms, showing a particularly strong interaction with Dcdc42V12. In the fly, lowering RotundRacGAP dosage specifically modifies eye defects induced by expressing Drac1 or Dcdc42 but not Drho1, confirming that Drac1 and Dcdc42 are indeed in vivo targets of RotundRacGAP. Furthermore, embryonic-directed expression of either RotundRacGAP, or dominant negative Drac1N17, transgenes induces similar defects in dorsal closure and inhibits Drac1-dependent cytoskeleton assembly at the leading edge. Expression of truncated forms of RotundRacGAP shows that the GAP domain of RotundRacGAP is essential for its function. Unexpectedly, transgenes encoding Drac1N17, Dcdc42N17, or RotundRacGAP do not affect the c-Jun N-terminal kinase-dependent gene expression of decapentaplegic and puckered, indicating that another Drac1-independent signal redundantly activates this pathway. Finally, in a situation where Drac1 is constitutively activated, RotundRacGAP greatly reduces the ectopic expression of decapentaplegic, possibly by negatively regulating Dcdc42.
RhoGTP酶受GTP酶激活蛋白（GAPs）的负调控。在此我们证明，果蝇的RotundRacGAP在体外对Drac1和Dcdc42具有活性，但对Drho1无活性。同样，在酵母中，RotundRacGAP特异性地与Drac1和Dcdc42及其激活的V12形式相互作用，与Dcdc42V12的相互作用尤为强烈。在果蝇中，降低RotundRacGAP的剂量会特异性地改变由表达Drac1或Dcdc42而非Drho1所诱导的眼部缺陷，证实Drac1和Dcdc42确实是RotundRacGAP在体内的作用靶点。此外，RotundRacGAP或显性负性Drac1N17转基因的胚胎定向表达会在背侧闭合中诱导类似的缺陷，并抑制前缘处依赖Drac1的细胞骨架组装。RotundRacGAP截短形式的表达表明，RotundRacGAP的GAP结构域对其功能至关重要。出乎意料的是，编码Drac1N17、Dcdc42N17或RotundRacGAP的转基因并不影响decapentaplegic和puckered的c-Jun N末端激酶依赖性基因表达，表明另一种不依赖Drac1的信号冗余地激活了该途径。最后，在Drac1组成型激活的情况下，RotundRacGAP可能通过负调控Dcdc42而极大地降低decapentaplegic的异位表达。
