Knetsch M L, Schäfers N, Horstmann H, Manstein D J
Department of Biophysics, Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany.
EMBO J. 2001 Apr 2;20(7):1620-9. doi: 10.1093/emboj/20.7.1620.
Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.
盘基网柄菌（Dictyostelium discoideum）的DdRacGap1（DRG）同时含有Rho鸟苷酸交换因子（Rho-GEF）和Rho鸟苷酸酶激活蛋白（Rho-GAP）结构域，这一特征与哺乳动物的Bcr和Abr相同。为阐明这种多功能蛋白的生理作用，我们在体外对重组DRG片段的酶活性进行了表征，构建了DRG基因缺失细胞，并通过分析DRG缺失细胞和用DRG或DRG片段互补的DRG基因缺失细胞所表现出的表型变化，在体内研究了该蛋白的功能。我们的结果表明，DRG-GEF通过Rac1依赖的信号通路调节F-肌动蛋白动力学和cAMP诱导的F-肌动蛋白形成。DRG的RacE-GAP活性是正确进行胞质分裂所必需的。此外，我们提供的证据表明，DRG的特异性并不局限于小GTP酶Rho家族的成员。一种重组DRG-GAP在体外可使RabD的GTP水解加速30倍，我们的互补研究表明，DRG-GAP活性是盘基网柄菌中RabD依赖的收缩泡系统调节所必需的。
