Xu X, Wang Y, Barry D C, Chanock S J, Bokoch G M
Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 1997 Jan 21;36(3):626-32. doi: 10.1021/bi962059h.
The Rac GTPases are currently being subjected to intensive study due to their involvement in a wide array of cellular phenomena. Many studies of Rac function have relied upon the use of relatively uncharacterized Rac dominant active, dominant negative, and effector domain mutants on the basis of the analogy to Ras structure. We have generated and purified such Rac2 mutants and characterized their guanine nucleotide binding properties in vitro. The Rac2 G12V and Q61L activating mutations were shown to hydrolyze bound GTP very slowly and were unresponsive to p190 Rac GTPase-activating protein. Distinct differences in the kinetics of nucleotide binding to individual mutant proteins were observed, accounting for the behavior of these proteins in biological assays. The structural features required for the responsiveness of Rac2 to the guanine nucleotide exchange protein smgGDS were examined. We show that guanine nucleotide exchange by smgGDS is dependent upon intact switch 1 and switch 2 regions in Rac2. Functional interactions between the switch 1 and switch 2 regions and the G12V mutation of Rac2 are described. These data form the basis for rational use of Rac mutants in biological studies.
由于Rac GTP酶参与了广泛的细胞现象，目前正受到深入研究。许多关于Rac功能的研究基于与Ras结构的类比，依赖于使用相对未充分表征的Rac显性激活、显性阴性和效应器结构域突变体。我们已经生成并纯化了此类Rac2突变体，并在体外表征了它们的鸟嘌呤核苷酸结合特性。Rac2 G12V和Q61L激活突变显示出结合的GTP水解非常缓慢，并且对p190 Rac GTP酶激活蛋白无反应。观察到核苷酸与各个突变蛋白结合动力学的明显差异，这解释了这些蛋白在生物学测定中的行为。研究了Rac2对鸟嘌呤核苷酸交换蛋白smgGDS反应性所需的结构特征。我们表明，smgGDS介导的鸟嘌呤核苷酸交换依赖于Rac2中完整的开关1和开关2区域。描述了开关1和开关2区域与Rac2的G12V突变之间的功能相互作用。这些数据构成了在生物学研究中合理使用Rac突变体的基础。
