1 Section of Molecular Pathology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet2 Department of Biomedical Sciences, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet3 Brakebusch Group, BRIC Research Groups, BRIC, Københavns Universitet4 Institute of Toxicology; Hannover Medical School; Hannover, Germany.5 Helmholtz Centre for Infection Research6 Section of Molecular Pathology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet
During mitotic entry, the centrosomes provide a scaffold for initial activation of the CyclinB/Cdk1 complex, the mitotic kinase Aurora A, and the Aurora A-activating kinase p21-activated kinase (PAK). The activation of PAK at the centrosomes is yet regarded to happen independently of the Rho-GTPases Rac/Cdc42. In this study, Rac1 (but not RhoA or Cdc42) is presented to associate with the centrosomes from early G 2 phase until prometaphase in a cell cycle-dependent fashion, as evidenced by western blot analysis of prepared centrosomes and by immunolabeling. PAK associates with the G 2/M-phase centrosomes in a Rac1-dependent fashion. Furthermore, specific inhibition of Rac1 by C. difficile toxinB-catalyzed glucosylation or by knockout results in inhibited activation of PAK1/2, Aurora A, and the CyclinB/Cdk1 complex in late G 2 phase/prophase and delayed mitotic entry. Inhibition of PAK activation at late G 2-phase centrosomes caused by Rac1 inactivation coincides with impeded activation of Aurora A and the CyclinB/Cdk1 complex and delayed mitotic entry.