Clostridium botulinum C2 toxin delays entry into mitosis and activation ofp34(cdc2) kinase and cdc25-C phosphatase in HeLa cells

The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, an d rounding of cells. After treatment,vith C2 toxin, cells stop to prolifera te but remain viable for about 2 days. In view of reported correlations bet ween the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cy cle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protei n kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were ex amined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34(cdc2) kinase subunits. Fo r its activation at the G(2)/M border, p34(cdc2) needs to be associated,vit h cyclin B and additionally dephosphorylated at Tyr-15 by the specific phos phatase cdc25-C. Treatment of synchronized cells in S or G(2) phase with C. botulinum C2 toxin prevented p34(cdc2) protein kinase activation by inhibi ting its tyrosine dephosphorylation at the G(2)/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34(cdc2) kinase and cdc25-C phosphatase represents the final dow nstream events in the action of C2 toxin resulting in a G(2) phase cell cyc le delay in synchronized HeLa cells.