ADP-RIBOSYLATION OF THE RHOA GENE-PRODUCT BY BOTULINUM C3 EXOENZYME CAUSES SWISS 3T3 CELLS TO ACCUMULATE IN THE G(1) PHASE OF THE CELL-CYCLE

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the r ho gene products (rho proteins), we examined the role of these protein s in cell cycle progression in Swiss 3T3 cells. Incubation of cell lys ates with C3 exoenzyme revealed a single [P-32]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by i soelectric focusing and peptide mapping. When C3 exoenzyme was added t o the culture, it ADP-ribosylated the substrate protein in the celts a nd reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribo sylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concent ration-dependently accumulated the cells in the G1 phase of the cell c ycle. When G1-enriched cells were treated with C3 exoenzyme and cell c ycle progression initiated by the addition of serum was monitored, inh ibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes th is activity and causes the cells to accumulate in G1 phase.