TI - Results DNA damage induces Net1 activation . AB - We have previously shown that exposure to DNA damaging agents induces activation of the small GTPase RhoA , which delays cell death [6] . In order to investigate how the signal delivered by DNA damage is transduced from the nucleus to the cytosol , we have studied the activation of the RhoA specific GEF Net1 in response to CDT or IR . These genotoxic agents were chosen since they both induce DNA double strand breaks and activate identical DNA damage checkpoint responses in mammalian cells [4] -[6] . Decreased PHOSphorylation of Net1 on the key negative regulatory site Ser152 was used as the hallmark of Net1 activation [15] . Endogenous Net1 was immunoprecipitated from HeLa cells left untreated or exposed to CDT for 12h , and the levels of Ser152 phosphorylaTION were assessed by western blot using a PHOSphorylated specific antibody . As shown in Figure 1A , the PHOSphorylation of endogenous Net1 on Ser152 ( pS152-Net1 ) was significantly decreased in intoxicated cells . To assess whether dePHOSphorylation of Net1 is a reproducible effect of DNA damage , HeLa cells were transfected with a plasmid expressing an HA-epitope-tagged Net1A , the major Net1 isoform expressed in these cells [16] . The transfected cells were then exposed to CDT or IR and the expression of total and PHOSphorylated Net1 was monitored over time . A 70% decrease in the levels of pS152-Net1A was observed within 30 min after irradiation . The effect was similar to that achieved by CDT intoxication where low levels of p152-Net1A were maintained for atleast 12h ( Figure 1B and data not shown ) . Thus , exposure to DNA damage induces dePHOSphorylation of Net1 on its negative regulatory site .