TI - Net1 is activated and regulates RhoA -dependent actin stress fiber formation upon induction of DNA damage . AB - Alberts et al . have previously shown that PHOSphorylation of Net1 on Ser152 prevents RhoA activation and concluded therefore that pSer152 inhibits the GEF activity of Net1 [15] . We now show that exposure to CDT or IR decreases the levels of pSer152 PHOSphorylation of the endogenous as well as an ectopically expressed Net1 ( Figure 1 ) , thus identifying genotoxic stress as a signal for Net1 activation in vivo . The mechanisms involved in Net1 de-PHOSphorylation remain unknown . A constitutively active form of the Rac1 -activated protein kinase PAK1 ( PAK1* ) was identified as the Ser152-specific Net1 kinase in vitro , and expression of PAK1* prevented Net1 -induced stress fiber formation in Swiss 3T3 cells [15] . We did not observe any significant change in the level of Rac1 or Cdc42 activation in HeLa cells or primary fibroblasts exposed to CDT or IR [6] and PAK1 activity was not changed within 30 min after irradiation , a time when the dePHOSphorylation of pS152-Net1 was maximal ( data not shown ) . This suggests that the decrease in pSer152-Net1 observed in our experiments does not involve inactivation of PAK1 . Conceivably , a different , as yet unknown Net1 specific kinase may be down-regulated . Alternatively , exposure to CDT or IR may enhance the rate of p-S152-Net1 de-PHOSphorylation by activation of a phosphatase . Inhibition of endogenous Net1 by RNAi and expression of a dominant negative Net1 demonstrated that this GEF is required for RhoA activation and for the subsequent re-organization of the actin cytoskeleton in response to intoxication or irradiation ( Figures 2 and 3 ) . It is noteworthy that stress fiber formation is detected in epithelial cells that are arrested in G1 following treatment with TGF-beta [19] -[21] , and similar changes occur in cells exposed to bacterial toxins that inhibit , Cycle inhibiting factor ( Cif ) [22] , or promote , Pasteurella multocida toxin ( PMT ) [23] , cell cycle progression . Net1 is a major player in the morphological changes that characterize both TGF-beta [21] and DNA damage ( in this work ) . It is thereby tempting to speculate that Net1 -regulated cytoskeleton rearrangements may be a common feature of the response to stress signals that deregulate the cell cycle .