TI - Net1/RhoA -dependent survival signals . AB - Our RNAi experiments demonstrate that Net1 and RhoA are critical for protecting intoxicated and irradiated cells from cell death induced by DNA damage ( Figure 4 ) , and identify p38 MAPK as a key mediator in the delivery of the survival signals ( Figures 5 to 9 ) . The mechanism by which RhoA controls p38 MAPK PHOSphorylation remains still unclear . Marinissen at al .demonstrated that RhoA stimulates c-jun expression via activation of the p38gamma MAPK isoform , resulting in aberrant cell growth and malignant transformation [24] . Interestingly , we have found that the activation of p38 MAPK in response to DNA damage was abrogated by SB203580 and SB202190 that are specific inhibitors for the alpha - and beta-isoforms of p38 MAPK [25] , suggesting that different isoforms may be targeted by RhoA depending on the triggering stimulus . A large number of effector proteins mediate signalling downstream of RhoA [26] . Since the RhoA activated kinases ROCKI and ROCKII regulate many of the cytoskeletal effects of RhoA , we examined whether they transduced the signal from RhoA to p38 MAPK . Interestingly , pre-treatment with pharmacological inhibitors of ROCK did not prevent p38 MAPK activation , but inhibited stress fiber formation ( Figure 9 ) , suggesting that RhoA may utilize different sets of effector proteins to control these cellular responses . This is likely to require the selective clustering of the downstream effectors on distinct scaffold proteins , as suggested by the finding that the RhoA -dependent activation of JNK involves the association of Net1 and the JNK activators MLK2 , MLK3 and MKK7 with the scaffold protein CNK1 , while this is not required for induction of stress fibers [27] . The current literature identifies JNK as the main MAPK induced by irradiation [17] . Consistent with our previous results showing that CDT or IR do not activate the JNK regulators Rac and Cdc42 [6] , we detected only low levels of JNK PHOSphorylation and AP1 activation 4h and 12h after intoxication in HeLa cells ( data not shown ) . In contrast , up to 10 fold increase of p38 MAPK PHOSphorylation was consistently observed both in HeLa and HCT116 cells ( Figures 5 and 6 ) . This result is not surprising since , depending on the experimental models , p38 MAPK was shown to contribute to either survival or death signals in response to DNA damage [17] . The downstream signals involved in the survival response remain unclear . Reinhardt at al .recently showed that the survival in p53-deficient fibroblasts exposed to cisplatin and doxorubicin is enhanced by ATM -dependent activation of p38 MAPK and its downstream effector MK2 [28] . This pathway has been defined as the third cell cycle -dependent checkpoint , in addition to the well-characterized ATM/Chk2 and ATR/Chk1 responses [28] . Our findings demonstrate that these signals operate also in tumor cells , such as HeLa and HCT116 , since blockage of p38 MAPK activation by specific inhibitor abrogates their capacity to survive irradiation or intoxication ( Figures 5 and 6 ) . Furthermore , we have identified Net1 and RhoA as key molecules controlling the activation of this novel checkpoint pathway ( Figures 7 and 8 ) . A schematic illustration of the Net1 -regulated signal cascade identified in this work is shown in Figure 11 .