a Chk1 activity is required for translocation of RhoB to lysosomes. sumoylation by PIAS1, which is required for RhoB/TSC complex (??)-Huperzine A to translocate to lysosomes. As a result, mTORC1 is usually inhibited, (??)-Huperzine A and autophagy is usually activated. Knockout of severely attenuates lysosomal translocation of TSC complex and the DNA damage-induced (??)-Huperzine A autophagy. Reintroducing wild-type but not sumoylation-resistant RhoB into significantly inhibits DNA damage-induced apoptosis35, suggesting that RhoB suppresses tumorigenesis through promoting cell death. Our previous study showed that E3 ubiquitin ligase Smurf1 targets RhoB for degradation to maintain a relative low RhoB level in the basal state. Activation of ATR/Chk1 signaling upon DNA damage induces self-degradation of Smurf1, and therefore prevents RhoB from Smurf1-mediated degradation36. In this study, we found that RhoB is usually phosphorylated by Chk1 after DNA damage, which promotes its binding to SUMO E3 ligase PIAS1 and subsequent sumoylation. Meanwhile, this phosphorylation also enhances the binding of RhoB to TSC complex. Therefore, the sumoylated phospho-RhoB functions as a carrier protein to translocate TSC complex to lysosomes, initiating autophagy by inhibiting mTORC1 activity. Results PIAS1 mediates sumoylation of small GTPase RhoB Our previous study showed that Smurf1 targets RhoB for ubiquitination to control its abundance in cells under basal conditions. Upon DNA damage, ATR/Chk1 signaling triggers Smurf1 self-degradation and leads to an accumulation of RhoB to promote apoptosis36. To further investigate the role of RhoB in DDR, we carried out a yeast-two-hybrid screen using RhoB as the bait to identify novel RhoB-binding proteins. Interestingly, we found that among the identified candidates several clones encode ubiquitin-conjugating enzyme 9 (Ubc9), the only known SUMO-conjugating E2 enzyme in mammalian cells. To verify this conversation, we performed coimmunoprecipitation assay (Fig.?1a) and in vitro GST pull-down assay (Supplementary Fig.?1a), confirming that RhoB interacts with Ubc9 in cells and in vitro. Open in a separate windows Fig. 1 RhoB is usually sumoylated by PIAS1. a RhoB interacts with Ubc9. HEK293T cells transfected with indicated combinations of Flag-tagged Ubc9 (F-Ubc9) and HA-tagged RhoB (HA-RhoB) were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting assay to detect associated RhoB. b RhoB is usually sumoylated. HEK293T transfected with indicated combinations of His-tagged RhoB (His-RhoB), HA-tagged SUMO (HA-SUMO) 1 or 2 2, and Myc-tagged Ubc9 (Myc-Ubc9) were lysed with 6?M guanidine-HCl followed by Ni-NTA agarose beads pull-down (Ni pull-down) assay. SUMO-conjugated RhoB was detected by immunoblotting with anti-HA. Conjugation of mono-SUMO and multi-SUMO to RhoB are indicated as RhoB-SUMO and RhoB-(SUMO)n, respectively. c PIAS1 promotes sumoylation of RhoB. HEK293T cells cotransfected with His-RhoB, HA-SUMO2, and indicated Myc-tagged PIAS (Myc-PIAS) family member 1C4 were subjected to sumoylation assay as described in panel b. d Knockdown of PIAS1 attenuates RhoB sumoylation. HEK293T cells transfected with indicated combinations of His-RhoB, HA-SUMO2, and shRNAs against PIAS1 were subjected to sumoylation assay as described in panel b. e The E3 catalytic activity is required for PIAS1-mediated RhoB sumoylation. HEK293T cells were transfected with His-RhoB, HA-SUMO2, and Myc-PIAS1 wild-type (WT) or catalytically inactive mutant (C351S) as indicated. The cells were subjected to sumoylation assay as described in panel b. f PIAS1 interacts with endogenous RhoB. HeLa cells transduced with lentivirus encoding HA-tagged PIAS1-C351S mutant (HA-PIAS1-C351S) were subjected to anti-RhoB IP followed by immunoblotting with rat anti-HA to detect associated HA-PIAS1-C351S. g Sumoylation sites mapping on RhoB. HEK293T cells transfected with HA-SUMO2 and indicated His-RhoB mutants were subjected Flt1 to sumoylation assay as described in panel b. h PIAS1 enhances sumoylation of WT but not 4KR RhoB. HEK293T cells transfected with indicated combinations of HA-SUMO2, Myc-PIAS1 (WT or C351S), and His-RhoB (WT or 4KR) were subjected to sumoylation assay as described in panel b We therefore examined whether RhoB could be sumoylated. We immobilized His-tagged RhoB using NickelCnitrilotriacetic acid (??)-Huperzine A (Ni-NTA) agarose beads followed by immunoblotting to detect the conjugation (??)-Huperzine A of SUMO. Indeed, we found that RhoB could be sumoylated with a preference for SUMO2 conjugation, and coexpression of Ubc9 enhanced RhoB sumoylation (Fig.?1b). In addition, we carried out in vitro sumoylation assay and confirmed that Ubc9 could directly target RhoB for SUMO2 conjugation (Supplementary Fig.?1b). We next examined the effects of PIAS family of SUMO E3 ligases on RhoB sumoylation. As shown in (Fig.?1c), PIAS1 significantly enhanced the sumoylation of RhoB, whereas other PIAS family members did not. Meanwhile, knockdown of endogenous PIAS1.