Fig. 6

Chk1 phosphorylates RhoB to promote its sumoylation and translocation to lysosomes. a Chk1 activity is required for translocation of RhoB to lysosomes. HeLa cells pretreated 0.5 h with or without Chk1 inhibitor (0.2 μM) were subjected to immunofluorescence assay 2 h after UV (80 Jm⁻²) or 4 h after MMS (0.5 mM) treatment to examine the localization of endogenous RhoB and LAMP1. Scale bar, 10 μm. b Chk1 activity is essential for UV or MMS-induced sumoylation of endogenous RhoB. HeLa cells with expression of His-SUMO2 were treated as in panel a and then subjected to sumoylation assay. SUMO2 conjugation of RhoB was detected by immunoblotting with RhoB antibody. c Chk1 interacts with endogenous RhoB. HeLa cells transduced with Flag-tagged Chk1-D130A mutant (F-Chk1-D130A) were subjected to anti-RhoB IP followed by immunoblotting with anti-Chk1 to detect associated F-Chk1-D130A. d Chk1 phosphorylates RhoB at its threonine residues. In vitro kinase assay was carried out as described in Methods. Phosphorylated RhoB was detected by immunoblotting using phospho-threonine or phospho-serine antibodies. e Chk1 phosphorylates RhoB at Thr173 and Thr175. RhoB WT or T173,175A mutant (2A) purified from bacteria were used to perform in vitro kinase assay. f Phosphorylation of RhoB by Chk1 promotes its binding to PIAS1. HEK293T cells with expression of indicated combination of Flag-tagged PIAS1 (F-PIAS1) and HA-tagged wild-type (WT), T173,175E (2E), or T173,175 A (2A) RhoB were subjected to coimmunoprecipitation assay 1 h after treated with or without UV (80 Jm⁻²) to detect associated RhoB. g Phosphorylation of RhoB by Chk1 is required for UV or MMS-induced sumoylation. HEK293T cells with expression of indicated combination of HA-tagged SUMO2 (HA-SUMO2) and His-tagged RhoB (WT, 2E, or 2A) were subjected to sumoylation assay 1 h after treated with UV (80 Jm⁻²) or 2 h after treated with MMS (0.5 mM) to detect the SUMO2 conjugation of RhoB. h Chk1-mediated phosphorylation is essential for UV or MMS-induced lysosomal translocation of RhoB. U2OS cells with expression of HA-tagged RhoB (WT, 2E, or 2A) were subjected to immunofluorescence assay 4 h after treated with UV (80 Jm⁻²) or MMS (0.5 mM). Scale bar, 10 μm