Fig. 4

CHK1 K132 was ubiquitinated by TRAF4. a Alignment of conserved CHK1 lysine residues among different species. Kinase, kinase domain; SQ, Ser/Gln cluster; CM, conserved motif. b Ubiquitination of CHK1 mutants by TRAF4 was determined by an in vivo ubiquitination assay in 293T cells transfected with various constructs. c Lysine (K) 132 is the ubiquitination site in CHK1. 293T cells were transfected with various constructs and subjected to in vivo ubiquitination analysis. d CHK1 phosphorylation in CHK1-null HT29 cells is rescued by exogenous WT CHK1. CHK1 WT or CHK1 knockout HT29 cells transfected with CHK1 WT or CHK1 K132R mutant were treated with 5-Fu and subjected to IB analysis. e In vitro kinase assay of WT and mutant CHK1. HT29 cells were transfected with various constructs and treated with 5-Fu. WCEs were immunoprecipitated with Flag-tag antibody. The IP proteins were incubated with CDC25C and subjected to an in vitro kinase assay. f In vivo ubiquitination of WT CHK1 and the S317/345 mutant by TRAF4 in 293T cells with 5-Fu treatment. g Chromatin-associated CHK1 ubiquitination in HT29 cells with 5-Fu treatment. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and chromatin and non-chromatin fractions were isolated and subjected to immunoprecipitated and ubiquitination analysis. Chro, chromatin faction. Non-Chro, cytoplasmic proteins and soluble nuclear proteins. h Co-IP analysis of ATR and CHK1 interaction in chromatin fraction. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to Co-IP analysis. i Chromatin-associated CHK1 WT and K132R mutant ubiquitination in HT29 cells with 5-Fu treatment. HT29 cells were transfected with Flag-CHK1 or Flag-CHK1 K132R and treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to immunoprecipitated and ubiquitination analysis. j Co-IP analysis of ATR and CHK1 interaction in chromatin fraction. HT29 cells were transfected with Flag-CHK1 or Flag-CHK1 K132R and treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to Co-IP analysis