Figure 5: CHK1 phosphorylates SSBP1 blocking its mitochondrial localization to decrease nuclear H₂O₂ levels.

(A) Comparison of CRISPRi scores following AUR treatment with CHK1 phosphorylation sites characterized in Blasius et al.⁶⁷ identifies SSBP1•S67 as a potential CHK1 target mediating resistance to AUR. (B) SSBP1 is a direct target of CHK1 that is phosphorylated in a H₂O₂-dependent manner in vitro (see methods). (C) CHK1•C408D has heightened levels of activity towards SSBP1. (D) SSBP1 regulates nuclear H₂O₂ levels downstream of CHK1. Left, immunoblot of SSBP1 levels in K562-dCas9-KRAB cells expressing the indicated sgRNAs. Right, Measurement of nuclear H₂O₂ with HyPer7 in K562 cells depleted of the indicated genes. (E) SSBP1 phosphorylation by CHK1 blocks its mitochondrial localization. Left, the localization of SSBP1-HA was determined by immunofluorescence analysis of K562 cells following the indicated treatments. Right, quantification of mitochondrial colocalization of HA-SSBP1. (F) SSBP1•S67D phosphomimetic mutant does not localize to the mitochondria. (G) CHK1•C408D is sufficient to drive SSBP1 re-localization. (H-I) SSBP1•S67D phosphomimetic mutant decreases nuclear H₂O₂ following CHK1 inhibition. Immunoblot analysis of the indicated proteins reintroduced into SSBP1-depleted cells (H) and corresponding levels of H₂O₂ levels measured with nuclear HyPer7 (I). Scale Bar=10 μm. Data are represented as mean ± SEM. **p < 0.001, ***p< 0.0001. Statistical significance was determined by Student’s t-test (two-tailed, unpaired).