Fig. 6

Fig. 6

CHK1 phosphorylates SPRTN and regulates its recruitment to chromatin. a, b siRNA SPRTN-depleted HEK293 or haploinsufficient SPRTN HeLa cells (ΔSPRTN) still contain a residual amount of SPRTN (10, 30%, respectively). Data are representative immunoblots (a) and quantification of SPRTN normalised to vinculin (b). Mean ± SEM; n = 3, two-tailed Student's t-test. c, d CHK1 promotes SPRTN recruitment on chromatin. HeLa-wt or -ΔSPRTN cells ectopically expressing CHK1-wt, or treated with formaldehyde (FA) as a positive control, were fractionated and endogenous SPRTN protein level was assessed in the chromatin fraction. Residual SPRTN in ΔSPRTN cells were also recruited to chromatin. Representative immunoblots (c) and quantification of SPRTN on chromatin (d). Mean ± SEM; n = 3, two-tailed Student's t-test. e, f Overexpression of CHK1-full length (FL) or N-terminal products (CP1, 2 or 3) promote SPRTN retention on chromatin. Doxycycline-inducible stable cells expressing SPRTN-wt-cSSH cells were fractionated and SPRTN was assessed in chromatin fractions. Data are representative immunoblots (e) and quantification of HA (SPRTN) on chromatin (f). Mean ± SEM; n = 3, two-tailed Student's t-test. g Schematics to identify the CHK1 phosphorylation sites on SPRTN by Mass-Spectrometry. h Diagram depicting human SPRTN with the CHK1 phosphorylation sites on Ser 373, 374 and 383 identified in this study. SPRTN domain and motifs; DBS: DNA binding site; SHP box: p97 interacting region; PIP box: PCNA interacting peptide; UBZ: ubiquitin binding domain. Sequence below (from SwissProt: Q9H040) shows the location of these sites highlighted in red and amino acids surrounding the region. i, j The degree of phosphorylation at a CHK1 consensus target sequence is diminished in the phospho-deficient SPRTN variants. Different SPRTN variants were expressed in HEK293 cells and purified under denaturing conditions. An antibody recognizing the epitope Rxxp (S/T) (phospho-Ser/Thr preceded by Arg at position -3) was used to visualise CHK1 phosphorylated targets. The CHK1 inhibitor UCN-01 was used as a control. Data are representative immunoblots (i) and quantification of CHK1-mediated phosphorylation of SPRTN normalised to total SPRTN protein level in denatured sample (j). Mean ± SEM; n = 3, two-tailed Student's t-test. Source data for (a–f, i, j) are provided as a Source Data file