Fig. 5

Fig. 5

SPRTN cleaves CHK1 and releases kinase-active CHK1 fragments. a Purified Flag-CHK1 was incubated with recombinant SPRTN-wt or SPRTN-E112A (a protease-dead variant). Anti-Flag immunoblotting detected N-terminal CHK1 fragments released by the SPRTN proteolytic activity, referred to here as CHK1 Cleavage Products (CPs) -1, -2 and -3 and indicated by blue arrows. Representative immunoblots from three replicates. b Graphical representation of the three CPs released by SPRTN activity. The approximate size of these CPs was estimated from the immunoblots; CHK1 amino acids 1–338, 1–293 and 1–237, respectively. Red ticks represent the locations of Ser296, Ser317 and Ser345. Inh: autoinhibitory domain. c, d Endogenous CHK1 from T24 cells synchronised in G₀- or S-phase (c) or from HEK293 cells ectopically expressing SPRTN-wt or SPRTN-E112A (d) was immuno-purified under denaturing conditions using a CHK1 antibody against an N-terminal CHK1 epitope. SE: short exposure, LE: long exposure. Quantification of the CHK1 fragments for (d). Mean ± SEM; n = 3, two-tailed Student's t-test. e Lysates from HEK293 cells ectopically expressing Flag-CHK1-wt or Flag-CHK1-S317A were denatured and CHK1 was then immuno-purified. Representative immunoblots from three replicates. f Ectopic expression of CHK1-CPs or CHK1-FL (full-length) in SPRTN-inactivated HEK293 cells. Left: mean ± 25–75 percentile range (box) ± 10–90 percentile range (whiskers); centre and right: mean ± SEM. >100 DNA fibers were analysed per condition and experiment; n = 3 experiments, two-tailed Student's t-test. g Ectopic expression of CHK1-CP3 in zebrafish embryos. Ref: reference value for statistics. Mean ± SEM; n = 3, two-tailed Student's t-test. h Graphical description of experimental setting. In the first reaction, GST-CHK1 was mixed with SPRTN-E112A or SPRTN-wt to generate CHK1 fragments (o/n, 37 °C); in the subsequent reaction, the product of the first reaction was mixed with Cdc25A in a kinase buffer to induce its phosphorylation (30 min, 37 °C). i Top panel: cleavage of CHK1 by SPRTN-wt after the first reaction. Bottom panel: phosphorylation of Cdc25A by the products of the first reaction. j quantification of phospho-Cdc25A in (i). Experiment was repeated 3 times with similar results. Source data for (a, c–g, i, j) are provided as a Source Data file