Cdk activities in checkpoint-arrested egg extracts. MPF (□; cyclin B–cdk1) and cdk2 (▪) kinase activities and autoradiographs of [³⁵S]-labeled extracts proteins (the position of cyclin B is indicated) from portions of a cycling Xenopus egg extract during incubation at 23°C. (A) Control extract (+nocodazole). (B) Checkpoint-arrested extract (+demembranated sperm nuclei + nocodazole). Portions of the checkpoint-arrested extract received after 22 min of incubation: (C) GST–Cip1 (100 nM). (D) Roscovitine (2 μM; in DMSO). (E) MAPK activity in a checkpoint-arrested extract. (F) MAPK activity in a portion of the checkpoint-arrested extract that received GST–Cip1 (100 nM) after 22 min of incubation. The addition of GST or DMSO to checkpoint-arrested extracts did not reverse cell cycle arrest (data not shown).

Requirement for cdk activity in Mad2-induced mitotic arrest. MPF(□) and cdk2 (▪) kinase activities and autoradiographs of [³⁵S]-labeled extracts proteins (the position of cyclin B is indicated) from portions of a cycling Xenopus egg extract during incubation at 23°C. (A) Control extract, buffer added prior to incubation. (B) Recombinant human Mad2 (80 μg/μL of extract; + Mad2) added prior to incubation. GST–Cip1 (C) or Roscovitine (D) were added to samples of the Mad2-treated extract portion after 40 min incubation. GST or DMSO did not reverse Mad2-dependent cell cycle arrest (data not shown).

Cdk inhibition overrides checkpoint-dependent arrest in HeLa cells. Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition. DMSO, as control, or Roscovitine were then added in the continuous presence of nocodazole and samples taken at the indicated time points. (A) Chromosomes were stained with Giemsa. Arrowheads (Control) or brackets (Roscovitine) indicate maintenance or loss of sister chromatid cohesion. (B) Securin and cyclin B1 were visualized by immunoblot from total cell lysates; securin and cyclin B1 were also visualized from cells taken after nocodazole washout (NWO). In A, sister chromatid cohesion was lost in about 80% of cells 20 min after Roscovitine addition. (C) Cdc20 or Cdc27 were immunoprecipitated from samples taken at the indicated time points from Roscovitine addition (Ip) and associated Cdc27, Cdc20, and Mad2 were detected by immunoblot (Ib); the total amount of Mad2 is also shown. (Mk Ip) Mock precipitations; (Ig) immunoglobulin. (D) Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition and split into seven dishes. Then, DMSO (lane 1), Alsterpaullone (800 nM; lane 2), Indirubin 3′ monoxime (6 μM; lane 3), Purvalanol A (600 nM; lane 4), UO 126 (30 μM; lane 5), SB 203589 (30 μM; lane 6), and H89 (30 μM; lane 7), were individually added in the continuous presence of nocodazole, and cells harvested after additional 40 min incubation. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 and Mad2 were detected by immunoblot (Ib), the total amount of Mad2, cyclin B1, and cdk1 is also shown. UO 126, SB 203589, and H89 were also tested in a range of concentration from 30 nM to 50 μM giving similar results (not shown).

Cdk1 inhibition affects Cdc20 phosphorylation and binding to Cdc27 and Mad2 in Hela cells. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 or Mad2 were detected by immunoblot (Ib) from checkpoint-arrested cells taken at the indicated time points from Roscovitine addition (A) and nocodazole washout (B). Optical density values of Cdc27 bound to Cdc20 are indicated (Cdc27 od; arbitrary units). (Mk Ip) Mock precipitations. (C, left) Immunoblot of Cdc20 from thymidine-arrested (T) and from nocodazole-arrested HeLa cells taken at the indicated time points after Roscovitine addition. (Right) Immunoblot of Cdc20 from thymidine-arrested cells (T), from nocodazole-arrested cells nontreated (0) and treated (λPP) in vitro with λ protein phosphatase and from nocodazole-arrested cells after 20 min from Roscovitine addition (20).

Cdk1-dependent phosphorylation of Cdc20 is required for Mad2-dependent arrest. (A) [³⁵S]-labeled Cdc20 wild-type (wt) was produced in reticulocyte lysates and incubated with portions of a CSF-arrested egg extract in the presence of cycloheximide. Samples were taken at time 0 (lane 1), after 30 min incubation without further additions (lane 2), after 30 min incubation in the presence of 100 nM GST–Cip1 added from time 0 (lane 3; Cip1 low), and after 30 min incubation in the presence of 2 μM GST–Cip1 added from time 0 (lane 4; Cip1 high). Autoradiographs show [³⁵S]-labeled Cdc20 in the extract samples (top), histone H 1 phosphorylated (P-HH1) by cdk2 (middle), or cdk1 (bottom) immunoprecipitated (Ip) from extract samples 1, 2, 3, and 4. (B) [³⁵S]-labeled Cdc20 wild type (wt) and seven-phosphorylation-sites mutant (7A) were produced in reticulocyte lysates (-Cdk1) and incubated with active cdk1 (+Cdk1). Phosphorylated wild-type and 7A Cdc20 were then incubated with portions of a CSF-arrested extract containing recombinant human Mad2 (80 μg/μL of extract) and cycloheximide for 30 min at 23°C. Cdc27 or Mad2 were immunoprecipitated (Ip) and the amount of bound wild-type (wt) and 7A Cdc20 detected by autoradiography. (Mk) Mock precipitations. (C) A CSF-arrested extract was labeled with [³⁵S]-methionine and cysteine for 30 min, then cycloheximide and buffer (Control) or recombinant human Mad2 (+Mad2) were added and incubation prolonged for 30 min at 23°C. Subsequently, cdk1 phosphorylated Cdc20 wild type and 7A were added to Mad2 containing extract portions (1 reticulocyte lysate/40 extract) and incubation further prolonged for 5 min. CaCl₂ was then added, and samples taken at the indicated time points. The addition of nonprogrammed reticulocyte lysate (up to 1/10 extract volume) did not affect Mad2-dependent arrest.