AGC kinases phosphorylates Mad1 in vitro and in cells. (A) Alignment of the amino acid sequences of the putative AGC kinase phosphorylation site in Mad1 and previously reported AGC kinase substrates. (B) 293T cells were transfected with expression vector of Flag-S6K1. Cells were stimulated with insulin (10 μg/ml) for 15 min. Activated Flag-S6K1 was immunoprecipitated from the cell lysate by using anti-Flag M2 beads and was eluted with Flag peptide. Purified GST, GST-Mad1, or GST-Mad1S145A protein (1 μg) was incubated with activated Flag-S6K1 or recombinant RSK1 in the presence of [γ-³²P] ATP. ³²P-labeled proteins were resolved by SDS/PAGE and visualized by autoradiography. The input of GST proteins was shown by Western blot analysis with anti-GST antibody. (C) The experiment was performed as in B except by using recombinant activated Akt. (D) 293T cells were transfected with vectors encoding Flag-tagged Mad1 with kinase-dead, wild-type, or activated alleles of S6K1. Cells were serum-starved for 18 h, pretreated with MG132 (10 μM) and with or without rapamycin (Rap; 20 nM) for 1 h and stimulated with insulin (0.5 μg/ml) for 15 min. Flag-Mad1 was immunoprecipitated and immunoblotted with anti-phospho-Mad1 and anti-Mad1 antibodies. Total cell extracts were analyzed by Western blotting with the indicated antibodies. (E) 293T cells were transfected with vectors encoding Flag-tagged Mad1 wild-type or kinase-dead RSK1. After serum starvation for 18 h, cells were treated with MG132 (10 μM) for 1 h and stimulated with PMA (50 nM) for 15 min. Analysis was performed as in D. (F) 293T cells were transfected with vectors encoding Flag-tagged Mad1 and wild-type RSK1. After serum starvation for 18 h, cells were treated with MG132 (10 μM) and with or without PD98059 (50 μM) for 1 h and stimulated with PMA (50 nM) for 15 min. Analysis was performed as in D.

Ser-145 phosphorylation of Mad1 promotes serum-induced Mad1 degradation. (A) HeLa-Flag-Mad1 cells were serum-starved for 18 h, pretreated with MG132 (10 μM) for 1 h, and stimulated with serum for indicated amount of time. Flag-Mad1 was immunoprecipitated and eluted with Flag peptide. The elution was analyzed by Western blotting with the indicated antibodies. (B) The experiment was performed as in A except that cells were pretreated with MG132 and with or without a combination of PD98059 (50 μM) and rapamycin (20 nM). The immunoprecipitates and lysates were analyzed by Western blotting with the indicated antibodies. (C) HeLa-Flag-Mad1 cells were starved (SD) for 24 h, pretreated with various drugs as indicated for 1 h, and stimulated with serum (SS) for the indicated times. The lysates were analyzed by Western blotting with the indicated antibodies. (D) HeLa cells expressing wild-type or S145A mutant Mad1 were starved in serum-free medium overnight. The cells were then stimulated with serum for the indicated amount of time. The lysates were analyzed by Western blotting with the indicated antibodies. (E) 293T cells were transfected with vectors encoding wild-type or S145A mutant Mad1 and S6K1 or RSK1 as indicated. After 24 h, cells were lysed and the lysates were analyzed by Western blotting with the indicated antibodies. LY, LY294002; PD, PD98059; Rap, rapamycin.

Phosphorylation of Mad1 by RSK and S6K1 promotes cell proliferation and Myc-mediated transcription. (A) Equal numbers of MCF10A cells stably expressing indicated proteins were seeded, and cell proliferation was measured by counting cell number with a Levy hemacytometer. Data are presented as the mean ± SD of three independent experiments. (B) The cell cycle distributions of MCF10A cells expressing vector (MSCV), wild-type Mad1, or S145A mutant Mad1 were analyzed by FACS. (C) The total RNA from MCF10A cells stably expressing empty vector, wild-type Mad1, or S145A mutant Mad1 were extracted and analyzed by semiquantitative RT-PCR for the levels of ODC1 or CCND2 mRNAs. β-actin was used as an internal control. (D) MCF7 stable cells expressing empty vector, wild-type Mad1, or S145A mutant Mad1 were seeded at equal numbers (5 × 10⁴ cells per plate) and cultured for 12 days. The colonies were stained with crystal violet. The experiment was repeated three times, and the representative images were shown. (E) Signaling pathways involved in Mad1 degradation. Activated RSK and S6K1 leads to Mad1 degradation in a phosphorylation-dependent manner, whereas c-IAP1 promotes phosphorylation-independent ubiquitination and degradation of Mad1.

The cellular levels of Mad1 are rapidly reduced upon mitogen or insulin stimulation. (A) HeLa cells were cultured in the presence (S) or absence of serum (SD) for 48 h before stimulation with serum (SS) for the indicated amount of time. The lysates were fractionated and analyzed by Western blotting with the indicated antibodies. (B) HeLa cells were starved for 48 h and pretreated with DMSO or MG132 (10 μM) for 1 h before serum stimulation for 3 h. The Mad1 levels were analyzed by Western blotting. (C) The experiment was performed as in A except by using HeLa-Flag-Mad1 cells and stimulating with insulin (10 μg/ml) as indicated. (D) HeLa-Flag-Mad1 cells were starved overnight and pretreated with DMSO or MG132 (10 μM) for 1 h before stimulation with insulin (10 μg/ml) for 30 min. The Mad1 levels were analyzed by Western blotting. (E) HeLa cells were starved (SD) with serum-free medium for indicated amount of time. The cell lysates were analyzed by using Western blotting with the indicated antibodies.

Mad1 is phosphorylated by endogenous RSK and S6K1 in vivo upon serum stimulation. (A) HeLa-Flag-Mad1 cells were starved for 18 h, pretreated with MG132 (10 μM) and with LY294002 (50 μM) and/or PD98059 (50 μM) as indicated for 1 h before stimulation with serum or insulin (10 μg/ml) for 15 min. Flag-Mad1 was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-phospho-Mad1 and anti-Mad1 antibodies. Total cell extracts were analyzed by Western blotting with the indicated antibodies. (B) 293T cells were transfected with vectors of Flag-Mad1 and kinase-dead Akt (Akt-DN) and/or kinase-dead RSK1 (RSK1Δ/Δ). The cells were serum-starved for 18 h, pretreated with MG132 (10 μM) for 1 h and stimulated with serum for 15 min. Analysis was performed as in A. (C) The experiment was performed as in B except that the cells were transfected with kinase-dead S6K1 (K/R) as indicated. (D) The experiment was performed as in A except with rapamycin (20 nM) as indicated. (E) HeLa-Flag-Mad1 cells were transfected with siRNAs targeting S6K1 and/or RSK1/2 as indicated. The total amount of siRNA was normalized by addition of a nontarget siRNA. Forty hours after transfection, cells were starved for 18 h, pretreated with MG132 (10 μM), and stimulated with serum for 15 min. Analysis was performed as in A. (F) HeLa cells (≈10⁸) were starved, pretreated, and stimulated as in A. Mad1 was immunoprecipitated by using anti-Mad1 antibody. The immunoprecipitates and cell lysates were analyzed by Western blotting with the indicated antibodies. PD, PD98059; LY, LY 294004; Rap, rapamycin.

S145A Mad1 mutant is more stable than wild-type Mad1. (A) Lysates were prepared from MCF10A cells stably expressing empty vector (MSCV), wild-type Mad1, or S145A mutant Mad1 and analyzed by using Western blotting with the indicated antibodies. The mRNA levels of Mad1 and β-actin were analyzed by RT-PCR. (B) HeLa cells expressing Mad1 or S145A mutant Mad1 were treated with cycloheximide (CHX; 100 ng/ml) and harvested at the indicated time points. The cell lysates were analyzed by Western blotting with anti-Mad1 and anti-tubulin antibodies. (C) 293T cells were transfected with vectors encoding Flag-tagged Mad1 (wt) or S145A mutant Mad1 (sa) and HA-tagged ubiquitin for 20 h and then treated with or without MG132 (10 μM) for 4 h before harvesting. Anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (D) HeLa cells expressing Mad1 or S145A mutant Mad1 were starved with serum-free medium (SD) and harvested at indicated time points. The cell lysates were analyzed by Western blotting with the indicated antibodies.