Volume 50, Issue 5, Pages 699-710 (June 2013) A Posttranslational Modification Cascade Involving p38, Tip60, and PRAK Mediates Oncogene-Induced Senescence  Hui Zheng, Alim Seit-Nebi, Xuemei Han, Aaron Aslanian, John Tat, Rong Liao, John R. Yates, Peiqing Sun  Molecular Cell  Volume 50, Issue 5, Pages 699-710 (June 2013) DOI: 10.1016/j.molcel.2013.04.013 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 PRAK Interacts with Tip60 In Vitro and in Senescent Cells (A) Schematic diagram of the Tip60α protein. p38 phosphorylation site is denoted by asterisk. The hatched bar represents the PRAK-interacting Tip60 fragment identified from the yeast two-hybrid screen using PRAK as bait. (B) FLAG-Tip60α was immunoprecipitated from 293T cells transfected with Myc-PRAK or Myc-PRAK-K51M, FLAG-Tip60α or vector, and MKK3E or vector, and subjected to western blotting along with lysates. (C) V5-tagged PRAK was immunoprecipitated from 293T cells transfected with V5-PRAK or vector and FLAG-Tip60 isoforms (1, a, or b) or vector using an anti-V5 antibody, and subjected to western blotting along with lysates. (D) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with HA-PRAK or vector (BP), FLAG-Tip60α, FLAG-Tip60β, or vector (WHF), and HaRasV12 or vector, and subjected to western blotting along with lysates. (E) Endogenous PRAK was immunoprecipitated from BJ cells transduced with HaRasV12 using an anti-PRAK or anti-actin antibody, and subjected to western blotting along with lysates. (F) FLAG-Tip60α or HA-PRAK was immunoprecipitated from 293T cells transfected with HA-PRAK, and WT or indicated deletion mutants of FLAG-Tip60α or vector, and subjected to western blotting. See also Figure S1. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Tip60 Is Essential for Oncogenic ras-Induced Senescence (A) Semiquantitative RT-PCR analysis of Tip60 mRNA levels in BJ cells transduced with GFP or indicated Tip60 shRNA using isoform-specific (D+C) or universal (Tot 5′+3′) primers (Figures S2A–S2C). (B) Western blot analysis of BJ cells transduced with GFP or indicated Tip60 shRNA. (C) Population doubling of BJ cells transduced with GFP (shGFP) or Tip60 (shTip) shRNA and Ha-RasV12 or vector (WH). (D) Population doubling of BJ cells transduced with GFP (shGFP) or Tip60 (shTIP887) shRNA, mouse Tip60 (mTip60) or vector (VT), and Ha-RasV12 or vector (WH). Inset, western blotting of BJ cells transduced with GFP or Tip60 shRNA (shTip887) and FLAG-mTip60 or vector (VT). (E) BJ cell populations in (C) and (D) were stained for SA-β-gal on day 18 post-ras transduction. (C–E) Values are mean ± SD for triplicates. See also Figure S2. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 p38 Phosphorylates Tip60 at Thr158/106 In Vitro and during ras-Induced Senescence (A) FLAG-p38α was immunoprecipitated from 293T cells expressing FLAG-p38αWT or -p38αAF, and MKK3E or vector, using an agarose-conjugated anti-FLAG antibody M2, and subjected to kinase assay toward recombinant ATF2 or Tip60α in the presence of [γ-32P]ATP. Western blotting was performed with part of the IPs and whole-cell lysates. (B) Recombinant His-Tip60α or ATF2 was incubated with recombinant His-p38α, His-p38β, His-p38γ, His-p38δ, or buffer and GST-MKK6E (+) or buffer (−) in the presence of [γ-32P]ATP. Phosphorylation of Tip60 and ATF2 were detected by autoradiography. (C) Wild-type or indicated mutant of His-Tip60α was incubated with MKK6E and p38α in the presence of [γ-32P]ATP and resolved on SDS-PAGE. The phosphorylation of Tip60 is detected by autoradiography. (A–C) Substrate input was stained with Coomassie brilliant blue R. (D) Western blotting of BJ cells transduced with wild-type or indicated mutant of FLAG-Tip60α and HaRasV12 or vector (BP) on day 8 post-ras transduction. (E) Western blotting of BJ cells on day 8 posttransduction with HaRasV12, MKK3E (K3E), or vector (WH) (left panel) or with shRNA for GFP or Tip60 (shTIP887) and HaRasV12 (right panel). (F) Western bloting of BJ cells on day 8 posttransduction with HaRasV12, MKK3E, or vector (BP) and treated with 10 μM SB203581 for 7 days. Numbers are relative levels of pTip60αT158 and pTip60βT158 after normalizing to the levels of total Tip60α and Tip60β, respectively. (G) Western blotting of BJ cells on day 8 posttransdcution with wild-type or indicated active mutant of p38 isoforms. See also Figure S3. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Phosphorylation of Tip60-T158 by p38 Induces the Acetyltransferase Activity of Tip60 and Is Required for the Ability of Tip60 to Mediate Senescence (A) Increasing amounts of Tip60α or buffer (−) was incubated first with p38α and MKK6E with (+) or without (−) ATP and then with histone and acetyl-CoA. (B) Increasing amounts of wild-type or indicated mutant of Tip60α or buffer (−) was incubated first with p38α, MKK6E, and SB203580 (SB) or vehicle control (Ctrl) and then with histone and acetyl-CoA. (C) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α (WT) or FLAG-Tip60α-A158 or vector (Ctrl) and HaRasV12 (Ras), MKK3E (K3E) or vector (VT), and incubated with histone and acetyl-CoA. (D) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α, shRNA for GFP or p38α (756, 785) or p38δ (386, 695), and HaRasV12 (Ras) or vector (VT), and incubated with histone and acetyl-CoA. (A–D) Acetylated histone H4 was detected by western blotting using an anti-acetyl-Lys antibody. Substrate input and recombinant proteins were stained with Ponceau. Part of the IPs and lysates were analyzed by western blotting (C and D). (E) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α (WT), FLAG-Tip60α-D98, FLAG-Tip60α-D158, FLAG-Tip60β (WT), or FLAG-Tip60β-D106 and incubated with histone and [14C]acetyl-CoA. Reactions were resolved by SDS-PAGE. Acetylation of histone H4 was detected by autoradiography. Histone input was stained with Coomassie brilliant blue. Part of the IPs was analyzed by western blotting. (F) Population doublings of BJ cells transduced with shRNA for GFP or Tip60 (sh887); mTip60 (left panel), mTip60-A155 (middle panel), mTip60-A158 (right panel), or vector (WN); and HaRasV12 (Ras) or vector (WH) over 10 days, starting on day 4 post-ras transduction. (G) Percentage of SA-β-gal-positive cells in cell populations described in (F). (F and G) Values are mean ± SD for triplicates. See also Figure S4. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Tip60 Acetylates PRAK in a Phosphorylation-Dependent Manner (A) His-PRAK was incubated with Tip60α, p38α, MKK6E, ATP, and acetyl-CoA for an indicated period. (B) His-PRAK was incubated with increasing amounts of Tip60α or buffer (−), p38α, MKK6E, and acetyl-CoA with or without ATP. (C) His-PRAK was incubated first with p38α, MKK6E, and ATP, and then with wild-type Tip60α (WT), Tip60α-A158, or buffer (−) and acetyl-CoA with or without SB203580. (D) Wild-type PRAK or PRAK-A182 was incubated with increasing amounts of Tip60α or buffer (−), p38α, MKK6E, ATP, and acetyl-CoA. (E) PRAK was incubated first with p38α and MKK6E with or without ATP, and then with Tip60α and acetyl-CoA with or without SB203580. (A–E) Acetylation of PRAK and phosphorylation of PRAK and Tip60 were detected by western blotting using anti-acetyl-Lys, anti-pPRAK-T182, and anti-pTip60-T158 antibodies, respectively. Input of recombinant proteins was stained by Ponceau. See also Figure S5. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Tip60 Acetylates PRAK at K364 In Vitro and during Oncogenic ras-Induced Senescence (A) Wild-type (WT) or indicated mutant of recombinant PRAK was incubated with wild-type (WT) or HAT-defective (HD) Tip60α and acetyl-CoA in the presence of p38α and MKK6E. Acetylation of PRAK was detected by western blotting using an anti-acetyl-Lys antibody. Recombinant proteins were stained by Ponceau. (B) HA-PRAK was immunoprecipitated from 293T cells transfected with wild-type (WT) or indicated mutant of HA-PRAK or vector (VT) and WT or HAT-defective (HD) Tip60α. (C) HA-PRAK was immunoprecipitated from BJ cells transduced with HA-PRAK, shRNA for GFP or Tip60 (887, 1506), and HaRasV12 or vector (VT). (D) HA-PRAK was immunoprecipitated from BJ cells transduced with wild-type (WT) or indicated mutant of HA-PRAK, and HaRasV12 or vector (WH). (E) Endogenous PRAK was immunoprecipitated from 293T cells transfected with wild-type (WT) or HAT-defective (HD) Tip60α or vector, MKK3E, and p38α. (F) Endogenous PRAK was immunoprecipitated from BJ cells transduced with shRNA for GFP (shGFP) or Tip60 (887 or 1506) and HaRasV12 (Ras) or vector (VT). (B–F) Acetylated PRAK and total immunoprecipitated PRAK were detected by western blotting using an anti-acetyl-Lys antibody and an anti-HA (B–D) or anti-PRAK (E and F) antibody, respectively. Part of the lysates was also analyzed by western blotting. (C and D) Numbers are fold induction of acetyl-PRAK signals by ras, after normalizing to the levels of total HA-PRAK. See also Figure S5. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Acetylation of PRAK-K364 by Tip60 Stimulates PRAK Kinase Activity In Vitro and in Cells and Is Required for the Induction of the Kinase Activity and Prosenescent Function of PRAK during ras-Induced Senescence (A) Wild-type (WT) or indicated mutant of His- PRAK was incubated first with WT or HAT-defective (HD) Tip60α, ATP, acetyl-CoA, p38α, and MKK6E, and then with HSP27. Phosphorylated HSP27 (S82) was detected by western blotting. HSP27 input and recombinant proteins were stained by Ponceau. (B) HA-PRAK was immunoprecipitated from 293T cells transfected with wild-type (WT) or K354R mutant of HA-PRAK or vector (VT), WT or HAT-defective (HD) Tip60α, and MKK3E and p38α. (C) HA-PRAK was immunoprecipitated from BJ cells transduced with HA-PRAK, shRNA for GFP or Tip60 (887, 1506), and HaRasV12 or vector (VT). (D) HA-PRAK was immunoprecipitated from BJ cells transduced with wild-type (WT) or K364R mutant of HA-PRAK, and HaRasV12 or vector (WH). (B–D) Immunoprecipitated HA-PRAK was incubated with HSP27 and ATP. Phosphorylated HSP27 (S82) and total immunoprecipitated HA-PRAK were detected by western blotting. HSP27 input was stained by Ponceau. Part of the lysates were also analyzed by western blotting. (E) Population doubling of BJ cells transduced with PRAK shRNA (shPK89); mPRAK (WT), mPRAK-K364R (364R), or vector (BP); and HaRasV12 (Ras) or vector (WH) over 12 days, starting on day 5 post-ras transduction. (F) Percentage of SA-β-gal-positive cells in the BJ cell populations described in (E) on day 12 post-ras transduction. (G) 1 × 104 of BJ cells transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887); human PRAK (WT), PRAK-K364Q (Q364), or vector (WH); and HaRasV12 (ras) or vector (V) were seeded in 12-well plates. Cell numbers were counted 3 days later. (H) Percentage of SA-β-gal-positive cells in the BJ cell populations described in (G) on day 10 post-ras transduction. (E–H) Values are mean ± SD for triplicates. See also Figure S5. Molecular Cell 2013 50, 699-710DOI: (10.1016/j.molcel.2013.04.013) Copyright © 2013 Elsevier Inc. Terms and Conditions