Volume 54, Issue 6, Pages 932-945 (June 2014) PP2A-Mediated Regulation of Ras Signaling in G2 Is Essential for Stable Quiescence and Normal G1 Length  Nana Naetar, Velmurugan Soundarapandian, Larisa Litovchick, Kelsey L. Goguen, Anna A. Sablina, Christian Bowman-Colin, Piotr Sicinski, William C. Hahn, James A. DeCaprio, David M. Livingston  Molecular Cell  Volume 54, Issue 6, Pages 932-945 (June 2014) DOI: 10.1016/j.molcel.2014.04.023 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 PP2A Is Required during G2 in Order to Allow Stable G0 (A) T98G cells expressing wild-type SV40 small t (WT st), mutant small t (C103S; Mut st), or an empty vector (Ctrl) were serum-deprived for 72 hr and then labeled with BrdU. (B) Synchronized T98G cells were treated with okadaic acid (OA; 60 nM) in different cell-cycle phases (dotted line). Directly after OA treatment, cultures were deprived of serum. BrdU labeling was performed for 6 hr after completion of mitosis. (C) Synchronized T98G cells were treated with OA and nocodazole (Noco; 400 ng/ml). Proliferative activity was determined as in (B) after release from the nocodazole block. Aliquots of the cultures were analyzed with fluorescence-activated cell sorting for the presence of phosphorylated histone H3 (P-H3; right). (D) st-Gem or st-Cdt1 fusion proteins were expressed in T98G or BJ hTERT cells. Expression was monitored throughout the cell cycle by western blot. Synchronized cultures were serum deprived in late G2 and then labeled with BrdU as in (A). Numbers within the gates (A–D) depict the percentage of BrdU-positive cells in the population (mean ± SEM from three independent experiments). See also Figures S1–S3. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Inhibition of PP2A in G2 Shortens G1 Phase (A) T98G cells were synchronized and treated with OA (60 nM) in G2/M (dotted line). After treatment, cultures were deprived of mitogens, and BrdU was added for a 6 hr time interval starting either directly after completion of mitosis (0–6 hr) or 6 hr after mitosis (6–12 hr). (B) T98G cells were synchronized as before but left untreated and kept in 10% FBS. BrdU was added as in (A). (C) T98G cells were synchronized and treated with OA as in (A) but were kept in 10% FBS after treatment (left). Alternatively, T98G cells were transduced with st-Gem or an empty vector, synchronized, and kept in 10% FBS (right). BrdU was added as in (A). (D) Asynchronously growing T98G and BJ hTERT cells expressing st-Gem or st-Cdt1 were labeled with BrdU for 2 hr. Numbers in (A–D) depict the percentage of BrdU-positive cells in the population (mean ± SEM from three independent experiments). Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 The PP2A B Subunit B56γ Is Required for Stable G0 T98G cells were transduced with lentiviral vectors expressing shRNAs targeting individual B subunits and then serum deprived for 48 hr and analyzed by BrdU incorporation. Each B subunit was targeted by a pool of five or more shRNAs. See also Figure S4. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 PP2A Inhibition in G2 Disrupts Pocket Protein Function in the Next G1 (A) Synchronized T98G cells were treated with OA (60 nM) in G2 (left and middle) or in G1 (right) and then processed for chromatin isolation and western blot analysis. G2 OA-treated cells were also allowed to proceed to the next G1 in the absence of serum and analyzed (2 and 5 hr after mitotic exit). Relevant western blot bands were quantitated (normalized to histone H1 loading control and shown as fold difference over G2 control). (B–D) G2 OA-treated T98G cells were analyzed in G2 and the next G1 by chromatin immunoprecipitation with antibodies to E2F4 or p130 (B). Graphs display relative fold enrichment over an unspecific promoter (Actin). Alternatively, RNA was isolated and processed for qRT-PCR (C). Fold differences over mock-treated controls are shown. Data in (B) and (C) are mean ± SD from experimental replicates representative of at least three independent experiments. Assembly of DREAM was assessed by coimmunoprecipitation in G2 and G1 (D). Asterisks mark bands from IgG heavy chains. See also Figure S5. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 PP2A-Inhibition-Dependent G0 Escape Is Mediated by Cyclin E Upregulation in Mitotic Cells (A) mRNA expression levels were determined by qRT-PCR after OA treatment in G2. G2 OA-treated T98G cells were also allowed to proceed to M in the presence of nocodazole (400 ng/ml) and analyzed by qRT-PCR (left) or western blot (right) after mitotic shakeoff. Western blot bands were quantitated (normalized to loading control and shown as fold difference over control). (B) G2 OA-treated cells were harvested in the presence of nocodazole either 1 hr before mitosis (pre-M) or in mitosis by mitotic shakeoff (M). Graphs display relative fold enrichment of regions in the cyclin E1 gene body over a nontranscribed region after phospho-Ser2 RNA polymerase II ChIP. (C) T98G cells transduced with cyclin E1 or empty vector were deprived of serum for 72 hr and then labeled with BrdU. (D–F) T98G cells stably expressing shRNAs targeting luciferase (Luc) or Cdk2 and vectors encoding WT HA-tagged Cdk2 (HA-Cdk2wt) or the analog-sensitive Cdk2 F80G mutant (HA-Cdk2as) were transduced with SV40 st and then mitogen deprived for 48 hr in the presence or absence of 3-MB-PP1 (10 μM). Cells were analyzed by BrdU labeling (D), qRT-PCR (E), or coimmunoprecipitation (F). (G) Alternatively, cells were synchronized, and treated with OA (60 nM) in G2 and 3-MB-PP1 (10 μM) in M and G1. Cultures were deprived of serum and BrdU-labeled for 6 hr after completion of mitosis. Data in (A), (B), and (E) are presented as mean ± SD from experimental replicates and are representative of at least three independent experiments. Data in (C), (D), and (G) are represented as mean ± SEM from three independent experiments. See also Figure S6. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Hyperactivation of Ras Signaling Mediates PP2A-B56γ-Dependent G0 Escape and Cyclin E Deregulation via c-Myc (A) Synchronized T98G cells were treated with OA at the indicated concentrations in G2 and then analyzed by western blot (left). Alternatively, T98G cells expressing SV40 st were transduced with H-RasAsn17 and analyzed by western blot along with empty vector controls (Ctrl; middle). Cells expressing shRNA targeting luciferase (shLuc) or B56γ were also analyzed by western blot (right). asynch, asynchronous. Relevant western blot bands were quantitated (normalized to loading control and shown as fold difference over control). (B) T98G cells expressing st or shRNAs targeting B56γ were transduced with H-RasAsn17. Cells were serum-deprived for 72 hr and then labeled with BrdU. (C) T98G cells were transduced as in (A), treated with 400 ng/ml nocodazole for 10 hr, and analyzed with mitotic shakeoff, and qRT-PCR. Data are presented as mean ± SD from experimental replicates and are representative of three independent experiments. (D) T98G cells transduced with c-Myc or empty vector were either deprived of serum for 72 hr and then labeled with BrdU (top) or treated with nocodazole and analyzed as in (C; bottom). (E) T98G cells expressing st were transduced with empty vector or a dominant-negative Myc fragment (Omomyc). Cells were serum-deprived for 72 hr and then labeled with BrdU. Numbers in (B), (D), and (E) depict the percentage of BrdU-positive cells in the population (mean ± SEM from three independent experiments). (F) G2 OA-treated cells were harvested in the presence of nocodazole ∼1 hr before M (pre-M). Graphs display relative fold enrichment of regions in the cyclin E1 promoter over an unspecific region after c-Myc ChIP. Data are presented as mean ± SD from experimental replicates and are representative of three independent experiments. See also Figure S7. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Model for a PP2A-B56γ-Mediated Pathway Controlling G0 and G1 Left, PP2A-B56γ is functional for modulating Ras signaling in G2. Normal G0 and G1 control can be achieved. Right, PP2A-B56γ is inhibited in G2, causing cyclin E upregulation via c-Myc and pocket protein malfunction. Cells are no longer able to reach stable G0 and go into G1 overdrive. Molecular Cell 2014 54, 932-945DOI: (10.1016/j.molcel.2014.04.023) Copyright © 2014 Elsevier Inc. Terms and Conditions