1. Cyclin G Recruits PP2A to Dephosphorylate Mdm2
Koji Okamoto, Hongyun Li, Michael R Jensen, Tingting Zhang, Yoichi Taya, Snorri S Thorgeirsson, Carol Prives 
Molecular Cell 
Volume 9, Issue 4, Pages (April 2002)
DOI: /S (02)00504-X

2. Figure 1 Cyclin G and PP2A Form a Stable Quaternary Complex
Top panel: cyclin G-HA (pCMV cyclin G, 5 μg) was cotransfected with Flag-tagged B′α1 (CMV FB′α1, 2.5 μg) into COS-1 cells, and cell lysates were fractionated by glycerol gradient centrifugation. Immunoprecipitates of gradient fractions were probed for components of PP2A or cyclin G by Western blotting.
Middle panel: lysates from transfected cells were immunoprecipitated with normal rabbit serum (NRS), anti-HA antibody, or anti-cyclin G antibody. Western blot analyses of immunoprecipitates were performed to detect cyclin G or the PP2A C subunit.
Bottom panel: lysates from transfected cells were immunoprecipitated with NRS, anti-Flag, or anti-cyclin G antibodies. Cyclin G in both supernatant and immunoprecipitate was detected by Western blotting with anti-cyclin G antibody.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

3. Figure 2 Cyclin G Is Associated with Enzymatically Active PP2A
(A) Phosphatase activity is coimmunoprecipitated with cyclin G in vivo. Cyclin G-HA and Flag-B′α1 were cotransfected into COS-1 cells, and cell lysates were immunoprecipitated with either normal rabbit serum (NRS) or anti-cyclin G antibody (cyc G) in the presence or absence of cyclin G peptide. Phosphorylase b was the substrate for phosphatase assays. Data are expressed as fold stimulation over control immunoprecipitate with NRS. Value 1.0 corresponds approximately to the release of 10 pmol phosphate per hour from labeled phosphorylase b.
(B) The PP2A-cyclin G complex is active as a phosphatase in vitro. Left panel: silver stained proteins purified from baculovirus-infected Sf9 cells. Right panel: in vitro phosphatase assays using immunoprecipitated proteins at left. Purified cyclin G and PP2A complex were incubated alone or together and then immunoprecipitated with anti-cyclin G antibody. Associated phosphatase activity in immunoprecipitates was determined using phosphorylase b as substrate.
(C) Cyclin G-associated phosphatase activity is induced after p53 activation. Clone 6 cells were harvested at 38°C or 2 hr after shift to 32°C, and lysates prepared from cells were either analyzed by Western blotting with anti-cyclin G, anti-PP2A C subunit, or actin antibodies (total, top left panels) or immunoprecipitated with normal rabbit serum (IgG) or anti-cyclin G antibody. Immunoprecipitates were used for Western analysis probing for cyclin G or the C subunit of PP2A (IP, top right panels) or to determine associated phosphatase activity using 32P-labeled phosphorylase b as substrate.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

4. Figure 3 Cyclin G Associates with Mdm2 and p53 In Vivo
(A) Association between cyclin G and Mdm2 in H1299 cells. H1299 cells were transfected with cyclin G-HA (8 μg) and/or Mdm2 (12 μg). Lysates from transfected cells were subjected to immunoprecipitation with a mixture of anti-Mdm2 monoclonal antibodies (SMP14 and 2A10). Western blot analyses of lysates (10% of amount precipitated) were probed with anti-cyclin G polyclonal antibody (top), or immunoprecitates were probed with anti-mouse Mdm2 polyclonal antibody or anti-cyclin G polyclonal antibody (bottom).
(B) Cyclin G-p53 interaction is mediated by Mdm2. H1299 cells were cotransfected with cyclin G (6 μg) either in the presence or in the absence of Mdm2 (9 μg) and/or HA-p53 (3 μg). Immunoprecipitation with anti-p53 antibody (pAb421) and Western blot analyses of the immunoprecipitates with anti-HA antibody p53 and anti-cyclin G antibody were performed. The asterisk indicates a cross-reacting IgG heavy chain.
(C) Cyclin G is found in immunoprecipitates with Mdm2 and p53 in clone 6 cells at 32°C. Clone 6 cells were incubated at 38°C or 32°C for 5 hr and then further incubated at each temperature for 3 hr with 50 μM MG132. Cell lysates were immunoprecipitated with control antibody (cont, mouse IgG), anti-p53 antibody (pAb421), or anti-Mdm2 antibody (SMP14). Total lysates and immunoprecipitates were used for Western blot analyses with SMP14, pAb421, and anti-cyclin G antibody.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

5. Figure 4
Regions of Cyclin G and Mdm2 Involved in Their Interactions In Vitro and In Vivo
(A) Upper panel: 35S-labeled Mdm2 translated in vitro was incubated with Glutathione S transferase (GST) or GST-fusion proteins as described. The N-terminal domain of cyclin G (amino acids 1–45), (GST-cyc G [N-ter]), cyclin G lacking amino acids 46–293, (GST-cyc G[del-N]), and human GST-cyclin A (GST-cyc A) were used for the assays. Mdm2 bound to GST-fusion proteins was resolved by SDS-PAGE and detected by autoradiography. Lower panel: 35S-labeled cyclin G translated in vitro was incubated with GST or GST-fusion proteins as indicated, and bound cyclin G was detected as above.
(B) Cyclin G-HA protein immunopurified from Sf9 insect cells and incubated with purified GST or GST-Mdm2 was detected by probing with anti-HA antibody.
(C) H1299 cells were transfected with HA-cyclin G (3.6 μg) or cotransfected with HA-cyclin G (3.6 μg) and different Flag-MDM2 plasmids (3.6 μg) as indicated for 42 hr. Lysates from transfected cells were immunoprecipitated with anti-Flag mAb M2 (M2 lanes). Lysate with cyclin G alone was immunoprecipitated with anti-HA mAb (HA lane). Western blotting analyses of immunoprecipitates (upper two panels) and lysates (7.5% of total; bottom panel) were performed with anti-cyclin G antibody or anti-Flag antibody M2 followed by the anti-mouse Fc-specific secondary antibody. Asterisk indicates the IgG heavy chain that is detected by this antibody. Arrows indicate positions of wild-type or truncated Flag-Mdm2 proteins.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

6. Figure 5 Cyclin G Stimulates Dephosphorylation of Mdm2 by PP2A
(A) Cyclin G enhances the ability of PP2A to dephosphorylate GST-Mdm2 in vitro. Left panel: GST-Mdm2 was incubated with cyclin A/cdk2 either in the absence (left lane) or the presence (right lane) of ATP, repurified on Glutathione Sepharose resin, and subjected to SDS-PAGE. Right panel: GST-Mdm2 prephosphorylated with cyclin A/CDK2 was incubated alone or with purified cyclin G and PP2A, separately or together as indicated, and then subjected to SDS-PAGE. Western blots were probed with anti-Mdm2 mAbs SMP14 or 3G5.
(B) Cyclin G stimulates dephosphorylation of GST-Mdm2 specifically by PP2A containing a B′ subunit. Top panel: GST-Mdm2 was phosphorylated by cyclin A/cdk2 as in (A) and then incubated with purifed PP2A containing either B′α1 (PP2A-B′) or Bα (PP2A-B) subunits in the absence or presence of cyclin G-HA for 15 min at 30°C. GST-Mdm2 proteins were separated by SDS-PAGE and probed with mAb SMP14. Bottom panel: results were quantified by densitometry.
(C) Expression of cyclin G leads to decreased dephosphorylation of Mdm2 in vivo. H1299 cells were cotransfected with Flag-Mdm2 (4 μg) and HA-cyclin G (3.6 μg) or control vector PCDNA3 (3.6 μg) for 24 hr. Transfected cells were not treated (NT) or treated with hydroxyrea (1.5 μM; HU) for 17 hr and then released by extensive washing. At the indicated times, extracts were prepared and immunoprecipitated with anti-Flag (M2). Western blots of immunoprecipitates were probed with anti-Mdm2 antibodies 3G5 or SMP14 as indicated, and total lysates (7.5%) were probed with anti-cyclin G antibody.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

7. Figure 6 Cyclin G Impact on Mdm2 and p53 in Mouse and Human Cells
(A) Mdm2 phosphorylation and p53 levels are increased in cyclin G−/− cells. Fibroblasts (MEFs) from wild-type (+/+) or cyclin G-deficient (−/−) mice were lysed directly in E SB followed by SDS-PAGE. Mdm2 was detected with mAb SMP14, 2A10, or 3G5 by Western blotting as indicated. A mixture of monoclonal antibodies (pAb240, pAb242, pAb246, and pAb248) was used to detect p53. Endogenous cyclin G was probed with anti-cyclin G antibody.
(B) Mdm2 is underphosphorylated in cyclin G null MEFs after reintroduction of cyclin G. MEFs from wild-type (+/+) or cyclin G-deficient (−/−) mice were infected with either control or cyclin G retroviruses. Infected cell lysates were separated by SDS-PAGE, and Western blots were probed with SMP14, 2A10, anti-cyclin G, and anti-actin antibodies. Densitometric analysis was used to calculate the ratio of SMP14-reactive Mdm2 per amount of actin in each case.
(C) Reactivity with SMP14 antibody is increased after dephosphorylation of Mdm2. MEFs derived from cyclin G-deficient mice were infected with control or nontagged or myc-tagged cyclin G retroviruses (mt-cyclin G). Lysates were separated by 10% SDS-PAGE and transferred onto PVDF membranes that were incubated in the presence or the absence of shrimp alkaline phosphatase as indicated. Membranes were then used for Western analyses with SMP14, anti-actin, or anti-cyclin G antibodies.
(D) Overexpression of cyclin G increases the interaction between p53 and Hdm2. COS-1 cells were transfected with 0, 1, and 2 μg of HA-cyclin G construct for 42 hr. Transfected cell lysates were immunoprecipitated with anti-p53 mAb Western blot analyses of immunoprecipitates were performed with anti-Mdm2/Hdm2 mAb 3G5 or anti-p53 antibody Lysates (10%) were directly probed with anti-cyclin G pAb.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)

8. Figure 7
Cyclin G Expression Causes Dephosphorylation of Ser166 of Hdm2
(A) Specific recognition of phosphorylated Mdm2 by anti-P-S166 antibody. COS-1 cells were incubated with 1 μM campthotecin for 24 hr, and cell lysates were immunoprecipitated with anti-Hdm2 antibody (IF-2). Immunoprecipites were then incubated with buffer alone or with calf intestine alkaline phosphatase either in presence or absence of sodium orthovanadate (Na3VO4) at 20°C for 30 min prior to SDS-PAGE and Western blotting analyses with anti-Mdm2 antibodies (IF-2 or the anti-P-S166 antibody).
(B) Cyclin G expression is associated with dephosphorylation of Ser166 of Hdm2. CV-1 cells were transfected with Flag-tagged human Hdm2 (9 μg) alone or with human cyclin G (3 or 9 μg). Transfected DNA amounts were adjusted to 18 μg with empty vector. Lysates were immunoprecipitated with anti-Mdm2 antibody (IF-2) followed by SDS-PAGE and Western blotting with anti-Mdm2 antibodies (IF-2, 2A10) and the anti-P-S166 antibody.
Molecular Cell 2002 9, DOI: ( /S (02)00504-X)