Increasing knockdown of B56α results in a robust linear increase in c-Myc protein levels. (A) siRNA knockdown of B55α does not affect c-Myc protein levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-HA-c-Myc, 0.5 μg pD40-His/V5-B55α, and siRNA (100 nM final concentration) targeted to either PP2A-C, B55α, or scramble control. Whole-cell lysates were collected at 48 h posttransfection and normalized for transfection efficiency by β-gal activity, and c-Myc, PP2A-C, and B55α proteins were visualized by Western blot (WB) analysis with anti-HA.11, anti-PP2A-Cα, and anti-V5, respectively. (B) Vector-expressed shRNAs targeted to PP2A-A and B56 family members specifically knock down intended targets. HEK-293 cells were cotransfected with 50 ng CMV-βgal; 0.5 μg of either pD30-PP2A-FLAG-A or pCEP4HA-B56α, -β, -γ1, -γ3, -δ1, or -ɛ (targets); and 2 μg pSUPER-shRNA expression vector [empty (−) or targeted to PP2A-A or B56α, -β, -γ1, -δ1, or -ɛ, as indicated]. Cells were maintained in DMEM supplemented with 2% FBS and l-glutamine for 72 h. Lysates were prepared and normalized as for panel A. Immunoblots were probed for PP2A-A with anti-M2-FLAG or for the indicated B56 family members with anti-HA.11. (C) Increasing knockdown of B56α results in increased c-Myc expression. HEK-293 cells were transfected with 50 ng CMV-βgal, 0.5 μg CMV-HA-c-Myc, and increasing amounts from 0.25 μg to 1.5 μg of pSUPER-shRNA expression vector [empty (−) or targeted to PP2A-A or B56α, -β, -γ1, -δ1, or -ɛ, as indicated]. Cells were maintained and lysates prepared and normalized as for panel B. c-Myc was visualized with anti-HA.11 by Western blot analysis. (D) Knockdown of PP2A-A or B56α results in a linear increase in c-Myc protein levels. c-Myc protein levels were quantified from panel C and two repeat experiments, and average c-Myc expression and error bars were calculated (see “Western blotting and quantitation” in Materials and Methods). Changes in c-Myc protein levels were then graphed as change relative to the shRNA control (panel C, lane 1). (E) Schematic of B56 family members. The B56 family of PP2A regulatory subunits share 71% and 73% amino acid sequence homology in the A subunit binding domains 1 and 2 (ASBD1 and -2), respectively, but significantly lower homology, less than 39% and 34%, in the N and C termini, respectively, which are believed to dictate substrate specificity (33, 39, 79). RNAi target sites used in the shRNA expression vectors are indicated by underlined regions for each B56 member. Splice variations in B56γ and B56δ are shown.

c-Myc specifically associates with B56α. (A) c-Myc complexes with PP2A-HA-C subunit. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 3 μg CMV-Myc, and 3 μg of either pD30-PP2A-HA-C (+) or CMV-empty (−), as indicated. Cleared lysate volumes were normalized by β-gal activity and subjected to immunoprecipitation (IP) with anti-HA.11. Ten percent input volumes and 50% IP samples were analyzed by Western blotting (WB) for c-Myc with anti-N262 and for PP2A-HA-C with anti-HA.11. (B) PP2A-HA-C associates with the transactivation domain of c-Myc. HEK-293 cells cotransfected with 50 ng CMV-βgal and 3 μg either pD40-His/V5-control, -c-Myc, or -c-Myc^ΔTAD (input shown in lanes 1 to 3 of the lower panel) were used to generate Ni-NTA-control, -c-Myc, or -c-Myc^ΔTAD columns (see “Ni-NTA columns” in Materials and Methods). Ni-NTA columns were incubated with cleared lysates from HEK-293 cells transfected with 3 μg pD30-PP2A-HA-C (input shown in lane 4). Column-bound proteins were eluted, and 50% eluate and 10% input samples were subjected to Western blot analysis with anti-V5 for c-Myc and anti-HA.11 for PP2A-HA-C. (C) PP2A-B56α interacts with c-Myc. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 3 μg pD40-His/V5-c-Myc, and 3 μg either CMV-empty (−) or pCEP4HA-B56α, -B56β, -B56γ1, -B56γ3, -B56δ1, or -B56ɛ, as indicated. Anti-HA.11 immunoprecipitations were carried out on cleared lysates adjusted for β-gal activity. Two percent input and 50% IP samples were analyzed by Western blotting with anti-V5 for c-Myc, anti-rabHA for B56 family members, and anti-PP2A-Cα for endogenous PP2A-C. (D) Endogenous PP2A-B56α holoenzymes associate with endogenous c-Myc. Endogenous c-Myc was immunoprecipitated from HEK-293cells with agarose-conjugated C-33 antibody. Control immunoprecipitation was done using agarose A+G beads. Two percent input and 50% IP samples were subjected to Western blot analysis with anti-PP2A-B′α for endogenous B56α, anti-N262 for endogenous c-Myc, and anti-PP2A-Cα for endogenous PP2A-C subunit.

Increased PP2A-B56α activity reduces c-Myc protein levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 1 μg CMV-Myc and 1 μg of pD30-PP2A-FLAG-A, pD30-PP2A-HA-C, and/or pD40-His/V5-B56α, as indicated. Whole-cell lysates were collected at 48 h posttransfection and normalized for transfection efficiency by β-gal activity, and samples were subjected to Western blot (WB) analysis with anti-N262 for c-Myc, anti-M2-FLAG for PP2A-A, anti-HA.11 for PP2A-C, and anti-V5 for B56α. c-Myc protein levels were quantified from three separate experiments, and average protein levels with error bars were graphed relative to control protein levels (see “Western blotting and quantitation” in Materials and Methods).

c-Myc protein levels are negatively regulated by PP2A holoenzyme activity. (A) Increased cellular PP2A activity decreases c-Myc protein levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg pD40-His/V5-c-Myc, and increasing amounts from 0.5 μg to 2.5 μg of pD30-PP2A-HA-C, as indicated. Whole-cell lysates were collected at 36 h posttransfection, normalized for transfection efficiency by β-gal activity, and visualized by Western blot analysis with anti-V5 for c-Myc, anti-PP2A-Cα for total endogenous and ectopic PP2A-C, and anti-HA.11 for ectopic PP2A-HA-C. (B) Limited reduction of c-Myc protein levels by increased expression of PP2A-C subunit. c-Myc and PP2A-HA-C protein levels were quantified from panel A and two repeat experiments using LI-COR software (see “Western blotting and quantitation” in Materials and Methods). Average protein levels and error bars were calculated and graphed relative to the maximum level seen for c-Myc or PP2A-HA-C. (C) PP2A holoenzyme formation is required to negatively regulate c-Myc protein levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-Myc, or 0.5 μg pCEP-SV40 small T antigen, plus either 1.5 μg or 2.5 μg of pD30-PP2A-HA-C, as indicated. Whole-cell lysates were prepared and normalized as for panel A. Immunoblots were probed for c-Myc with anti-N262 and for PP2A-HA-C with anti-HA.11. (D) Adenovirus E4orf4 expression causes an increase in c-Myc protein levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-Myc and increasing amounts from 0.5 μg to 2.5 μg of pCAN-E4orf4, as indicated. Lysates were prepared and normalized as for panel A, and c-Myc protein was visualized by Western blot analysis with anti-N262.

PP2A-B56α regulates c-Myc protein stability. (A) Knockdown of B56α does not affect endogenous c-myc mRNA levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal and 3 μg pSUPER-shRNA-empty (−) or pSUPER-shRNA-B56α under 10% FBS conditions for 24 h and then starved in 0.2% FBS for 48 h. Cells exhibiting transfection efficiencies by β-gal assay within 5% of each other were used for RT-PCR analysis of endogenous c-myc and GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene) mRNA levels. Endogenous c-Myc and β-tubulin protein levels were also examined in the same experiment by Western blotting (WB) with anti-N262 and anti-β-tubulin, respectively. (B) c-Myc protein stability is increased upon B56α knockdown. One-hundred-millimeter dishes of HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg pD40-His/V5-c-Myc, and 4 μg pSUPER-empty or p-SUPER-B56α under 10% FBS conditions for 24 h. Each transfection mixture was split into six 60-mm dishes with 10% FBS and then starved in 0.2% FBS for 48 h. Cells were treated with 100 μg/ml cycloheximide (CHX), and cell lysates were prepared at the indicated time points after treatment. c-Myc and β-tubulin were visualized from samples at each time point with anti-V5 and anti-β-tubulin by Western analysis. c-Myc protein levels were quantified relative to β-tubulin levels and graphed as percent c-Myc protein remaining after cycloheximide treatment. Protein half-life was calculated using the Excel (Microsoft) graphing function.

PP2A-B56α holoenzyme associates with the transactivation domain of c-Myc. (A) PP2A-B56α requires the transactivation domain of c-Myc for association. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 3 μg pCEP4HA-B56α, and 3 μg of either pD40-His/V5-contol, -c-Myc^WT, or -c-Myc^ΔTAD, as indicated. Cleared lysates were collected at 36 h posttransfection and normalized for transfection efficiency by β-gal activity, and immunoprecipitations (IP) were performed with anti-V5. Five percent input and 50% IP samples were subjected to Western blot (WB) analysis with anti-V5 for c-Myc, anti-rabHA for B56α, and anti-PP2A-Cα for endogenous PP2A-C. (B) PP2A-B56α shows a stronger association with c-Myc T58A and S62D point mutants. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 3 μg of either CMV-control (−) or pCEP-4HAB56α (+), and 3 μg pD40-His/V5-c-Myc^WT or pD40-His/V5-c-Myc point mutants (T58A, S62A, T58E, and S62D, as indicated). Lysates were prepared and normalized as for panel A and subjected to immunoprecipitation with anti-HA.11. Immunoprecipitates were washed with a stringent buffer (see “Coimmunoprecipitation” in Materials and Methods). Ten percent input and 50% IP samples were analyzed by Western analysis with anti-V5 for c-Myc, anti-rabHA for B56α, and anti-PP2A-Cα for endogenous PP2A-C.

Knockdown of PP2A-B56α increases S62- and T58-phosphorylated c-Myc as well as c-Myc-driven transcription. (A) c-Myc S62 and T58 phosphorylation increases upon PP2A-B56α knockdown. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-Myc-HA-WT, and 1.5 μg of pSUPER-shRNA expression vector [empty (−) or targeted to B56α, B56β, B56γ, B56δ, or B56ɛ, as indicated]. Whole-cell lysates were collected at 72 h posttransfection. Samples were normalized for β-gal activity and analyzed by Western blots (WB) simultaneously probed with anti-HA.11 for total c-Myc and either anti-T58phospho for T58 phosphorylation or anti-S62phospho for S62 phosphorylation. Anti-HA.11 was detected with secondary anti-mouse Alexa Fluor 680, and anti-T58phospho and anti-S62phospho were detected with secondary anti-rabbit IRDye800. The Western blots shown are representative of S62 phosphorylation, T58 phosphorylation, and total c-Myc protein levels from three separate experiments. (B) S62- and T58-phosphorylated c-Myc is enriched upon B56α knockdown. S62 or T58 phosphorylation levels along with total c-Myc levels, from simultaneously probed Western blots, were quantitated using LI-COR software (see “Western blotting and quantitation” in Materials and Methods). Ratios of S62-phosphorylation/total c-Myc and T58-phosphorylation/total c-Myc were calculated. Average ratios with error bars from the experiment shown in panel A and two repeat experiments were graphed relative to control (−) levels, and statistically significant differences are indicated (*). (C) B56α knockdown selectively increases c-Myc-driven transcription. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-HA-c-Myc, and 10 ng of either E2F2-Luc or E2F2(-E-box)-Luc, along with 1.5 μg pSUPER-shRNA expression vector [empty (−) or targeted to B56α B56β, B56γ, B56δ, or B56ɛ, as indicated]. Whole-cell lysates were collected at 72 h posttransfection. c-Myc protein levels are shown by Western analysis with anti-HA.11. Luciferase activity was measured and corrected for transfection efficiency based on β-gal activity. Average corrected luciferase activity from three separate experiments was graphed with error bars, and statistically significant differences are indicated (*). (D) Knockdown of B56α increases endogenous E2F2 mRNA levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal and 3 μg pSUPER-empty, pSUPER-B56α, or pSUPER-PP2A-A under 10% FBS conditions for 24 h and then starved in 0.2% FBS for 48 h. Cells exhibiting transfection efficiencies by β-gal assay within 5% of each other were used for RT-PCR analysis of endogenous E2F2, c-myc, and GAPDH mRNA levels.