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Articles

PHLPP-Mediated Dephosphorylation of S6K1 Inhibits Protein Translation and Cell Growth

Jianyu Liu, Payton D. Stevens, Xin Li, Micheal D. Schmidt, Tianyan Gao
Jianyu Liu
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0509
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Payton D. Stevens
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0509
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Xin Li
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0509
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Micheal D. Schmidt
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0509
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Tianyan Gao
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0509
2Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0509
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  • For correspondence: tianyan.gao@uky.edu
DOI: 10.1128/MCB.05799-11
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  • Fig. 1.
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    Fig. 1.

    Overexpression of PHLPP results in dephosphorylation of both Akt and p70S6K. (A) Dephosphorylation of Akt and S6K1 was analyzed in stable HT29 (lanes 1 to 3), KM20 (lanes 4 to 6), and 293E (lanes 7 to 9) cells overexpressing PHLPP1 or PHLPP2. The phosphorylation status of Akt at S473 and that of p70S6K at T389 in cell lysates were detected using the phospho-S473 (p473) and phospho-T389 (p389) antibodies, respectively. (B) Western blotting results as shown in panel A were quantified using the FluoChem digital imaging system. The relative phosphorylation of Akt and S6K1 was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibodies to that of total Akt or S6K1 proteins, and the quantitative results were expressed graphically. Data shown in the graph represent the means ± standard errors of the means (n = 3).

  • Fig. 2.
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    Fig. 2.

    PHLPP dephosphorylates S6K1 independently of Akt. (A) Knockdown of PHLPP1 or PHLPP2 resulted in an increase in S6K1 phosphorylation. Stable PHLPP1 or PHLPP2 knockdown cells were generated in HT29 (lanes 1 to 3), KM20 (lanes 4 to 6), and 293E (lanes 7 to 9) using lentivirus-mediated shRNA. The cell lysates were analyzed for the phosphorylation of Akt, S6K1, and rpS6 using phospho-specific antibodies. (B) Western blotting results as shown in panel A were quantified using the FluoChem digital imaging system. The relative phosphorylation of Akt and S6K1 was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibodies to that of total Akt or S6K1 proteins, and the quantitative results were expressed graphically. Data shown in the graph represent the means ± standard errors of the means (n = 3). (C) The stable sh-P1 and sh-P2 293E cells were transiently infected with shRNA lentivirus targeting PHLPP2 and PHLPP1, respectively. The sh-Con, sh-P1, and sh-P2 cells were infected with control virus. Cell lysates were prepared 36 h postinfection and analyzed using Western blotting. (D) Stable sh-Con, sh-P1, sh-P2, and sh-PPP2CA 293E cells were analyzed for the phosphorylation status of S6K1 and Akt. The relative phosphorylation was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibody to that of total protein and shown below the phosphoblots.

  • Fig. 3.
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    Fig. 3.

    Knockdown of PHLPP activates the negative feedback regulation of PI3K/Akt signaling. (A) Cell lysates prepared from stable control and PHLPP-knockdown cells, including HT29 (lanes 1 to 3), KM20 (lanes 4 to 6), and 293E (lanes 7 to 9) were analyzed for the total expression of IRS-1 and the phosphorylation status of Akt (Thr308), PRAS40 (Thr246), mTOR (Ser2481), and 4E-BP1 (Thr37/46) using Western blotting. (B) The stable control and PHLPP-knockdown HT29 cells were treated with rapamycin (20 nM) for 0, 8, and 12 h, and cell lysates were prepared and analyzed by using Western blotting. The relative phosphorylation of Akt was quantified by normalizing enhanced chemiluminescence signals generated by the p473 antibody to that of total Akt and shown below the p473 blot.

  • Fig. 4.
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    Fig. 4.

    Insulin-induced activation of Akt is reduced in PHLPP1-knockout MEF cells. (A) WT and PHLPP1-knockout (P1−/−) MEF cells were treated with rapamycin (20 nM) for 0, 1, or 24 h, and the expression levels of IRS-1 were determined using the IRS-1 antibody. (B) Western blotting results as shown in panel A were quantified, and the relative expression of IRS-1 was obtained by normalizing enhanced chemiluminescence signals of IRS-1 to that of tubulin. The expression of IRS-1 in untreated WT MEF cells was set to 1, and all other conditions were normalized accordingly. Data shown in the graph represent means ± standard deviations (n = 3). (C) WT and P1−/− MEF cells were subjected to one of the following treatments: (i) serum starvation for 6 h (lanes 1 and 5), (ii) serum starvation for 6 h and subsequent stimulation with insulin (100 nM) for 10 min (lanes 2 and 6), (iii) pretreatment with rapamycin (20 nM) for 24 h and deprivation of serum during the last 6 h prior to the stimulation with insulin (100 nM) (lanes 3 and 7), and (iv) serum starvation for 6 h and subsequent stimulation with EGF (10 ng/ml) for 10 min (lanes 4 and 8). The protein and phosphoprotein levels were analyzed by Western blot analysis. (D) Western blotting results as shown in panel C were quantified using the FluoChem digital imaging system. The relative phosphorylation of Akt and S6K1 was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibodies to that of total Akt or S6K1 proteins, and the quantitative results were expressed graphically. Data shown in the graph represent the means ± standard deviations (n = 3).

  • Fig. 5.
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    Fig. 5.

    S6K1 is a direct substrate of PHLPP. (A) Endogenous PHLPP and S6K1 interact. Cell lysates prepared from HT29 or 293E cells were immunoprecipitated with the anti-S6K1 antibody coupled to protein A/G beads or beads alone. The immunoprecipitates were analyzed for the presence of PHLPP1 and PHLPP2 using immunoblotting. (B) PHLPP-mediated dephosphorylation of S6K1 in vitro. The HA-tagged S6K1 was expressed and immunoprecipitated from 293T cells using the anti-HA affinity matrix. Dephosphorylation reactions were carried out by incubating the immunoprecipitates with the purified PP2C domain of PHLPP1 for 30 min at room temperature. The beads were then washed and incubated with purified GST-S6 in the presence of ATP. Phosphorylation of S6K1 and GST-S6 was detected using the corresponding phospho-specific antibodies. The total GST-S6 was visualized on the membrane using Ponceau S staining. The relative phosphorylation of S6K1 and GST-S6 was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibodies to that of total protein and shown below the phosphoblot. (C) The time course of S6K1 dephosphorylation. The dephosphorylation reactions were carried out as described for panel B for 0 to 30 min. (D) The quantitative representation of data shown in panel C. The relative phosphorylation was quantified by normalizing enhanced chemiluminescence signals generated by the phospho-specific antibodies to that of total S6K1. The data were fitted to a single exponential curve. (E) WT and P1−/− MEF cells were subjected to amino acid starvation for 0 to 45 min. At each time point, the phosphorylation status of S6K1 and S6 was analyzed in cell lysates. (F) Western blotting results as shown in panel E were quantified, and the relative phosphorylation of S6K1 was quantified by normalizing enhanced chemiluminescence signals generated by p389 to that of total S6K1. Data shown in the graph represent the means ± standard deviations (n = 3).

  • Fig. 6.
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    Fig. 6.

    PHLPP regulates the phosphorylation of rpS6 and the association of S6K1 with the translation initiation complex. (A) Knockdown of PHLPP promotes the phosphorylation of rpS6 bound with the 7-methylguanosine (m7GTP) cap complex. The stable control and PHLPP-knockdown HT29 (lanes 1 to 9) and 293E (lanes 10 to 18) cells were serum starved overnight and subsequently treated with insulin in the presence or absence of rapamycin pretreatment. The cell lysates were incubated with m7GTP beads to pull down cap-associated proteins. The amounts of proteins and phosphoproteins in precipitates and lysates were detected by Western blot analysis. (B) Knockdown of PHLPP decreases the binding between S6K1 and eIF3b. The stable control and PHLPP-knockdown HT29 cells grown in serum-containing medium (+S) were incubated with serum- and amino acid-free medium for 50 min (−AA) and subsequently incubated with amino acid-containing medium (+AA) for 1 h. The cell lysates were immunoprecipitated with the anti-eIF3b antibody, and the presence of S6K1 in the immunoprecipitates was determined by Western blot analysis. (C) WT and P1−/− MEF cells were incubated with serum- and amino acid-free medium for 50 min (−AA) and subsequently incubated with amino acid-containing medium (+AA) for 1 h. The cell lysates were immunoprecipitated with the anti-eIF3b antibody and analyzed using immunoblotting. The relative amount of S6K1 bound to eIF3b was quantified by normalizing S6K1 found in the immunoprecipitates to that in the input, and the quantitative results are shown below the S6K1 blot.

  • Fig. 7.
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    Fig. 7.

    PHLPP negatively regulates cell growth and cap-dependent translation. (A) Knockdown of PHLPP increases cell size. The stable control and PHLPP-knockdown KM20 cells, 293E cells, and WT and P1−/− MEF cells were cultured in regular growth medium to ∼50% confluence. Cell numbers were plotted against cell diameters. The average cell sizes and the relative cell volumes are shown in the tables below each graph. The cell volume was calculated based on the average diameter of each cell type. (B) Knockdown of PHLPP increases protein content in cells. Equal numbers of control and PHLPP-knockdown cells were lysed in lysis buffer, and protein concentrations were determined using Bradford assays. Each experimental point was done in duplicate, and three independent experiments were averaged and shown in the graph (mean ± standard deviation). The asterisks in all graphs indicate P < 0.01 as determined by two-sample t tests compared to the control cells. (C) Knockdown of PHLPP increases the cap-dependent translation. The control and PHLPP-knockdown KM20 and 293E cells were transfected with a dual-luciferase reporter. The transfected cells were subjected to one of the following treatments: (i) maintenance in serum-containing medium (+S), (ii) serum starvation overnight (−S), and (iii) serum starvation overnight followed by treatment with insulin for an additional 24 h (+Ins). Cells were collected at the same time, and luciferase activities were measured using dual-luciferase assays. The cap-dependent translation was determined using the ratio of Renilla to firefly luciferase light units. The value of control cells in the serum-containing medium was set to 1. Data shown in the graph represent the means ± standard deviations (n = 3). The asterisks in all graphs indicate P < 0.01 as determined by two-sample t tests compared to the control cells. (D) Diagram showing that PHLPP-mediated regulation of S6K1 alters the balance of PI3K/Akt signaling. Activation of S6K1 results in downregulation of IRS-1 expression and subsequent inhibition of Akt activity downstream of insulin/IGF-1 receptors. Our study here demonstrates that PHLPP dephosphorylates and inactivates S6K1 directly. Loss of PHLPP expression leads to activation of the S6K1-dependent negative feedback loop.

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PHLPP-Mediated Dephosphorylation of S6K1 Inhibits Protein Translation and Cell Growth
Jianyu Liu, Payton D. Stevens, Xin Li, Micheal D. Schmidt, Tianyan Gao
Molecular and Cellular Biology Nov 2011, 31 (24) 4917-4927; DOI: 10.1128/MCB.05799-11

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PHLPP-Mediated Dephosphorylation of S6K1 Inhibits Protein Translation and Cell Growth
Jianyu Liu, Payton D. Stevens, Xin Li, Micheal D. Schmidt, Tianyan Gao
Molecular and Cellular Biology Nov 2011, 31 (24) 4917-4927; DOI: 10.1128/MCB.05799-11
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