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. 2013 Jun 28;288(32):23225–23233. doi: 10.1074/jbc.M113.492371

FIGURE 1.

FIGURE 1.

Identification of PPM1G as a phosphatase of 4E-BP1. A, phosphorylation of 4E-BP1 is not sensitive to the treatment of the phosphatase inhibitors OA and calyculin A. 293E cells grown in regular growth medium containing 10% FBS were treated with dimethyl sulfoxide (DMSO), OA (100 nm), or calyculin A (10 nm) for 15 min (lanes 1–3, respectively), and cell lysates were analyzed using immunoblotting. The phosphorylation status of 4E-BP1 at the Thr-37/46 and Ser-65 sites was detected using the p37/46 and p65 antibodies, respectively. The phosphorylation of Akt at the Thr-308 site was used as a control. The total 4E-BP1, Akt, and tubulin proteins were detected using respective antibodies. B, to screen for phosphatases of 4E-BP1, a small library of knockdown 293E cell lines were generated using shRNAs targeting different PPM phosphatases including PPM1A, PPM1B, PPM1D, PPM1E, PPM1F, and PPM1G as well as PPP2CA and PPP2CB. Cell lysates were prepared and analyzed for phosphorylation of 4E-BP1 at the Thr-37/Thr-46, Ser-65, and Thr-70 sites using corresponding phosphospecific antibodies. The total protein expression of 4E-BP1 was detected by the 4E-BP1 antibody. β-actin was used as the loading control. C, knockdown of PPM1G promotes 4E-BP1 phosphorylation. Stable control (sh-Con) and two different PPM1G knockdown (sh-PPM1G#1 and sh-PPM1G#2) 293E and HCT116 cell lines (lanes 1–3 and 4-6, respectively) were cultured in regular growth medium containing 10% FBS until they reached ∼60–70% confluency. Cell lysates were prepared and analyzed for the phosphorylation status of 4E-BP1 by immunoblotting. The phosphorylation of 4E-BP1 at the Thr-37/46 and Ser-65 sites was detected by the corresponding phosphospecific antibodies. The phosphorylation of p70S6K at the Thr-389 site (p-S6K) was shown as a negative control. D, the relative phosphorylation of 4E-BP1 was obtained by normalizing ECL signals generated by the p37/46 or p65 antibody to that of total 4E-BP1 in both 293E and HCT116 cells. The value of control cells was set to 1, and data shown in the bar graphs represent the mean ± S.D. (n = 3). *, p < 0.05 as determined by two-sample Student's t tests compared with the control cells. Note that the quantified results were obtained from one of the stable PPM1G knockdown cell lines (Sh-PPM1G#1) and that this knockdown cell line was used in all subsequent experiments in our study. Similar results were obtained using the other knockdown cell line (Sh-PPM1G#2).