FIGURE 6.
During nutrient deprivation, PDCD4 stabilization and increased PRMT5 activity converge to increase methylated PDCD4 levels. A, MCF7 (PDCD4-PRMT5) cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) MG132 (10 μm, 1 h) under normal growth conditions (lanes 1 and 2) following culture in HBSS for 24 h (lanes 3 and 4) or following culture in HBSS for 24 h and then a period of recovery with media for 1 h (MG132 was added during recovery) (lanes 5 and 6). Immunoblot analyses of these cell lysates were probed with antibodies as indicated. B, quantification of methylated and total PDCD4 from three experiments (performed as in Fig. 2D), graphed as values relative to the 8-h time point. C, autoradiograph detecting tritiated proteins from in vitro methylation reactions where lysates from MCF7 expressing catalytically dead PRMT5 (mutation G367A/R368A) (lanes 3, 4, 7, and 8) or empty vector (lanes 1, 2, 5, and 6) were incubated with recombinant GST-PDCD4 (lanes 1–4) or GST-PDCD4R110K (lanes 5–8) and [3H]AdoMet. Immunoblot analyses were probed with antibodies as indicated.