Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.

<p>A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.