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. Author manuscript; available in PMC: 2016 Feb 5.
Published in final edited form as: Mol Cell. 2015 Feb 5;57(3):537–551. doi: 10.1016/j.molcel.2015.01.002

Figure 2. Erk2 phosphorylates Drp1 Serine 616.

Figure 2

(A) Alignment of the consensus Erk2 target sequence with amino acids 612–620 of human Drp1 (isoform 1) and the corresponding sequence from the indicated species. (B–C) Recombinant, active GST-Erk2R67S was incubated with either GST alone, GST-Drp1518–736 or GST-Drp1518–736, S616A in the presence of γ32P-ATP (B) or ATP (C) and resolved by SDS-PAGE. Drp1 phosphorylation was detected by autoradiography (B) or immunoblot (A). (D–J) Phosphorylation of Drp1 (P616) and Erk1/2 (Y202/T204) were monitored by immunoblot in the following cells: (D) HEK-TtH cells grown in serum-free DMEM supplemented with 10μM Mek inhibitor PD325901 for 16 hrs, then supplemented with 10% FBS over an 8-hour time course; (E) HEK-TtH cells supplemented with 10% FBS and treated with 0.78–200nM of PD325901 for 8 hrs. (F) HEK-TtH cells stably expressing HRasG12V treated with 0.78–200nM of PD325901 for 8 hrs; (G) HEK-TtH cells transfected with increasing amounts of Raf-22W; (H) HeLa cells transfected with increasing amounts of Raf-22W in the presence of DMSO or PD325901; (I) HEK-TtH cells transfected with increasing amounts of MEK-DD; (J) HeLa cells transfected with increasing amounts of active MEK-DD in the presence of DMSO or PD325901; Tom20, CoxIV, GAPDH: Loading Controls.