Figure 1.

Drp1 phosphorylation at S600 and S579 occurs simultaneously and prompts mitochondrial fission

(A) C57BL/6NTac male mice were intraperitoneally injected with saline (as vehicle), CL316,243 (1 mg/kg; 1 h), glucagon (1 mg/kg; 15 min), or isoproterenol (10 mg/kg; 15 min) in three experiments (n = 3 mice per condition). Then, tissues were extracted and snap frozen to evaluate Drp1 phosphorylation levels.

(B) 8-week-old mice were intraperitoneally injected with either saline (as vehicle) or CL316,243 (1 mg/kg; 1 h). Then, BAT was collected and flash frozen. After obtaining protein homogenates, 10 mg of proteins were used for immunoprecipitation, using antibodies against Drp1 P-S600. The immunoprecipitated material was digested with Glu-C and then used for LC-MS/MS analysis. As control, the isotopically labeled form of the doubly phosphorylated peptide KSKPIPIMPAS[Phos]PQKGHAVNLLDVPVPVARKLS[Phos]ARE was spiked in the samples. The graph illustrates the profiles of the 5 most intensely detected fragments from peptide precursors with a charge of +6 and how CL316,243 treatment exhibits similar detection patterns to the reference standard peptide.

(C) Drp1 KO MEFs were transfected with wild-type (WT), S600A, or S600E forms of Drp1. Then, 48 h later, protein extracts were obtained to evaluate Drp1 phosphorylation levels.

(D) Drp1 KO MEFs were transfected with either S600A or S600E forms of Drp1. Then, cells were treated with 60 μM roscovitine for the indicated time points, and protein samples were collected to examine the indicated markers. The percentage of Drp1 P-S579 levels is representative of n = 3 experiments.

All values are presented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (two-tailed Student’s t test) between the indicated groups. See also Figure S1.