Figure 1. PINK1 phosphorylates Drp1^S616 .

• A–F
  Decreased Drp1^S616 phosphorylation in PINK1‐null HEK293 cells, MEF cells, and mouse substantial nigra tissues. Lysates of PINK1‐null (PINK1^KO) and parkin‐null (parkin^KO) HEK293 cells (A), MEF cells (C), mouse substantial nigra tissues (E), and their matched wild‐type controls (WT) were immunodetected with antibodies against either phospho (Ser616)‐Drp1 (pS616), phospho (Ser637)‐Drp1 (pS637), or Drp1. β‐actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown (B, D, F, respectively). Two independent PINK1^KO and parkin^KO HEK293 cell lines (1, 2) and substantial nigra from two separate PINK1^KO and parkin^KO mice (1, 2) were shown. Student's test. *P < 0.05, ***P < 0.001, ns: no significance. Data were presented as mean ± SEM of three independent experiments.
• G, H
  Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1^S616 phosphorylation in PINK1^KO HEK293 cells. Lysates of PINK1^KO and PINK1^WT control (WT) HEK293 cells expressing PINK1 variants were immunodetected with an anti‐phospho(Ser616)‐Drp1 antibody (pS616), an anti‐Drp1 antibody (Drp1), and an anti‐PINK1 antibody (PINK1). Cells with mock transfection (Blank) and transfected with an empty plasmid (3.1) were included as controls (G). Quantitation of pS616/Drp1 is shown (H). PINK1: wild‐type PINK1; D384N: PINK1 kinase‐dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mitochondrial target‐deficient mutant. Student's test. ***P < 0.001, ns: no significance. Data were presented as mean ± SEM of three independent experiments.
• I, J
  PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1/2. Lysates of PINK1^KO and PINK1^WT control (WT) HEK293 cells were immunodetected with an anti‐phospho (Ser616)‐Drp1 antibody (pS616), an anti‐Drp1 antibody (Drp1), an anti‐PINK1 antibody (PINK1), an anti‐CDK1 antibody(CDK1), an anti‐p44/p42 MAPK antibody (ERK1/2), an anti‐phospho (Thr161)‐CDK1 antibody (pThr161), and an anti‐phospho(Thr202/204)‐p44/p42 MAPK antibody (pThr202/204). β‐actin was detected as a loading control (I). Quantitation of CDK1^Thr161 phosphorylation (pThr161) and ERK1/2^Thr202/204 phosphorylation (pThr202/204) was shown (J). Student's test. ns: no significance. Data were presented as mean ± SEM of three independent experiments.
• K
  Alignment of human Drp1^S616 in different species. Alignment of human Drp1 (isoform 1) amino acids 612–620 with indicated species. Serine residual (S) corresponding to human Drp1^Ser616 was labeled as red.
• L, M
  PINK1 phosphorylates Drp1^S616 in vitro. Recombinant TcPINK1 was incubated with either GST‐Drp1^518‐736 (WT) or GST‐Drp1^{518‐736, S616A} (S616A) in the presence (+)/absence (−) of ATP. After heat inactivation, reactions were treated with (+)/without (−) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1^S616 (pS616) was immunodetected (L, upper panel). Total Drp1 (L, middle panel) and protein loading stained with Coomassie blue staining (L, lower panel) were shown. Quantitation of pS616/Drp1 ratio was shown (M). One‐way ANOVA followed with Tukey's test. ***P < 0.001. Data were presented as mean ± SEM of three independent experiments.
• N, O
  Label‐free relative quantitative mass spectrum analysis of Drp1^S616 phosphorylation. Representative spectra for the Drp1 phospho‐peptide (S616) SKPIPMPA(p)SPQK and non‐phospho‐peptide SKPIPMPA(p)SPQK. Spectra obtained for peptides from PINK1^WT HEK293 cells are shown (N). The relative ratios of the phospho‐peptides containing phosphor‐Ser616 to peptides containing Ser616 from PINK1^WT and PINK1^KO HEK293 samples were calculated and plotted (O).
• P, Q
  Km and Kcat for PINK1‐mediated phosphorylation of ubiquitin and Drp1. In vitro kinase assays were performed with different concentrations of ubiquitin (P) or Drp1 (Q) with TcPINK1 at fixed ATP concentration (500 μM). Results were analyzed on phos‐tag gels and modeled to the Michaelis–Menten equation. The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently. Km: Michaelis constant. Kcat: catalytic constant. Bars represent the mean ± SEM.