Figure 1Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected 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 Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. Decreased Drp1S616 phosphorylation in PINK1-null MEF cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) MEF cells (c) and their matched wildtype controls (WT) were immuno-detected 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 Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. Decreased Drp1S616 phosphorylation in PINK1-null mouse substantial nigra tissues. Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) mouse substantial nigra tissues (e), and their matched wildtype controls (WT) were immuno-detected 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 Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. (g, h). Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells expressing PINK1 variants were immuno-detected 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: wildtype PINK1; D384N: PINK1 kinase-dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mito-target deficient mutant. Student's test. ***p<0.001, ns: no significance. Data was 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 PINK1KO and PINK1WT control (WT) HEK293 cells were immuno-detected 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 CDK1Thr161 phosphorylation (pThr161) and ERK1/2Thr202/204 phosphorylation (pThr202/204) was shown (j). Student's test. ns: no significance. Data was presented as mean ± SEM of three independent experiments. (k). Alignment of human Drp1S616 in different species. Alignment of human Drp1 (isoform 1) amino acids 612-620 with indicated species. Serine residual (S) corresponding to human Drp1Ser616 was labeled as red. (l, m). PINK1 phosphorylates Drp1S616 in vitro. Recombinant TcPINK1 was incubated with either GST-Drp1518-736 (WT) or GST-Drp1518-736, S616A (S616A) in the presence (+)/absence (-) of ATP. After heat inactivation, reactions were treated with (+)/without (-) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1S616 (pS616) was immuno-detected (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 was presented as mean ± SEM of three independent experiments. (n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p) SPQK and non-phospho-peptide SKPIPMPA(p) SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO 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.