Ed that dissipation of m triggers PINK1 autophosphorylation of Ser-228 and Ser-402. This autophosphorylation is critical for Parkin recruitment to the same mitochondria (10). When WT PINK1, PINK1 using a S228A/S402A double mutation (autophosphorylation-deficient form), or PINK1 having a S228D/S402D double mutation (autophosphorylation mimic type) have been expressed in PINK1 / MEFs at the proper expression level, we discovered that the S228D/S402D mutant promoted ubiquitin-oxyester formation on the Parkin C431S mutant comparable to WT PINK1 (Fig. 4B, lane 8). The S228A/ S402A mutant, in contrast, failed to support formation in the ubiquitin adduct on Parkin (lane 6). Taken collectively, the results shown in Fig. 4 suggest that mitochondrial localization, kinase activity, and autophosphorylation of PINK1 are necessary forJOURNAL OF BIOLOGICAL CHEMISTRYMechanism of Parkin ActivationAExogenous HA-Parkin + Endogenous Parkin +BPPase CCCP four + 30 +CCCCP-/+ +/+ -+ -/+Genomic PINK1 +/+ Exogenous PINK1 +Phos -tag Parkin -CCCP +Phos -tag*1 2 3*Parkin+Phos -tag 1*3*1 2 3 4 5*HAParkinDHA-Parkin in PINK1-/- MEFs with H271Q G309D E417G G386A G409V A168P E240K 534insQ L347P C92FEFraction CCCP Input + Cyt + Mt +WTPINK1 Phos-tagKD*HAParkin**IB: anti-Parkin + phos-tag IB: anti-Parkin non phos-tag IB: anti-LDH (cytosol marker) IB: anti-Tom22 (mitochondria marker)**anti-Parkin5164 Non Phos-tag(kDa)FL 1 2 anti-PINK(kDa)FIGURE 5. A, exogenous or endogenous Parkin underwent phosphorylation following CCCP therapy. The cell lysate of exogenous Parkin-expressing HeLa cells or intact HEK293T cells CCCP therapy had been subjected to Phos-tag Page. Note that mobility will not reflect the molecular weight of proteins in Phos-tag Page (58) and thus molecular weight markers are usually not shown. The blue asterisks indicate phosphorylated Parkin, whereas the arrows indicate the cross-reacting band unless otherwise specified.212127-80-5 uses B, protein phosphatase treatment in cell lysates caused the high-molecular shift of endogenous Parkin to disappear.1450879-67-0 Chemical name C, phosphorylation of Parkin following CCCP remedy in cells is determined by PINK1.PMID:24516446 Parkin was not phosphorylated in PINK1 / MEFs following CCCP therapy, whereas exogenous PINK1 complemented the aforementioned defect. D, phosphorylation of Parkin was severely compromised by different pathogenic mutations of PINK1. Parkin C431S mutant-expressing PINK1 / MEFs complemented by disease-relevant PINK1 were treated with CCCP and subjected to immunoblotting. FL, full-length PINK1; 1 and two, the amino-terminal processed kind as described in the legend to Fig. 4A. E, intact HEK293T cells CCCP therapy have been subjected to fractionation experiments. Cyt and Mt indicate the cytosol-rich supernatant along with the mitochondria-rich membrane pellet, respectively. Arrowheads indicate endogenous Parkin.formation with the ubiquitin-thioester intermediate on Parkin Cys-431. Parkin Phosphorylation Is Dependent on Both m Dissipation and PINK1–Because PINK1 is a Ser/Thr kinase, the simplest model is the fact that PINK1 phosphorylation of Parkin accelerates formation on the ubiquitin-thioester intermediate. Nonetheless, PINK1 phosphorylation of Parkin has, till recently, been controversial (7, 54 ?6). To detect a potentially phosphorylated kind of Parkin with high sensitivity, we performed electrophoresis applying polyacrylamide gels conjugated using a 1,3-bis[bis(pyridin-2-yl-methyl)amino]propan-2-olato diMn(II) complex (referred to hereafter as Phos-tag). Since two Phos-tag can capture phosphomonoester d.