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Tricornered/NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue maintenance - PubMed

  • ️Tue Jan 01 2013

Tricornered/NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue maintenance

Zhihao Wu et al. Genes Dev. 2013.

Abstract

Eukaryotes employ elaborate mitochondrial quality control (MQC) to maintain the function of the power-generating organelle. Parkinson's disease-associated PINK1 and Parkin actively participate in MQC. However, the signaling events involved are largely unknown. Here we show that mechanistic target of rapamycin 2 (mTORC2) and Tricornered (Trc) kinases act downstream from PINK1 to regulate MQC. Trc is phosphorylated in mTORC2-dependent and mTORC2-independent manners and is specifically localized to mitochondria in response to PINK1, which regulates mTORC2 through mitochondrial complex-I activity. Genetically, mTORC2 and Trc act upstream of Parkin. Thus, multiplex kinase signaling is acting between PINK1 and Parkin to regulate MQC, a process highly conserved in mammals.

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Figures

Figure 1.
Figure 1.

Genetic interaction between PINK1 and mTORC2. (A) PINK1 RNAi-induced ATP level drop was rescued by Rictor or Sin1 overexpression (OE) but enhanced by their LOF. (B) PINK1 RNAi-induced abnormal wing posture was rescued by Rictor overexpression or Rictor + Sin1 overexpression but enhanced by their LOF. (C) PINK1 RNAi-induced mitochondrial aggregation in muscle was strongly rescued by Rictor overexpression and moderately rescued by Sin1 overexpression but was enhanced by rictor LOF. Mitochondrial morphology was monitored with a mito-GFP reporter. Bar, 30 μm. (D) PINK1 LOF-induced mitochondrial aggregation in DNs was strongly rescued by Rictor overexpression and moderately rescued by Sin1 overexpression but enhanced by rictor LOF. Bar, 5 μm. (E) PINK1 LOF-induced loss of DNs in the PPL1 cluster was rescued by Rictor overexpression but enhanced by rictor LOF. A rictor deletion mutant (Δ2) and two independent rictor RNAi lines (#1 and #2) were used in LOF studies. (* or #) P < 0.05; (** or ##) P < 0.01; (*** or ###) P < 0.005 in one-way ANOVA tests when data from day 1 or day 14 were compared.

Figure 2.
Figure 2.

Effects of PINK1 and mitochondrial CI function on TORC2 activity. (A) Western blot analysis of Mhc-Gal4-driven control (wild type [WT]), PINK1 overexpression, and PINK1-RNAi muscle extracts with the indicated antibodies and data quantification. (B, top) In vitro phosphorylation of recombinant human GST-AKT-KD by Drosophila TORC2 affinity-purified from control, PINK1-overexpressing, and PINK1 LOF animals, all expressing a Sin1-Flag transgene. (Bottom) Similar amounts of Sin1 and dTOR were present in the extracts or immunoprecipitated TORC2. After kinase reaction, AKT phosphorylation was detected with anti-pS473-AKT, and total GST-AKT-KD was detected by Coomassie Blue staining (CBS). (P.C.) Positive control with extract added; (N.C.) negative control with no kinase added. Bar graph shows data quantification. (C–G) The rescuing effect of PIP3 treatment on in vivo p-S505-AKT level (C), in vitro mTORC2 kinase activity (D), lifespan (E), wing posture (F), and muscle mitochondrial morphology (G) in PINK1-RNAi animals. Values represent relative protein amounts after normalization with controls. Mitochondrial morphology was monitored with a mito-GFP reporter. In vitro mTORC2 kinase assay was performed as in B. Bar graphs show data quantification. Bar, 30 μm. (H) Reduction of pS505-AKT level by rotenone treatment of wild-type animals. (I) Effect of CI subunit (ND75 and CG9762) RNAi on pS505-AKT level and restoration of the p-S505-AKT level in PINK1-RNAi animals after yNDI1 coexpression. Values represent relative protein amounts after normalization with controls. Bar graphs show data quantification. (*) P < 0.05 in one-way ANOVA or Student's t-tests.

Figure 3.
Figure 3.

Evidence that Trc acts in the PINK1 pathway. (A,B) Mhc-Gal4>PINK1 RNAi-induced flight ability (A) and wing posture (B) defects were rescued by the coexpression of the short (S) or long (L) isoforms of wild-type Trc or constitutively active (S292E or T453E) Trc but enhanced by dominant-negative (K122A or K122A/T453A) Trc. (* or #) P < 0.05; (** or ##) P < 0.01; (*** or ###) P < 0.005 in one-way ANOVA tests when data from day 1 (*) or day 14 (#) were compared. (C) Mhc-Gal4>PINK1 RNAi-induced mitochondrial aggregation in indirect flight muscle was rescued by the coexpression of Trc-L or Trc-S292E but enhanced by Trc-K122A/T453A. Mitochondrial morphology was monitored with a mito-GFP reporter. Bar, 30 μm. (D,E) PINK1B9 mutation-induced mitochondrial aggregation in DNs (D) or loss of DNs in the PPL1 clusters (E) was strongly rescued by TH-Gal4-driven expression of wild-type or constitutively active Trc but enhanced by dominant-negative Trc. Bar, 5 μm. (F) Western blot analysis showing the effects of PINK1 LOF or overexpression on Trc phosphorylation. Trc-L overexpression served as a positive control. (G) In vitro kinase assay showing the effects of PINK1 LOF or overexpression on Trc kinase activity. Trc-L purified from control, PINK1 mutant, and PINK1-overexpressing animals was tested for kinase activity using AAK1-HA as a substrate in the presence of ATP-γ-S and phosphorylation detected by anti-thiophosphate ester antibody after esterification with p-nitrobenzylmesylate (PNBM). (P.C.) Positive control with extract added; (N.C.) negative control with no kinase added. Bar graph shows data quantification. (H) Western blot analysis showing that PINK1-induced Trc phosphorylation of T453 was Rictor-dependent, whereas that of S292 was Rictor-independent. (*) P < 0.05; (***) P < 0.005 in one-way ANOVA tests in E and G.

Figure 4.
Figure 4.

Genetic evidence that Parkin and MQC executors act downstream from TORC2 and Trc in the PINK1 pathway. (A,E) The enhancement of PINK1 RNAi-induced abnormal wing posture (A) or mitochondrial aggregation (E) by rictor deletion was suppressed by Parkin but not GFP overexpression. (B,F) The rescue of PINK1 RNAi-induced abnormal wing posture (B) or mitochondrial aggregation (F) by Rictor overexpression was blocked by Parkin LOF. (C,G) The enhancement of PINK1 RNAi-induced abnormal wing posture (C) or mitochondrial aggregation (G) by dominant-negative Trc was completely suppressed by Parkin but not GFP overexpression. (D,H) The rescue of PINK1 RNAi-induced abnormal wing posture (D) or mitochondrial aggregation (H) by wild-type or constitutively active Trc was blocked by Parkin LOF. (I,J) The enhancement of PINK1 RNAi-induced abnormal wing posture (I) or mitochondrial aggregation (J) by rictor deletion was suppressed by Miro-RNAi, Marf-RNAi, or Atg1 overexpression. (K,L) The enhancement of PINK1 RNAi-induced abnormal wing posture by dominant-negative Trc was suppressed by Miro-RNAi, Marf-RNAi, or Atg1 overexpression. (* or #) P < 0.05; (** or ##) P < 0.01; and (*** or ###) P < 0.005 in one-way ANOVA or Student's t-tests when data from day 1 (*) or day 14 (#) were compared. Bars: E–F,J,L, 30 μm.

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