Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria

Dulbecco-Telethon Institute, Padua, Italy.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2008; 105(41):15803-8. DOI: 10.1073/pnas.0808249105
Source: PubMed


Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca(2+) rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurin-dependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca(2+) rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.

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Available from: Luca Scorrano, Oct 02, 2015
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    • "We next asked if the elongated phenotype of SPG7 KD contributed to increased [Ca 2+ ] m retention. To test this, we measured [Ca 2+ ] m retention in wild-type cells overexpressing mitochondrial fission dominant-negative Drp1 K38A , known to result in elongated mitochondria (Cereghetti et al., 2008; Frank et al., 2001; Smirnova et al., 2001). As expected Drp1 K38A overexpression resulted in elongated mitochondria (Figures S2C–S2E). "
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    ABSTRACT: Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca2+- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca2+ retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca2+ and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca2+- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.
    Molecular Cell 09/2015; DOI:10.1016/j.molcel.2015.08.009 · 14.02 Impact Factor
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    • "HeLa-mtRFP cells were cultured as described (Cereghetti et al., 2008). Cells were transfected using Lipofectamine 2000 Reagent (Life Technologies, Burlington, Canada) for plasmids and HiPerfect Transfection Reagent (Qiagen) for siRNAs following manufacturers' instructions. "
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    ABSTRACT: Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking client-binding domain caused mitochondrial fragmentation. Together, data suggest that molecular ratio of mtHsp40-mtHsp70 is important for their chaperonic function and mitochondrial morphology. While mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short-isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study result from aberration of mitochondrial innermembrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS and ATP production. Altogether, our data propose that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1L cleavage leading to cristae opening, decreases OXPHOS and triggers mitochondrial fragmentation following reduction in their chaperonic function. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 04/2015; 26(12). DOI:10.1091/mbc.E14-02-0762 · 4.47 Impact Factor
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    • "The immunoprecipitated material was washed twice in CPBS and resuspended in SDS/PAGE loading buffer (NuPAGE), boiled, and loaded on 4%–12% gels (NuPAGE). For phosphorylation studies, total cell lysates were loaded on a phosphoprotein binding column (QIAGEN) as previously described (Cereghetti et al., 2008). Flow-through (unphosphorylated) and Molecular Cell 58, 244–254, April 16, 2015 ª2015 The Authors 251 eluted (phosphorylated) proteins were collected and concentrated and 20 mg of proteins were separated by 4%–12% SDS-PAGE. "
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    ABSTRACT: Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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