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Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1
Yumay Chen1, Maria Gaczynska, Pawel Osmulski
1Department of Medicine, Division of Endocrinology, University of California, Irvine, USA. yumayc@uci.edu
Abstract:
VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.
Insights
Never-in-mitosis A related kinase 1 (Nek1) phosphorylation of VDAC1 at Ser193 regulates mitochondrial permeability. This phosphorylation closes VDAC1, preventing cytochrome c release and excessive cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Voltage-dependent anion channel 1 (VDAC1) is crucial for mitochondrial permeability transition pore (mPTP) formation.
- Mitochondrial permeability allows pre-apoptotic molecules like cytochrome c to enter the cytosol, initiating apoptosis.
- The kinase Nek1 phosphorylates VDAC1 at Ser193, a modification linked to preventing excessive cell death.
Purpose of the Study:
- To elucidate the mechanism by which Nek1-mediated phosphorylation of VDAC1 regulates its activity.
- To investigate the structural and functional changes in VDAC1 upon phosphorylation by Nek1.
Main Methods:
- Atomic force microscopy (AFM) to determine VDAC1 configuration.
- Cytochrome c conductance studies in liposomes to assess VDAC1 channel activity.
- Analysis of wild-type VDAC1, VDAC1-Ser193Ala mutant, and VDAC1-Ser193Glu mutant.
Main Results:
- Wild-type VDAC1 is open but closes upon Nek1 phosphorylation, inhibiting cytochrome c efflux.
- The VDAC1-Ser193Ala mutant, unable to be phosphorylated, remains constitutively open, allowing cytochrome c leakage.
- The VDAC1-Ser193Glu mutant, mimicking constitutive phosphorylation, remains closed and prevents cytochrome c leakage.
Conclusions:
- Nek1 phosphorylation of VDAC1 at Ser193 induces a conformational change, closing the channel.
- This mechanism provides a direct link between Nek1 activity and the regulation of VDAC1-mediated cell death.
- Phosphorylation of VDAC1 by Nek1 serves as a critical switch controlling mitochondrial permeability and apoptosis.
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