Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1.
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.
Department of Medicine, Division of Endocrinology, University of California, Irvine, USA. yumayc@uci.edu
Yumay
Y
Chen
Maria
M
Gaczynska
Pawel
P
Osmulski
Rosaria
R
Polci
Daniel J
DJ
Riley
D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D003580:Cytochromes / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D018625:Microscopy, Atomic Force; D009154:Mutation; D000072380:NIMA-Related Kinase 1; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D050995:Voltage-Dependent Anion Channel 1 / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D003580:Cytochromes; D008081:Liposomes; D011994:Recombinant Proteins; C494762:VDAC1 protein, human; D012694:Serine; D050995:Voltage-Dependent Anion Channel 1; C000606914:NEK1 protein, human; D000072380:NIMA-Related Kinase 1; D017346:Protein Serine-Threonine Kinases