Nek1 regulates cell death and mitochondrial membrane permeability through phosphorylation of VDAC1.
The mammalian NIMA-related protein kinase 1 (Nek1) is important for keeping cells alive after DNA damage, but the mechanism by which injured cells die without functional Nek1 has not yet been demonstrated. Here we show that Nek1 regulates the pathway to mitochondrial cell death through phosphorylation of voltage dependent anion channel 1 (VDAC1) on serine 193. Nek1 associates with VDAC1 in a yeast two-hybrid system, as well as by GST pull-down assays and by reciprocal immunoprecipitation. A portion of Nek1 in cells also localizes at mitochondria. Ectopic expression of a kinase-dead Nek1 mutant results in cell death, which is immediately preceded by loss of the Nek1-dependent VDAC1-S193 phosphorylation. UV irradiation of Nek1-deficient cells or silencing of endogenous Nek1 expression similarly results in loss of the specific S193 phosphorylation before cells die. Nek1-deficient cells are characterized by exaggerated mitochondrial membrane permeability (MMP) and accelerated cell death. Ectopic expression of a VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1, also results in cell death. A VDAC1-Ser193Glu mutant, designed to mimic constitutive phosphorylation by Nek1, rescues exaggerated MMP and keeps cells alive after DNA damaging injury, but only transiently. The direct interaction between Nek1 and VDAC1 provides a mechanism to explain how Nek1 prevents excessive cell death, as well as the first direct evidence that a specific kinase regulates VDAC1 activity.
Chen
Yumay
Y
Department of Medicine, Division of Nephrology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA. cheny@uthscsa.edu
Craigen
William J
WJ
Riley
Daniel J
DJ
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000072380:NIMA-Related Kinase 1; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D050995:Voltage-Dependent Anion Channel 1 / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D050995:Voltage-Dependent Anion Channel 1; C000606914:NEK1 protein, human; D000072380:NIMA-Related Kinase 1; C000606920:Nek1 protein, mouse; D017346:Protein Serine-Threonine Kinases