Nek5 interacts with mitochondrial proteins and interferes negatively in mitochondrial mediated cell death and respiration.
Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2 μM). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in ROS formation after 4 h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.
Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil; Programa de Pós-graduação em Biologia Funcional e Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. Electronic address: talita.melo@lnbio.cnpem.br.
Talita D
TD
Melo Hanchuk
Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil; Programa de Pós-graduação em Genética e Biologia Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. Electronic address: priscila.papa@lnbio.cnpem.br.
Priscila Ferreira
PF
Papa
Departamento de Patologia Clínica, Campinas, SP, Brazil. Electronic address: pglgcoyote@bol.com.br.
Paolo G
PG
La Guardia
Departamento de Patologia Clínica, Campinas, SP, Brazil. Electronic address: anibal@unicamp.br.
Anibal E
AE
Vercesi
Programa de Pós-graduação em Biologia Funcional e Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil; Programa de Pós-graduação em Genética e Biologia Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil; Inst. de Biologia, Dep. Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas, Campinas, SP, Brazil; Universidade Estadual de Campinas, Faculdade de Ciências Farmacêuticas, Campinas, São Paulo, Brazil. Electronic address: jorg.kobarg@lnbio.cnpem.br.
Jörg
J
Kobarg
D017209:Apoptosis / Q000187:drug effects; D000079922:Copper Transport Proteins; D045304:Cytochromes c / Q000378:metabolism; D045222:Electron Transport Chain Complex Proteins; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D000072379:NIMA-Related Kinases; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D019284:Thapsigargin / Q000633:toxicity; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D000079922:Copper Transport Proteins; D045304:Cytochromes c; D045222:Electron Transport Chain Complex Proteins; D003576:Electron Transport Complex IV; D024101:Mitochondrial Proteins; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D019284:Thapsigargin; D025521:Tumor Suppressor Proteins; C119386:BCLAF1 protein, human; C484188:COX11 protein, human; C000603592:NEK5 protein, human
Mitochondria; Reactive oxygen species; Respiratory chain; hNek5