title: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress.
abstract: OBJECTIVE
  Retinitis pigmentosa (RP), a retinal neurodegenerative disorder characterized by apoptosis of photoreceptor cells, is caused by mutations in many different genes. We analyzed the RP gene ceramide kinase-like (CERKL) to determine CERKL function and contribution to pathogenesis.
  
  
  METHODS
  RT-PCR was performed to characterize CERKL expression in many human adult and fetal tissues, including retina. We analyzed the protein subcellular localization by confocal microscopy and further verified it by sucrose gradients. We performed lipid kinase activity assays. And finally, we studied the effects on cell apoptosis after CERKL overexpression in transiently transfected cultured cells by propidium iodide staining and poly-(ADP-ribose)-polymerase (PARP) caspase-dependent cleavage.
  
  
  RESULTS
  CERKL transcripts underwent alternative splicing. In the human retina, four different CERKL isoforms of 532, 558, 419, and 463 amino acids were expressed. CERKL proteins were mainly localized in the endoplasmic reticulum and Golgi compartments, but they also shifted localization to nuclei and nucleoli. We also found that CERKL prevented cells from entering apoptosis induced by oxidative-stress conditions.
  
  
  CONCLUSIONS
  CERKL remains a unique orphan lipid kinase in that no candidate substrate has been identified after intense research. The dynamic localization of CERKL suggests multiple sites of action. Remarkably, CERKL (but not the RP R257X mutant) exerts a protective role in cells against oxidative stress, consistent with RP mutations impairing the normal protein function in photoreceptors and thus tilting the balance toward apoptosis. These results provide valuable insights into the molecular mechanisms causing retinal degeneration.
authors.affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Tuson
authors.forename: Alejandro
authors.initials: A
authors.lastname: Garanto
authors.forename: Roser
authors.initials: R
authors.lastname: Gonzàlez-Duarte
authors.forename: Gemma
authors.initials: G
authors.lastname: Marfany
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D019556:COS Cells; D002518:Ceramides / Q000378:metabolism; D002522:Chlorocebus aethiops; D019610:Cytoprotection; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000201:enzymology / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D014162:Transfection
chemical_list: D015415:Biomarkers; D002518:Ceramides; D007527:Isoenzymes; D012333:RNA, Messenger; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase