Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress.
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.
Tuson
Miquel
M
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
Garanto
Alejandro
A
Gonzàlez-Duarte
Roser
R
Marfany
Gemma
G
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
D015415:Biomarkers; D002518:Ceramides; D007527:Isoenzymes; D012333:RNA, Messenger; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase