Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress

Abstract

Purpose: Retinitis pigmentosa (RP), a retinal neurodegenerative disorder characterized by apoptosis of photoreceptorcells, is caused by mutations in many different genes. We analyzed the RP gene ceramide kinase-like (CERKL) to determineCERKL function and contribution to pathogenesis.Methods: RT-PCR was performed to characterize CERKLexpression in many human adult and fetal tissues, includingretina. 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 CERKLoverexpression in transiently transfected cultured cells by propidium iodide staining and poly-(ADP-ribose)-polymerase(PARP) caspase-dependent cleavage.Results: CERKLtranscripts 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 andGolgi compartments, but they also shifted localization to nuclei and nucleoli. We also found that CERKL prevented cellsfrom entering apoptosis induced by oxidative-stress conditions.Conclusions: CERKL remains a unique orphan lipid kinase in that no candidate substrate has been identified after intenseresearch. The dynamic localization of CERKL suggests multiple sites of action. Remarkably, CERKL (but not the RPR257X mutant) exerts a protective role in cells against oxidative stress, consistent with RP mutations impairing the normalprotein function in photoreceptors and thus tilting the balance toward apoptosis. These results provide valuable insightsinto the molecular mechanisms causing retinal degeneration.