טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentVekslin Sharon
SubjectCharacterization of the CERKL (Cermide Kinase-Like) Gene in
the Mammalian Retina
DepartmentDepartment of Medicine
Supervisor Professor Tamar Ben-Yosef
Full Thesis textFull thesis text - English Version


Abstract

CERKL mutations are associated with severe retinal degeneration. CERKL encodes for a novel ceramide kinase (CERK)-like protein. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide in vitro or in vivo. CERKL’s function in the retina is unknown. The purpose of this work was to further characterize CERKL's expression and activity in the mammalian retina.

Our results show that Cerkl is expressed in the mouse eye as early as embryonic day 14. The expression of Cerkl in both mature and embryonic mouse retina indicate that this gene plays a crucial role in retinal activity, and that it may be important for retinal development as well. A total of seven different Cerkl splice-isoforms were identified in the mouse retina. The high complexity of the gene both in humans and in mice may imply for several functions of the translated proteins and different regulation processes. In the mouse retina CERKL is located in the cytoplasm of the ganglion cell layer, in amacrine cells and in cone photoreceptors, whereas in retina-derived cell lines its subcellular localization was variable and was distributed in the cytoplasm, perinuclear region and in the nucleus. The high expression level of CERKL in cones correlates with the CERKL-associated phenotype in humans, which involves severe cone degeneration. CERKL's ability to localize both in the cytoplasm and in the nucleus in cultured cells while in retinal tissue it is expressed only in the cytoplasm, is intriguing. Whether nucleocytoplasmic transport of CERKL actually occurs in vivo under certain conditions and its functional significance remain to be discovered.

Based on a calcium-overlay assay, CERKL does not appear to bind calcium directly. Co-immunoprecipitation revealed that CERKL binds CIB1 but not calmodulin, though immune-staining revealed that CERKL and CIB1 are not expressed in the same cell types in the mouse retina. CERKL manifested significant up-regulation under serum starvation of cells. In addition, morphological and spatial changes were noticed when cells were exposed to different stress treatments, as indicated by western blot analysis and immune-staining. CERKL up-regulation and morphological changes under these stress conditions may imply a role for CERKL in cell survival. Its up-regulation under apoptosis encouraging treatment may suggest a link between CERKL's function and apoptotic processes.