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Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

ABSTRACT: CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. 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 or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina.CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay.We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cation-binding domains (EF-hands 2-4).This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca(2+) signaling, providing important clues for future research required in this direction.

SUBMITTER: Nevet MJ

PROVIDER: S-EPMC3394741 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

Nevet Mariela J MJ Vekslin Sharon S Dizhoor Alexander M AM Olshevskaya Elena V EV Tidhar Rotem R Futerman Anthony H AH Ben-Yosef Tamar T

Investigative ophthalmology & visual science 20120710 8

<h4>Purpose</h4>CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. 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 or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the ma ...[more]

PMID: 22678504

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Project description:Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.

Project description:PurposeThe CERKL gene encodes for ceramide kinase-like, a novel protein of unknown function. CERKL mutations are associated with a severe retinal phenotype. The purpose of this work was to investigate alternative splicing, and the temporal and spatial expression pattern of CERKL in the mouse retina.MethodsReverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis of mouse retina RNA was used to study the expression of Cerkl at various developmental time points, and to identify its various splice-isoforms. A specific anti-CERKL antibody was developed and used for immunostaining to study the localization of the endogenous CERKL protein in retina-derived cell lines and in the mouse retina.ResultsCerkl is expressed in the mouse eye as early as embryonic day 14. A total of seven different Cerkl splice-isoforms were identified in the mouse retina. The subcellular localization of CERKL in retina-derived cell lines is variable: CERKL is diffusely distributed in the cytoplasm, and in many cells, it is highly concentrated in the perinuclear region. In most, but not all cells, CERKL is also highly concentrated in the nucleus. In the mouse retina, CERKL is located in the ganglion cell layer, in amacrine cells of the inner nuclear layer, and in photoreceptors. CERKL is highly expressed in cone photoreceptors; however, its expression level in rod photoreceptors is very low. In cultured cells, CERKL is detected in the nucleus, but in retinal cells in situ, it is mostly located in the cytoplasm.ConclusionsThe expression of Cerkl in both mature and embryonic mouse retina and the severe retinal phenotype associated with human CERKL mutations indicate that this gene plays a crucial role in retinal activity, and that it may be important for retinal development as well. The high expression level of CERKL in cones correlates with the CERKL-associated phenotype in humans. Whether nucleocytoplasmic transport of CERKL actually occurs in vivo under certain conditions and its functional significance remain to be discovered.

Dataset Information

Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

Publications

Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

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