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Invest Ophthalmol Vis Sci. 2012 Jul 10;53(8):4565-74. doi: 10.1167/iovs.12-9770.

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

Author information

1
Department of Genetics and The Rappaport Family Institute for Research in the Medical Sciences, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

Abstract

PURPOSE:

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.

METHODS:

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.

RESULTS:

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).

CONCLUSIONS:

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.

PMID:
22678504
PMCID:
PMC3394741
DOI:
10.1167/iovs.12-9770
[Indexed for MEDLINE]
Free PMC Article
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