Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.
OBJECTIVE
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.
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.
Mariela J
MJ
Nevet
Sharon
S
Vekslin
Alexander M
AM
Dizhoor
Elena V
EV
Olshevskaya
Rotem
R
Tidhar
Anthony H
AH
Futerman
Tamar
T
Ben-Yosef
D000818:Animals; D002412:Cations; D002417:Cattle; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D051604:Guanylate Cyclase-Activating Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051580:Neuronal Calcium-Sensor Proteins / Q000502:physiology*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism / Q000502:physiology*; D012160:Retina / Q000201:enzymology*
D002412:Cations; D051604:Guanylate Cyclase-Activating Proteins; D051580:Neuronal Calcium-Sensor Proteins; C098768:guanylyl cyclase-activating protein, bovine; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase