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

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.

Figure 1.

Identification of CERKL interaction with neuronal calcium sensor proteins in yeast. (A) pMet-Myc-Ras-CERKL bait constructs used for RRS. These constructs encode for chimeric proteins, composed of mouse CERKL amino acids 1 to 358 (CERKL A) or 272 to 525 (CERKL B), fused to a cytoplasmic Ras mutant, with a myc tag at the N-terminus. CERKL domains included in each construct are indicated. (B) cdc25-2 yeast were cotransformed with the indicated bait and prey combinations and grown on GAL–LUM or on GAL-LU plates incubated in the permissive (24°C) and the restrictive (36°C) temperatures. Cotransformation of yeast with empty bait and prey vectors (first row) or with CERKL-bait vectors and an empty prey vector (second and third rows) served as a negative control. Pak and ChpAc are two proteins known to interact with each other, and thus served as a positive control (fourth row). Yeast transformed with a combination of CERKL and GCAP2, recoverin (RCVRN), or CIB1 grew on GAL-LUM plates (on which both bait and prey proteins are expressed) at both the permissive and the restrictive temperatures, therefore indicating an interaction between these proteins. The same plasmid combinations did not allow growth of cdc25-2 yeast when grown at 36°C on GAL-LU plates (on which the bait protein is not expressed). (C) Protein extracts from cdc25-2 yeast transformed with CERKL A or CERKL B bait constructs were subjected to Western blot analysis with an anti-Myc tag antibody. Both constructs yielded proteins of the expected size (58 kDa for CERKL A and 50 kDa for CERKL B) in media lacking methionine (−Met), but not in methionine-rich media (+Met), indicating inducibility of the pMET425 promotor. The PAK protein (50 kDa) served as a positive control (PC).

Figure 2.

Verification of the interaction between CERKL and NCS proteins by co-IP. (A) COS-7 cells were transiently cotransfected with a combination of any of six different HA-tagged NCS proteins (GCAP1, GCAP2, Recoverin, CIB1, Neurocalcin D, or calmodulin) and Myc-tagged CERKL. Cell lysates were subjected to co-IP with an anti-Myc antibody, and eluted proteins as well as total cell lysates were separated by SDS-PAGE, followed by Western blotting (WB) with anti-HA and anti-Myc antibodies. The presence of each of the NCS proteins GCAP1, GCAP2, Recoverin, CIB1, and Neurocalcin D in the anti-Myc (CERKL) immunocomplexes supported their interaction with CERKL in mammalian cells. Calmodulin did not interact with CERKL. (B) Bovine POS extracts were subjected to IP with an anti-CERKL antibody, and eluted proteins as well as the total extract were separated by SDS-PAGE, followed by WB with anti-CERKL and anti-GCAP2 antibodies. The presence of GCAP2 in the anti-CERKL immunocomplexes demonstrates an endogenous interaction between these two proteins in photoreceptors.

Figure 3.

Mapping the interacting regions of CERKL and GCAP2. (A) Schematic representation of the different co-IP experiments performed to map the interacting regions. The scattered line represents the detected interacting parts of the two proteins. (B) COS-7 cells were transiently cotransfected with various combinations of partial GCAP2_HA constructs and full-length CERKL_MYC. Cell lysates were subjected to co-IP with an anti-Myc antibody, and eluted proteins as well as total cell lysates were separated by SDS-PAGE, followed by WB with anti-HA and anti-Myc antibodies. Each one of GCAP2 EF-hands 2, 3, and 4 was found to independently bind CERKL, while EF1 did not.

Figure 4.

The effect of mutations in CERKL and GCAPs on their mutual interaction. (A) Schematic representations of the different GCAP1, GCAP2, and CERKL mutated constructs tested. Each line represents a co-IP experiment. None of the disease-causing mutations tested (marked in red) affected the interaction. However, the D66N mutation in EF2 of GCAP2 (marked in blue), which was shown to affect binding of both Ca²⁺ and Mg²⁺, eliminated the interaction between the two proteins. (B) Examples of experimental data for the results summarized in panel A. COS-7 cells were transiently cotransfected with various combinations of wt or mutant CERKL_MYC, GCAP1_HA, and GCAP2_HA. Cell lysates were subjected to co-IP with an anti-Myc antibody, and eluted proteins as well as total cell lysates were separated by SDS-PAGE, followed by WB with anti-HA and anti-Myc antibodies.

Figure 5.

CERKL-GCAP interaction is cation dependent. (A) Schematic representation (produced with the UCSF Chimera software provided in the public domain at http://www.cgl.ucsf.edu/chimera/) of Ca²⁺–bound EF2 hand of GCAP1 (PDB ID 2R2I; resolution: 2.00Å). The two critical residues for cation binding (D66 and E77) are colored red. The Ca²⁺ ion is colored green. (B) COS-7 cells were transiently transfected with wt CERKL_MYC and either wt or mutant GCAP2_HA. Cell lysates were subjected to co-IP with an anti-Myc antibody, and eluted proteins as well as total cell lysates were separated by SDS-PAGE, followed by WB with anti-HA and anti-Myc antibodies. Note that while the E77Q mutation did not affect CERKL-binding, the D66N mutation eliminated it. (C) COS-7 cells were transiently transfected with full-length wt GCAP2_HA and CERKL_MYC. Cell lysates were subjected to co-IP with an anti-Myc antibody. Lysates were incubated and washed with a modified lysis buffer containing either 5 mM CaCl₂, 5 mM MgCl_2, or 5 mM of both EGTA and EDTA. Eluted proteins as well as total cell lysates were separated by SDS-PAGE, followed by WB with anti-HA and anti-Myc antibodies. Note that while binding was observed in the presence of either Ca²⁺ or Mg²⁺, it was eliminated in the presence of EDTA+EGTA.

Figure 6.

GCAPs are not required for proper localization of CERKL in cone photoreceptors. Immunofluorescence of retinal sections from wt (A) and GCAP1/GCAP2 double knockout mice (B) labeled with a CERKL-specific antibody (left panels). The anti-CERKL immunofluorescence (right panels) is superimposed on differential contrast image (DIC). ROS, rod outer segments; COS, cone outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; scale bar, 10 μm. Note that the anti-CERKL immunofluorescence is localized to cone outer segments in both wt and GCAP double knockout retinas.

Figure 7.

Ceramide kinase assay. Cells were transiently transfected with the indicated expression vectors and incubated with C6-NBD-ceramide. C6-NBD-Cer-1P was detected in cells transfected with CERK, but not with CERKL. The experiment was performed in duplicate and repeated twice.