Protein interactions among C53/LZAP, RCAD, and DDRGK1. A, co-immunoprecipitation of overexpressed proteins. The proteins indicated in the figure panel were overexpressed in 293T cells. Co-immunoprecipitation was performed as described using M2 anti-Flag EZ beads (Sigma). B, co-immunoprecipitation of endogenous proteins. Endogenous C53/LZAP was immunoprecipitated with C53 polyclonal antibody, and the presence of RCAD and DDRGK1 was detected with corresponding antibodies. C, RCAD domain responsible for RCAD-C53 interaction. D, C53 domain for C53-RACD interaction. Caspase-1 dominant negative (C1DN) was used as the negative control. E, DDRGK1 domain for DDRGK1-C53 interaction. F, C53 domain for C53-DDRGK1 interaction. G, size exclusion chromatography. HeLa S100 cell lysate was subject to Superose 6 gel filtration. Aliquots of the fractions were subject to SDS-PAGE and immunoblotting. The Superose 6 column was precalibrated with molecular weight markers (GE Healthcare).

RCAD knockdown results in dramatic reduction of C53 and DDRGK1 proteins. A, effect of RCAD knockdown on C53 and DDRGK1 proteins. The endogenous proteins were knockdown by indicated siRNAs, and the total lysates were subject to immunoblotting with specific antibody. B, quantitation of the immunoblots in A. The immunoblots of HeLa cells was quantified by OpenLab software. C, mRNA levels in siRNA-mediated knockdown cells. Real-time PCR was performed to evaluate the relative mRNA levels in specific knockdown cells.

RCAD is involved in regulation of NF-κB signaling. A, RCAD knockdown caused elevated basal and stimulated NF-κB activity in HeLa cells. Control and RCAD knockdown HeLa cells were transfected with NF-κB luciferase reporter, and then treated with TNFα for 8 h. The NF-κB activity was scored with dual luciferase assay. B, RCAD knockdown led to elevated basal NF-κB activity in U2OS cells. *, p = 0.004; **, p = 0.03. C, RCAD knockdown promoted cell invasion of U2OS cells. *, p = 0.02; **, p = 0.04. D, zymography of conditional medium of U2OS cells.

Two novel C53/LZAP-interacting proteins: RCAD and DDRGK1. A, silver staining of the immunoprecipitate of C53-Flag fusion protein. C53-Flag fusion protein was overexpressed in 293T cells, and immunoprecipitated with M2-agarose beads (Sigma). An aliquot was subject to SDS-PAGE and sliver staining. Identities of the bands were determined by LC/MALDI mass spectrometry. B, mRNAs of RCAD and DDRGK1 in mouse tissues. FristChoice Northern blot mouse blot I (Ambion) was blotted with either mouse RCAD or DDRGK1 probes. C, specificity of siRNAs and antibodies. Endogenous RCAD or DDRGK1 was knockdown with specific siRNAs, and the cell lysates were subject to SDS-PAGE and immunoblotting with affinity-purified antibodies. D, RCAD and DDRGK1 proteins in rat tissues. Rat tissue lysates were subject to SDS-PAGE and immunoblotting with specific antibodies. Nonspecific band (ns) is marked by a star. E, RCAD and DDRGK1 expression in cancer cell lines. The total cell lysates of various cancer cells were subject to immunoblotting.

DDRGK1 is an ER protein anchored by its N-terminal signal peptide. A, immunofluorescence staining of endogenous Myc-DDRGK1. HeLa cells were transfected with either negative control siRNA or DDRGK1 siRNA 1. At 48 h post-transfection, the cells were fixed and stained with DDRGK1 antibody along with an ER marker PDI. The confocal pictures were captured by Zeiss 510 Meta microscope. B, immunostaining of Myc-DDRGK1. HeLa cells were transfected with Myc-DDRGK1 construct along with YFP-ER marker. At 24 h post-transfection, the cells were fixed and stained with Myc antibody, and the pictures were taken by Zeiss 510 confocal microscope. C, electron micrographs of HeLa cells overexpressing Myc-DDRGK1. The left panels are the images with low magnification (×4,400), whereas the right panels are the images with high magnification (×20,000). The nuclei are marked by N. The electron dense “cloud” around the nucleus in DDRGK1-overexpressing cells indicated massive amplification of the ER network, which is better illustrated in the images in the right panels. D, subcellular fractionation. The cytosolic and ER fractions were isolated by discontinuous sucrose gradient, and the aliquots were subject to SDS-PAGE and immunoblotting. Calnexin was used as the ER marker, whereas α-tubulin was used as the cytosolic marker. E, deletion of DDRGK1 signal peptide. Monomeric AcGFP-DDRGK1 fusion protein and its mutant without the N-terminal signal peptide were transfected into HeLa cells, and the images were taken with OpenLab software at 24 h post-transfection.

RCAD regulates protein stability and ubiquitination of C53/LZAP and DDRGK1. A, RCAD knockdown promoted protein degradation of C53/LZAP and DDRGK1. Control and RCAD knockdown HeLa cells were treated with cycloheximide (100 μg/ml) for periods of indicated time. Total cell lysates were subject to immunoblotting. B, proteasome inhibitor MG132 prevented degradation of C53/LZAP and DDRGK1 in RCAD knockdown cells. Control and RCAD knockdown HeLa cells were treated with MG132 (20 μm) for the indicated time, and the protein levels were evaluated by immunoblotting. C, RCAD overexpression blocked ubiquitination of C53/LZAP and DDRGK1. HeLa cells were transfected with indicated constructs. After 24 h of transfection, Flag-tagged C53/LZAP (or DDGRK1) was immunoprecipitated with Flag antibody, and then immunoblotted with hemagglutinin antibody for detection of ubiquitinated proteins.