A Novel C53/LZAP-interacting Protein Regulates Stability of C53/LZAP and DDRGK Domain-containing Protein 1 (DDRGK1) and Modulates NF-κB Signaling^*

Tissue Culture Cells and Reagents

U-2 OS osteosarcoma cell (from ATCC) were grown in MaCoy's 5A medium supplemented with 10% fetal bovine serum (FBS),² while HeLa, MCF7, T47D, and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Cycloheximide, MG132, and TNFα were purchased from Sigma.

RCAD and DDRGK1 cDNA and Expression Constructs

The EST clones containing full-length RCAD and DDRGK1 cDNAs (human and murine) were purchased from Open Biosystems Inc. Murine RCAD cDNA was PCR-amplified using primers: 5′-GGGAATTCGGATGGCGGACGCCTGGGAG-3′ (forward) and 5′-AAGCGGCCGCTTAGGTACCCTCCTCTGTGACAGATGA-3′ (reverse), and verified by DNA sequencing. RCAD cDNA was further subcloned in-frame into pCMV-Myc (Clontech), and the resulting construct was used to overexpress Myc-tagged RCAD. Human DDRGK1 cDNA was PCR-amplified using primers: 5′-GGGAATTCGGATGGCGGAGTCTGTGGAGCGC-3′ (forward) and 5′-GGGCGGCCGCTCAGGCTGGGGCTTGGGCAGG-3′ (reverse), and further subcloned into pCMV-Myc or pCMV-Flag vectors. C53/LZAP constructs were described in our previous study (2).

siRNAs for Human C53/LZAP, RCAD, and DDRGK1 and Transfection

Silencer Select predesigned siRNAs were purchased from Ambion, Inc. The following are the sense sequences of siRNAs we used in this study: RCAD-1: GGAACUUGUUAAUAGCGGA; RCAD-2: GAGGAGUAAUUUUUACGGA; C53-2: GGCAGGAGAUUAUAGCUCU; C53-3: GGAUUGGCAGGAGAUUAUA; DDRGK1-1: GAAAAUUGGAGCUAAGAAA; DDRGK1-2: CCAUAAAUCGCAUCCAGGA. The negative control siRNAs were Silencer Select negative control 1 and 2 siRNAs that were purchased from Ambion.

Reverse transfections were performed using Hiperfect (Qiagen) following the manufacturer's instruction with minor modification. 10 nm siRNA was used as the standard concentration in our knockdown assays.

Generation of Rat Polyclonal Antibodies

Human RCAD fragment (residues 204–515) and DDRGK1 fragment (residues 1–150) were subcloned into pET-28C vector (Novagen), and His-tagged fusion proteins were purified from soluble fractions by nickel-NTA columns (Novagen). Purified proteins were injected into rats to make rat polyclonal antibodies according to the standard protocol (1). Polyclonal antibodies were affinity-purified using corresponding antigens.

Northern Blotting

The Ambion FristChoice Northern blot mouse blot I (purchased from Ambion) was used for our Northern blot assay. The DNA probes for murine RCAD and DDRGK1 were amplified from the EST clones containing their corresponding cDNAs and then labeled with [α-³²P]dATP using DECAprime II kit (Ambion). Hybridization of the blot was carried out in ULTRAhyb hybridization buffer (Ambion) according to the procedure provided by the manufacturer.

Antibodies, Co-immunoprecipitation, Immunoblotting, and Immunofluorescence Staining

The procedures for immunoblotting, immunoprecipitation, and immunofluorescence staining were described previously (1, 2). The following antibodies at indicated dilutions were used for immunoblotting: GAPDH (Santa Cruz Biotechnology, 1:10,000), anti-Flag M2 (Sigma, 1:2,000), Myc (9E10, Santa Cruz Biotechnology, 1:3,000), HA monoclonal antibody (Sigma, 1:2,000), C53 rat polyclonal and monoclonal antibody (1:1,000 dilution), RCAD rat polyclonal antibody (affinity purified, 1:500), DDRGK1 rat polyclonal antibody (affinity purified, 1:500). Species-specific horseradish peroxidase- and fluorophore-conjugated secondary antibodies were obtained from Jackson ImmunoResearch.

Subcellular Fractionation

Subcellular fractionation was performed according to the protocol of differential sucrose gradient isolation of ER and mitochondria (17). HeLa or HepG2 cells (from three 75-cm flasks) that had reached 90% confluency were harvested and washed with ice-cold phosphate-buffered saline, and then lysed mechanically with sonication in MTE buffer (10 mm Tris-HCl, pH 7.4, 270 mm d-mannitol, 0.1 mm EDTA) plus protease inhibitors. The following procedures were performed at 4 °C. A low-speed centrifugation (700 × g) was used to remove large cellular debris. The supernatant from this step was collected as a total lysed protein fraction, and further centrifuged at 15,000 × g for 15 min to separate mitochondria from ER and other light membrane fractions. The supernatant from 15,000 × g centrifugation (containing crude ER) was loaded onto a three-layered discontinuous sucrose gradient (1.3, 1.5, and 2 m sucrose from the top to the bottom in MTE buffer) and centrifuged at 152,000 × g for 70 min. The top layer was collected as the fraction of cytosol, while the banded fraction at the interface of the 1.3 m sucrose layer was collected as the ER fraction. The subcellular fractions were subjected to SDS-PAGE and followed by immunoblotting analysis using specific antibodies. We used calnexin as the ER marker, and α-tubulin as the cytosol marker.

Real-time PCR

Cells (1 × 10⁶) were collected and washed with ice-cold phosphate-buffered saline. mRNA was purified by RNeasy mini kit (Qiagen). The first-strand cDNA was synthesized by Superscript first-strand synthesis system for RT-PCR (Invitrogen), and used as the template for real-time PCR. QuantiTect SYBR Green PCR kit (Qiagen) was used for RT-PCR assays that were run on ABI 7500 RT-PCR system and analyzed by the relevant software. The primers were used for RT-PCR: human GAPDH: 5′-AAGGTGAAGGTCGGAGTCAA-3′ (forward); 5′-CCATGTAGTTGAGGTCAATGAAGG-3′ (reverse); human C53/LZAP: 5′-ATTTTTGGCCGATACTCTTCACA-3′ (forward); 5′-TCATAGTTGACATTCCGAACCAG-3′ (reverse); human RCAD: 5′-AGCAAACAGGCCTCAACTGT-3′ (forward); 5′-TTTCTGGTGCATCAGCTCAC-3′ (reverse); human DDRGK1: 5′-TGCTGGCTGAGGGGACTATAA-3′ (forward); 5′-CCGCTGTCGGATGAAGTTG-3′ (reverse).

NF-κB Reporter Assay

The NF-κB luciferase reporter plasmid (a gift from Dr. Junying Yuan) was transfected into HeLa or U2OS cells along with pRL-TK (Promega). At 24 h post-transfection, cells were treated with TNFα (10 ng/ml) for 8 h. Cells were lysed, and the luciferase activity was measured by Dual-Luciferase reporter assay kit (Promega).

Electron Microscopy

Cells were fixed with 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7. 4 at 4 °C for 30 min. Cells were then scraped from the tissue culture dish and harvested by centrifugation at 3000 rpm for 10 min. The cell pellet was further fixed in 2.5% glutaraldehyde for 2 h, and post-fixed with 1% osmium tetroxide for 2 h at 4 °C. The cell pellet was dehydrated in an ethanol gradient (50–100% for 10 min each) and embedded in Epon 812 at 60 °C for 2 days. Ultrathin sections were stained with uranyl acetate and lead citrate, and pictures were taken by a Zeiss 900 electron microscope.

Size Exclusion Chromatography

HeLa S100 extract was prepared as described by Dignam et al. (18). 0.5 ml of S100 extract was loaded on Superose 6 10/300 GL column (GE Healthcare) that was precalibrated with gel filtration calibration kit (GE Healthcare). The chromatography was performed using AKTApurifier (GE Healthcare) in the buffer of 20 mm HEPES, pH 7.4, 150 mm NaCl, 0.1 mm EDTA, 0.1 mm dithiothreitol plus protease inhibitors (0.3 ml/min flow rate). Fractions (0.5 ml) were collected, and aliquots of the fraction were subject to SDS-PAGE and immunoblotting with specific antibodies.

Invasion Assay

Cell invasion assays were performed in an invasion chamber, a 24-well tissue culture plate with 12 cell culture inserts. The inserts contain an 8-μm pore size polycarbonate membrane (Transwell Permeable Support, Corning Inc), coated with 60 μl of 1 mg/ml Matrigel (BD Sciences). U2OS cells were starved in serum-free medium overnight, trypsinized, and washed three times in DMEM containing 1% FBS. 2 × 10⁴ cells in 1% FBS-DMEM were seeded into the upper chamber, and 600 μl of DMEM containing 10% or 1% FBS was placed in the lower chamber. After a 16-h incubation, matrigel and cells remaining in the chamber were removed using cotton swabs. Invasive cells which cling to the bottom of the membrane were stained with 0.5 μm Calcein AM (Sigma-Aldrich). Pictures of the stained cells were taken under a fluorescent microscope with×1 magnification. Cells were then counted using the Openlab software. All experiments were run in duplicate and were repeated three times.

Zymography

Zymography of MMP9 was performed using 10% SDS-PAGE with 0.1% gelatin. U2OS cells were starved in serum-free MaCoy's 5A medium overnight. An aliquot (10 μl) of conditional medium was mixed with 10 μl of 2× Laemmli buffer without β-mercaptoethanol, incubated at 50 °C for 5 min, and then subject to SDS-PAGE. The gel was washed twice for 15 min in 100 ml of Triton X-100 (2.5% in H₂O) at room temperature, followed by incubation in 100 ml of development buffer (50 mm Tris-HCl, pH 8.0, 10 mm CaCl₂). After agitation for 15 min at room temperature, the gel was transferred to a 37 °C incubator with slow agitation (50 rpm) overnight. After development, the gel was incubated in fixing/destaining solution (45% methanol and 10% acetic acid) for 15 min at room temperature, and then stained with Coomassie Blue R-250 (0.1% in 50% methanol and 10% acetic acid) for 2–3 h at room temperature, and destained in destaining solution for at least 2 h at room temperature.

Two Novel C53/LZAP-interacting Proteins, DDRGK1 and RCAD, Are Highly Conserved and Ubiquitously Expressed in Multiple Tissues and Cell Lines

In an attempt to isolate C53/LZAP-interacting proteins, we performed co-immunoprecipitation using C-terminally Flag-tagged C53/LZAP as the bait, and identified potential interactors with mass spectrometry. As shown in Fig. 1A, there were at least three non-C53/LZAP proteins that were specifically immunoprecipitated with Flag-C53 protein, including heat shock protein 70 (Hsp70) and two uncharacterized proteins. A polypeptide with molecular mass of 40 kilodalton (kDa) was identified as DDRGK domain-containing protein 1 (DDRGK1, also named as C20orf116, CT116, and Dashurin) (4, 5), while a 90-kDa polypeptide was the uncharacterized protein KIAA0776 that was named hereafter as Regulator of C53/LZAP and DDRGK1 (RCAD) (Fig. 1A). Interestingly, our finding was in accordance with the data generated from a large-scale screening of protein-protein interactions (6). Both RCAD/KIAA0776 and DDRGK1/C20orf116 were among the potential C53 interactors in the screening (6) (also see the search result from the IntAct database using Cdk5rap3 as the keyword).

The human RCAD gene is located at chromosome 6q16.1. Similar to C53/LZAP, the human RCAD protein has 794 amino acid residues with no recognizable protein domains or motifs. Its orthologs can be found in the genomes of invertebrate, vertebrate, and plants, but not of yeast. Multiple sequence alignment indicates that its N-terminal portion is highly conserved (supplemental Fig. S1). RCAD is ubiquitously expressed in multiple tissues and cell lines. As shown in Fig. 1B, there are two major murine RCAD mRNA species with sizes of 2.8 and 4.2 kilobase (kb) in multiple mouse tissues. According to the EST data base, both mRNAs may give rise to the same protein with 793 amino acid residues. However, there are several human EST sequences with alternatively spliced exons that may encode RCAD isoforms with truncations. We generated an RCAD antibody that recognized an endogenous 90-kDa protein, while the antibody specificity was further confirmed by siRNA-mediated knockdown assay (Fig. 1C). Using this antibody, we examined RCAD expression in tissues and human cell lines. As illustrated in Fig. 1, D and E, RCAD was ubiquitously expressed in multiple tissues and cell lines. Interestingly, RCAD expression was remarkably lower in invasive breast cancer cell line MDA-MB-231 (Fig. 1E).

Human DDRGK1 protein has 314 amino acid residues with predicted molecular mass 35.6 kDa. Sequence analysis suggests that the first 1–28 residues are highly hydrophobic and may serve as a signal peptide. However, we could not detect any secretion of either endogenous or overexpressed DDRGK1 in tissue culture medium (data not shown). Furthermore, it contains a partial PCI (residues 229–273) domain that is frequently found in the subunits of the proteasome, COP-9 complex and translation initiation factors. DDRGK1 is a highly conserved protein, and its orthologs can be found in the genomes of vertebrates, invertebrates, and plants. Multisequence alignment suggests that its C-terminal portion contains several stretches of highly conserved sequences including the one with DDRGK pentapeptide (supplemental Fig. S2).

Northern blot analysis showed that murine DDRGK1 cDNA was about 1.3 kb, which is consistent with the size of both human and murine cDNA found in the full-length cDNA database (Fig. 1B). It was ubiquitously expressed in multiple tissues and organs, with relatively high-level expression in the liver, kidney, and testes. Our DDRGK1 antibody recognized a 42-kDa protein in SDS-PAGE, which is slightly larger than the predicted size (Fig. 1C). Similar to C53/LZAP and RCAD, DDRGK1 was expressed in multiple cancer cell lines with a relatively low expression in MDA-MB-231 cells.

RCAD Interacts with C53/LZAP and Forms a Large Complex

To further confirm our initial observation of the interactions among C53/LZAP, RCAD, and DDRGK1, we first performed co-immunoprecipitation (co-IP) assays using overexpressed proteins. As shown in Fig. 2A, both Myc-tagged RCAD and DDRGK1 were present in the Flag-tagged C53 immunoprecipitate. Endogenous RCAD and DDRGK1 were also co-immunoprecipitated with endogenous C53/LZAP in various cancer cells (Fig. 2B). Using co-IP assays, we briefly mapped the domains of both proteins for their interaction. It appeared that the N-terminal portion of RCAD (residues 1–400) was important for RCAD-C53 interaction, while the C (residues 269–506) terminal portion of C53/LZAP had a higher affinity to RCAD (Fig. 2, C and D). Additionally, we further mapped the interacting domains for the interaction between C53/LZAP and DDRGK1. As shown in Fig. 2, E and F, the N-terminal portion of DDRGK1 (1–114) was capable of binding to C53/LZAP, while both N- and C-fragments of C53/LZAP bound DDRGK1. It is not clear whether the C53-DDRGK1 interaction is direct or is mediated by RCAD.

To investigate the interaction of endogenous proteins, we performed a size exclusion assay to examine if they exist in a large protein complex. HeLa cell S100 fraction was subject to gel filtration chromatography, and the collected fractions were immunoblotted with specific antibodies. As shown in Fig. 2G, RCAD protein was exclusively present in a large complex that peaked around 550 kDa. Intriguingly, a small fraction of C53/LZAP was also present in a complex with similar size, while the remaining C53/LZAP appeared in the fractions ranging between 60 and 200 kDa. The co-peaking of endogenous RCAD and C53/LZAP in the same fractions of gel filtration strongly suggests that they may be the components of a large protein complex. In contrast to RCAD and C53/LZAP, DDRGK1 was absent in the S100 fraction but present in the membrane fraction (see below).

DDRGK1 Is an ER Protein Anchored by Its N-terminal Signal Peptide

Unlike RCAD and C53/LZAP, DDRGK1 has a putative N-terminal signal peptide, indicating that it may be an ER or secreted protein. Our efforts failed to detect any secreted DDRGK1 in culture medium. Meanwhile, immunofluorescence staining with our DDRGK1 antibody showed that DDRGK1 co-localized with the ER marker PDI (Protein Disulfide Isomerase), and the specificity of our DDRGK1 antibody was confirmed in DDRGK1 knockdown cells (Fig. 3A). Furthermore, overexpressed Myc-tagged DDRGK1 co-localized with another ER marker YFP-ER (Fig. 3B), and it appeared to be on the cytoplasmic side of the ER membrane (supplemental Fig. S3). Interestingly, DDRGK1 overexpression caused abnormal ER aggregation around the nucleus (Fig. 3B). Low-magnification electron micrographs showed that electron-dense “clouds” surrounded the nuclei of the cells transfected with DDRGK1, whereas such “clouds” were absent in the control cells. By examining the EM pictures, we found that 75% of DDRGK1-overexpressing cells contained abnormal ER aggregation, whereas none of the control cells displayed any abnormality (Fig. 3C). Examination at a high magnification revealed that the electron-dense structures corresponded to massive proliferation and aggregation of the ER network in DDRGK1-overexpressing cells (Fig. 3C). We also performed subcellular fractionation to biochemically confirm its ER localization. As shown in Fig. 3D, DDRGK1 was exclusively present in the enriched ER fraction, while C53/LZAP and RCAD were present in both cytosolic and ER fractions. Of note, RCAD appeared to be cleaved in the cytosolic fraction, and the possible cause remains unclear.

Except for its N-terminal putative signal peptide, DDRGK1 does not have any other recognizable transmembrane domains. We therefore speculated that this putative signal peptide may be responsible for its ER anchorage. As shown in Fig. 3E, deletion of the N-terminal 28 residues resulted in nuclear localization of AcGFP-DDRGK1 fusion protein, suggesting that the N-terminal signal peptide of DDRGK1 is indeed responsible for its ER localization.

RCAD Knockdown Causes Dramatic Reduction of C53/LZAP and DDRGK1 Proteins

To elucidate the biological function of RCAD, we examined the effect of RCAD depletion. As shown in Fig. 4A, two siRNAs effectively knocked down endogenous RCAD (more than 90%) in both HeLa and U2OS cells, and they did not significantly affect cell morphology and proliferation. However, RCAD knockdown in HeLa cells caused remarkable reduction of C53/LZAP (94% for RCAD siRNA-1 and 90% for RCAD siRNA-2, respectively) and DDRGK1 (94% for RCAD siRNA-1 and 2) (Fig. 4, A, left panel, and B). The similar phenotype was also observed in U2OS cells (Fig. 4A, right panel). More importantly, the reduction of protein levels was not caused by the change of mRNA levels (Fig. 4C). In comparison to the mock, transfection of either negative control or RCAD siRNAs modestly altered the mRNA levels of C53/LZAP and DDRGK1, and the alterations may be due to the nonspecific effect of siRNA transfection. Yet the variations on mRNA levels appeared random and were limited within the range of 2-fold change (Fig. 4C). Therefore, the dramatic drop of C53/LZAP and DDRGK1 protein levels in RCAD knockdown cells was unlikely to be caused by transcriptional change. Additionally, we examined if C53/LZAP can affect RCAD and DDRGK1 levels. In HeLa cells, C53/LZAP knockdown exerted a modest effect on the level of DDRGK1 (80% reduction of DDRGK1 in C53/LZAP knockdown cells) and minimal effect on RCAD (Fig. 4, A and B). In contrast, C53/LZAP knockdown in U2OS cells appeared to have more significant effect on both RCAD and DDRGK1, leading to reduction of both proteins. This result indicates that unlike RCAD, the effect of C53/LZAP appears to be cell type-specific.

RCAD Interferes with Ubiquitination of C53/LZAP and DDRGK1

To understand how RCAD influences the protein levels of C53/LZAP and DDRGK1, we first examined the kinetics of protein degradation. As shown in Fig. 5A, treatment of control HeLa cells with cycloheximide, a commonly used protein translation inhibitor, did not cause much degradation of either C53/LZAP or DDRGK1 during the time course of treatment. In contrast, cycloheximide treatment on RCAD knockdown cells led to more rapid degradation of C53/LZAP and DDRGK1 (Fig. 5A). Although we could not exclude the possibility that RCAD may affect microRNA-mediated pathways, our result provided indirect evidence that RCAD may influence protein stability of C53/LZAP and DDGRK1.

The ubiquitin/proteasome system (UPS) is the major cellular pathway for protein degradation. To further understand the molecular mechanism underlying RCAD function, we attempted to examine whether the UPS is involved in degradation of C53/LZAP and DDRGK1. HeLa cells were transfected with either control or RCAD siRNAs, and RCAD knockdown were confirmed by immunoblotting at 60 h post-transfection (Fig. 5B). The cells were then treated with proteasome inhibitor MG132 for various periods of time. As shown in Fig. 5B, in the cells transfected with negative control siRNA, MG132 treatment did not significantly affect the levels of three proteins. This result indicates that in the control cells, all three proteins are fairly stable, and the proteasome-mediated degradation of these proteins is not very robust. In contrast, protein levels of C53/LZAP and DDRGK1 were significantly elevated by MG132 treatment in RCAD knockdown cells, and the increase became obvious even at 2 h after treatment (Fig. 5B). This result strongly suggests that RCAD knockdown renders both C53/LZAP and DDRGK1 more susceptible to the UPS-mediated protein degradation. We further tested whether RCAD can affect ubiquitination of C53/LZAP and DDRGK1. As shown in Fig. 5C, both C53/LZAP and DDRGK1 can be ubiquitinated by HA-Ubiquitin, but the modification was dramatically inhibited by RCAD overexpression. This result strongly suggests that RCAD regulates degradation of C53/LZAP and DDRGK1 by affecting their ubiquitination.

RCAD Knockdown Causes Elevated NF-κB Activity and Cell Invasion

Wang et al. (3) have demonstrated that C53/LZAP acts as a suppressor of NF-κB signaling. Loss of C53/LZAP leads to the increase of NF-κB transcriptional activity and expression of its downstream targets such as MMP9. As shown above, RCAD knockdown resulted in a dramatic reduction of C53/LZAP level. Therefore, we speculated that RCAD may also influence the NF-κB activity. To test this hypothesis, we first used a luciferase reporter assay to measure the NF-κB activity in RCAD knockdown cells. In HeLa cells, knockdown of either RCAD or C53/LZAP led to elevation of both basal and TNFα-stimulated NF-κB activity (Fig. 6A). A similar result was obtained in U2OS cells (Fig. 6B). We consistently observed about a 2-fold increase of basal NF-κB activity (Fig. 6, A and B). Furthermore, like C53/LZAP, RCAD knockdown promoted cell invasion of U2OS cells (Fig. 6C), possibly due to the increased expression of matrix metalloprotease 9 (MMP9) (Fig. 6D). Taken together, our results suggest that RCAD plays an important role in NF-κB signaling.