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DAPK enhances DDX20 protein stability via suppression of TRIM25-mediated ubiquitination-based DDX20 degradation

Cancer Cell International volume 24, Article number: 382 (2024) Cite this article

Abstract

We have previously found that the DAPK-DDX20 signaling axis exerts an anti-cancer activity in hepatocellular carcinoma (HCC) by inhibiting the GTPase activity of CDC42, thereby reducing the invasive and migratory capabilities of cancer cells without affecting cell proliferation. DDX20 serves as an intermediate protein regulated by DAPK in the control of CDC42. Specifically, DAPK enhances DDX20 protein levels by suppressing DDX20 degradation. However, the mechanism underlying DAPK regulation of DDX20 remains unclear. In the current study, we discovered that DDX20 is degraded through the ubiquitin–proteasome pathway and identified TRIM25 as the E3 ubiquitin ligase of DDX20. TRIM25 mediates the proteasomal degradation of DDX20 by binding to, and ubiquitinating the 1-244 amino acid region of DDX20. Moreover, DAPK interacts with this 1-244 segment of DDX20, inhibiting its ubiquitination and enhancing its stability, despite the lack of direct physical interaction between DAPK and the 1-244 region of DDX20. Remarkably, DAPK, TRIM25, and DDX20 form a ternary protein complex in cells, and knockdown of TRIM25 leads to a reduction in the cellular levels of the binary DAPK-DDX20 complex, suggesting that TRIM25 acts as an important intermediate protein linking DAPK and DDX20. TRIM25 functions as an oncogene in liver cancer, as shRNA-mediated silencing of TRIM25 inhibits cell migration and invasion. Therefore, these novel findings of the interaction among these three proteins not only enhances our knowledge of the downstream molecular network of DAPK and its possible role in the development of HCC, but also provides potential druggable targets for the future development of novel anticancer drug therapeutics.

Introduction

Death-associated protein kinase (DAPK) is a central serine/threonine kinase that plays a vital role in various diseases including cancer, stroke, neurodegenerative diseases, etc. [1,2,3,4,5,6,7,8]. Since its discovery in 1995 [9], DAPK has been reported to participate in many physiologic and pathologic processes such as cellular necrosis, apoptosis and autophagy [10,11,12]. Recently, DAPK was found as one of the major regulators for the antiviral activity of pegylated IFN-α against hepatitis C virus (HCV) replication [13]. Multiple downstream targets of DAPK has been identified including p53 [14], tuberous sclerosis 2 (TSC2) [15, 16], NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) [17] as well as myosin light chain (MLC) [18]. In our previous study, we discovered the protein DEAD-box helicase 20 (DDX20), as a new downstream target of DAPK [19].

DDX20 (also known as Gemin3 or DP103) consists of 825 amino acids with 9 conserved motifs including the ASP-Glu-Ala-Asp motifs (or DEAD box motif) and belongs to DExD/H RNA helicase family [20]. DDX20 is involved in many cellular processes such as RNA metabolism including pre-mRNA splicing, ribosome biogenesis, RNA transport, translation initiation and RNA decay [20,21,22,23,24]. Our previous study revealed that DAPK suppressed hepatocellular carcinoma (HCC) cell migration and invasion which was dependent on its ability to upregulate DDX20 protein levels [19]. In contrast, DAPK did not affect HCC cell proliferation or colony formation. Further investigation demonstrated that DAPK enhanced the protein stability of DDX20, suggesting that DAPK could suppress the degradation of DDX20 protein [19]. Consistently, the proteasome inhibitor, MG132, increased the level and stability of the DDX20 protein, indicating that DDX20 is degraded via the ubiquitin-proteasomal pathway [19]. However, the mechanism underlying DDX20 protein degradation remained unknown, prompting us to decipher the mechanism underlying DDX20 protein degradation and how it is precisely regulated by DAPK.

In the present study, we identified Tripartite Motif Containing 25 (TRIM25) as the E3 ubiquitin ligase of DDX20 and investigated the molecular mechanisms underlying the regulatory interplays among DAPK, TRIM25, and DDX20. The current study expands our knowledge as to the downstream molecular network of DAPK and may possibly enhance our understanding of the molecular basis of HCC development.

Materials and methods

Cell culture

Hep3B cells were cultured in Eagle's Minimum Essential Medium [MEM, Biological Industries, Beit-Haemek, Israel (BI)]. SK-hep1, HEK-293T, SMMC-7721, HepG2(C3A) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, BI) supplemented with 10% fetal bovine serum (FBS, BI), 100 U/mL penicillin and 100 μg/mL streptomycin (1% Pen/Strep) (BBI life sciences, shanghai, China) at 37 °C in a humidified atmosphere containing 5% CO2.

All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and have been identified by short tandem repeats PCR genotyping.

Vector construction

The methodology for constructing the pCMV-DDX20-FLAG and pCDNA3.0-HA-DAPK plasmids has been previously described in our previous study [19]. The myc tag DNA sequence was linked to the cDNA sequence of TRIM25 and the fused sequence was cloned into the pCDH-CMV-MCS-EF1-puro vector through restriction enzyme site of EcoRI. The shRNAs were inserted into either PLVX-puro or pLKO.1-puro, with EcoRI and BamHI being the restriction enzyme sites for PLVX-puro and AgeI and EcoRI being those for pLKO.1-puro. The cDNA was inserted into the pCMV-N-mCherry vector (Beyotime) via restriction enzyme site BamH I, which was amplified from the pCMV-DDX20-FLAG vector through PCR. The full-length cDNA of DAPK was inserted into the pCDNA3.0-GFP vector through Kpn I and EcoR I site. Various mutant forms of DDX20 and in vitro protein-expressing plasmids were generated using specific primers with ClonExpress II One Step Cloning Kit (Additional file 1: Table S1). Further details can be found in the respective instructions manual. These plasmids were transferred into DH5α competent cells for further sequencing verification and use.

cDNA transfection or gene knockdowns in cells

VitaLGENE-II transfection reagent (Biocanaan, Kensington, USA) was utilized for the transfection of 1–2 plasmids, while Lipofectamine 3000 (Thermofisher, Waltham, USA) was employed for the co-transfection of more than two plasmids. Lipofectamine 2000 (Thermofisher, Waltham, USA) was utilized for the transfection of siRNAs and shRNAs (Additional file 1: Table S2). The transfection procedures were conducted following the corresponding instruction manuals, and all aforementioned transfections were performed under the following conditions: (1) Cells were transfected when confluence reached approximately 80% of the area of the petri dish. (2) Prior to transfection, the culture medium was replaced with serum-free medium 30 min in advance. (3) Following a period of 18–24 h after transfection, the original culture medium containing the transfections complex was replaced by fresh serum-containing medium. Cells were treated with cycloheximide (CHX) at a concentration of 100 μg/mL (150 µM; Sigma-Aldrich, Missouri, USA) for 4 h, G418 at a concentration of 0.38 μg/mL (Sigma-Aldrich), and MG132 at a concentration of 20 μM (Sigma-Aldrich) for 8 h. The DAPK kinase activity inhibitor TC-DAPK6 (MedChem Express, New Jersey, USA) was utilized at a concentration of 100 nM for 24 h.

RNA isolation and quantification

Total RNA was extracted using the Wizard® SV Gel and PCR Clean-Up System (Promega, Wisconsin, USA) while reverse transcription-PCR was conducted using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Kyoto, Japan) as per the manufacturer’s instructions. Quantitative RT-PCR analysis was performed to determine mRNA levels utilizing the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), following the manufacturer's instructions. Relative fold-change of target genes expression was normalized to the abundance of GAPDH and estimated by the 2^ΔΔCt method. The specific primer sequences were listed in Additional file 1: Table S1.

Cell lysis and western blotting

Cell lysis was prepared using RIPA protein extraction buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). The RIPA buffer consisted of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 0.1% SDS. Proteins present in this lysis buffer solution were subsequently separated by SDS-PAGE gel electrophoresis and then transferred onto a Cellulose nitrate membrane with pore size 0.2 μm (GE, Boston, USA). The membrane was blocked with 5% nonfat milk and then incubated with specific primary antibodies overnight at 4 °C. Thereafter, the membrane was incubated with secondary antibody for 1 h at 37 °C. Finally, the protein signals were visualized and quantified using Odyssey® CLx Infrared Imaging System (LI-COR Biosciences, Nebraska, USA). Information regarding the specific antibodies used in this study is deoicted in the key resources table.

Co-immunoprecipitation

Cells were lysed using 1 mLNET lysis buffer containing 50 mM Tris–HCl (PH = 8.0), 150 mM NaCl, 1% NP40, and 5 mM EDTA (PH = 8.0). The lysate was incubated on ice for 30 min with intermittent shaking to ensure sufficient cell lysis. Subsequently, the cell extracts were obtained through centrifugation at 16,000 rpm for 10 min. Before mixing with the cell extracts, 60 μL of protein A/G magnetitic beads were employed for each IP. The beads were incubated with 5% BSA for 8 h to block the non-specific binding sites on the magnetitic beads. Next, 20 μL blocked beads were added to the supernatants and incubated with rotation for 40 min to eliminate the non-specific binding proteins in cell extracts. The beads were then isolated and the liquid from the cell extract was transferred to a new test tube. The proteins in the new test tube were determined using the BCA protein Assay kit, and 1600 μg of total proteins were diluted in NET lysis buffer to a final volume of 500 μL. Subsequently the diluted proteins were incubated with 2 μg of antibody in the presence of 40 μL of blocked protein A/G magnetitic beads overnight at 4 °C with rotation. Following an overnight incubation, the beads were washed 6 times with the CO-IP buffer, and centrifuged at 5000 g for 2 min. The washed beads were resuspended in 50 μL SDS-PAGE buffer containing 5% SDS, 25% glycine, 0.01% bromophenol blue, and 200 mmol/L DTT, and eluted by boiling at 95 °C. A volume of 25 μL eluent was then separated by SDS-PAGE and immunoblotted.

Confocal microscopy

293T cells were transfected with expression plasmids for fluorescent fusion proteins, such as GFP-DAPK, mCherry-DDX20 or GFP-TRIM25 and subsequently analyzed using LSM780 Confocal Microscopy (Carl Zeiss, Oberkochen, Germany). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images were captured using a Plan-Apochrom at 63x/1.4 oil objective and processed using ZEN 2012 (Carl Zeiss).

In vitro binding assay

The cDNAs encoding DAPK (1–364) or DDX20 (1–244) were cloned into the pET28-HIS-sumo plasmid to generate pET28-HIS-sumo-HA-DAPK (1–364) or pET28-HIS-sumo-DDX20-FLAG (1–244) proteins, which were subsequently purified from BL21 bacteria cells transformed with pET28-HIS-sumo, pET28-HIS-sumo-HA-DAPK (1–364), or pET28-HIS-sumo-DDX20-FLAG (1–244). The purification procedure followed the instructions provided by the manufacturer of BeyGoldTM His-tag purification resin. For in vitro protein interaction experiments, BeaverBeads™ His-Tag Protein Purification beads (His beads) were employed. The HIS-sumo-DDX20 (1–244) was treated with the Ulp1 enzyme in order to remove the HIS-sumo tag. Purified HIS-sumo-HA-DAPK (1–364) and DDX20 (1–244) proteins were incubated at 4 °C for 2 h in pull-down buffer (600 μL), containing 50 mM Tris–Cl, 150 mM NaCl, 0.5 mM EDTA, 5% glycerol, and 50 mM ATP (pH7.5). The beads were then isolated and washed 4 times with the pull-down buffer, with each wash lasting for 5 min at 4 °C. At last, the protein on beads were eluted using pull-down buffer containing 500 mM imidazole and analyzed by western blotting.

Ubiquitination assay

In order to evaluate the ubiquitylation status of endogenous DDX20 in vivo, IP of endogenous DDX20 was performed from cells that treated with the proteasome inhibitor R-MG132 (MedChemExpress). Cell lysis and IP experiments were performed using TNTE buffer, which consisted of 50mM Tris-Cl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.5% (v/v) Triton X-100, 60 mM N-Ethylmaleimide, and protease inhibitor cocktail (Roche). The IP procedure was previously described [19]. The first round of immunoprecipitated beads underwent three washes with TNTE buffer and was then transferred into a new tube containing 60 μL of 1% SDS, followed by a 5-min boiling water bath treatment. This retrieved supernatant was expanded to 600 µL for a second IP with TNTE buffer following the same method as the first round.

For in vivo ubiquitylation assay, in order to detect the ubiquitylation status of exogenous protein, all ubiquitinated proteins were pulled down using the universal nickel magnetic beads and examined for the presence of the target protein. This methodology is applicable for cells that have been transfected with HIS-ubiquitination. The cell supernatant and nickel beads were incubated in ubiquitin binding buffer [comprising 6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole, 20 mM Sodium Phosphate, 500 mM NaCl, and 5 mM Imidazole (PH7.4)] at 4°C for 4 h. The cell supernatant was incubated with fresh beads twice. Following the two incubations, magnetic beads were collected and washed three times using a ubiquitin washing buffer (20 mM Na3PO, 500 mM NaCl, PH7.4), which includes 50 mM imidazole. The ubiquitin modified protein was eluted using 120 µL of ubiquitin washing buffer containing 500 mM imidazole. Western blot was performed on protein samples obtained from immunoprecipitation, nickel beads pull down, and total lysate samples to detect signals of substrate protein ubiquitination using corresponding antibodies.

Mass spectrometry analysis

The samples were prepared following Co-IP protocols. Subsequently, the samples were separated on SDS-PAGE and subjected to silver staining using Rapid silver dye for mass spectrometry kit (BBI life sciences).

The protein gels were cut into 0.5–0.7 mm cubes using a clean blade. The reduction reaction condition for protein cubes was 10 mM DTT, water bath at 56 °C for 30 min. Then, the blocks were put into 55 mM IAM (iodoacetamide)in a dark room at room temperature for 30 min with low speed of centrifugation for alkylation reaction. Decolorizing solution was used for washing the gel spots between each step. After the last washing step, 500 μL of acetonitrile were added to the gel blocks until the colloidal particles are completely white and the gel blocks were vacuum dry for 5 min. Then 0.01 μg/μL trypsin was added according to the volume of the gel. Next, the gel was ice bathed for 30 min before an appropriate amount of 25 mM NH4HCO3 PH8.0 enzymatic hydrolysis buffer was added. The gel was then enzymatic hydrolyzed overnight at 37 °C. After the enzyme hydrolysis was completed, 300 μL extract aliquot was added and the mixture was sonicated for 10 min and centrifuged at low speed, before the supernatant was collected and vacuum dried. The sample was then re-dissolved in 10–20 μL 0.2% trifluoroacetic acid and desalted by zip-tip chromatographic column (Merck Millipore). The dried peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analyzed using a Triple TOF 5600 plus mass spectrometer coupled with the Eksigent nanoLC system (AB SCIEX).

The original MS/MS data from the mass spectrometer was submitted to ProteinPilot (https://sciex.com.cn/products/software/proteinpilot-software, version 4.5) for data analysis. For protein identification, the Paragon algorithm in ProteinPilot was used to search the uniprot database. The parameters are set as follows: the instrument is TripleTOF 5600, and the cysteine is modified with iodoacetamide; the biological modification is selected as the ID focus. For the identified protein results, dependent on certain filtering criteria, peptides with an unused score > 1.3 (a credibility of more than 95%) are considered credible peptides and proteins containing at least one unique peptide are retaine.

CCK8 assay

The transfected cells (2 × 104 cells per well) were inoculated in 96-well plates and cell viability was estimated with CCK8 (TransGen Biotech, Beijing, China) at 0, 24, 48 and 72-h post transfection, respectively. More details can be found in our previous study [25].

Transwell assay

The Transwell assay was performed to evaluate the migration and invasion capacity of cells. Matrigel-coated membrane inserts were used for invasion assays, while inserts without Matrigel were used for migration assays. Following transfection with plasmids, SMMC-7721 cells were harvested and seeded at a density of 1 × 104 cells/well in medium containing 300 μL of serum-free medium in the upper chamber. 500 μL of medium containing 10% FBS was added to the lower chamber. After a specified incubation period, cells were fixed with 4% paraformaldehyde for 20 min and subsequently stained with 0.1% crystal violet for 30 min. The stained cells were then imaged and quantified using ImageJ software.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8. Unpaired two-tailed t-test was used for comparison between two groups, whereas one-way or two-way ANOVA was used for multigroup comparisons.

Results

Identification of TRIM25 as a ubiquitin ligase for DDX20

In order to investigate the proteasomal degradation of DDX20 protein, we first co-transfected 293T cells with DDX20-FLAG and HIS-Ubiquitin expression plasmids and performed IP assays using nickel magnetic beads purification or ani-FLAG antibody. The ubiquitination of exogenous DDX20-FLAG was detected using both HIS-IP (Fig. 1a) and FLAG IP assays (Additional file 1: Fig. S1A). Next, the ubiquitination of endogenous DDX20 was also confirmed via an IP assay using a DDX20-specific antibody and cell lysates isolated from Huh7 cells exposed to the proteasome inhibitor MG132 (Fig. 1b, Additional file 1: Fig. S1B). Then the K11 and K48 chain were found to be conjugated to DDX20 via Ni-bead IP using lysates of 293T cells co-transfected with DDX20-FLAG and various HIS-Ub mutants harboring the disruption of ubiquitin acceptor sites (Additional file 1: Fig. S1C, D).

Fig. 1
figure 1

Identification of TRIM25 as an E3 ubiquitin ligase of DDX20. A IB analysis of total ubiquitination of FLAG-tagged DDX20 in 293T cells co-transfected with plasmids expressing DDX20-FLAG; immunoprecipitates were pull down by nickel beads, and the ubiquitylated target protein was detected using an anti-FLAG antibody. B IB analysis of total ubiquitination of endogenous DDX20 in 293T cells incubated with the proteasome inhibitor MG132 or not. Whole cell lysates were immunoprecipitated with anti-DDX20 antibody and blotted with an anti-Ubiquitin antibody. The working concentration of MG132 was 20 μM. C Proteins interacting with DDX20 were identified by co-IP and mass spectrometry analyses using 293T cells as a cell model. The yellow star in Group: DDX means that DDX20-FLAG was overexpressed. D MYC-TRIM25 and DDX20-FLAG were transfected into 293T cells for 48 h, and then immunoprecipitation assays were performed with an anti-FLAG antibody, whereas MYC-TRIM25, DDX20-FLAG and GAPDH were detected with the indicated antibodies respectively. E Upper: Schematic representation of GFP tag fused (GFP-TRIM25) and mCherry tag fused DDX20(mCherry-DDX20); Lower: 293T cells were co-transfected with GFP-TRIM25 and DDX20-mCherry, the co-localization of GFP-TRIM25(green) and DDX20-mCherry (red) were detected. Scale bar denotes 10 μm. F 293T cells expressing HIS-Ubiquitin and DDX20-FLAG were transfected with MYC-TRIM25 or the empty myc-vector for 48 h. HIS ubiquitylation assays were performed as described in Materials and Methods. Left: The ubiquitylation level of DDX20-FLAG was detected using an anti-FLAG antibody. Right: Quantitative analysis of relative ubiquitinated DDX20-FLAG in cell expressing MYC-TRIM25 or not. G 293T cells were co-transfected with DDX20-FLAG and HIS-Ubiquitin together with anti-TRIM25 shRNA for 48 h. the polyubiquitination level of DDX20-FLAG was detected. INPUT: Whole cell lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. Relative ubiquitination level of target protein = gray value of ubiquitination band in each lane/(gray value of target protein/gray value of internal reference gene). *p < 0.05, between indicated groups. Data are shown as the mean ± SD of three independent experiments

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After confirming the ubiquitination of the DDX20 protein, we attempted to screen potential E3 ubiquitin ligases which may possibly target DDX20 as a substrate using IP combined with MS analysis (Additional file 1: Fig. S1E). The specificity of FLAG IP was confirmed by sliver staining and Western blotting (Fig. 1c). Wayne diagram results revealed that TRIM25 was the only overlapping E3 ubiquitin ligase when comparing MS results with the Biogrid database and published literature (Additional file 1: Fig. S1E).

The E3 ubiquitin ligase binds its substrates, thereby facilitating the conjugation of a ubiquitin chain to the substrate. To verify whether TRIM25 interacts with DDX20 in cells, we constructed plasmids overexpressing TRIM25 with a MYC tag and discovered that DDX20-FLAG and MYC-TRIM25 formed a complex in cells (Fig. 1d). We also generated expression plasmids harboring a GFP-TRIM25 and DDX20-mCherry fusion proteins and confirmed the co-localization of TRIM25 and DDX20 in cells using confocal microscopy (Fig. 1e). Moreover, protein interaction assay revealed that endogenous DDX20 also forms a complex with endogenous TRIM25 (Additional file 1: Fig. S1F). In addition, overexpression of MYC-TRIM25 increased the ubiquitination of the DDX20 protein (Fig. 1f), whereas knockdown of TRIM25 decreased the ubiquitination of DDX20 protein (Fig. 1g, Additional file 1: Fig. S1G). Collectively, these findings indicate that TRIM25 functions as an E3 ubiquitin ligase of DDX20. The effects of TRIM25 and DDX20 on cell function were further verified in SMMC-7721 cells, and it was found that the alterations of DDX20 and TRIM25 had no significant effect on cell proliferation (Additional file 1: Fig. S2A, E). When DDX20 was knocked down, the invasion and migration abilities of cancer cells were significantly enhanced (Additional file 1: Fig. S2B–D), indicating that DDX20 inhibited the metastasis of liver cancer cells. When TRIM25 was present at lower cellular levels, the invasion and migration capacities of cells markedly decreased (Additional file 1: Fig. S2F–H). In the TCGA_LIHC dataset, TRIM25 levels were higher in tumor tissues when compared to the adjacent normal tissues (Additional file 1: Fig. S2I), indicating that TRIM25 can promote cancer progression.

TRIM25 regulates DDX20 via its 1–244 domain

Next, we established lentiviral shRNA-mediated knockdown of TRIM25 in 293T cells and confirmed its efficiency through qPCR (Additional file 1: Fig. S3A). Expectedly, downregulation of TRIM25 led to an upregulation of endogenous DDX20 protein levels (Fig. 2a, b). Similar findings were observed in Hep3B cells (Additional file 1: Fig. S3B). Conversely, MYC-TRIM25 overexpression resulted in a significant reduction in the leves of both exogenous (Additional file 1: Fig. S3C) and endogenous (Additional file 1: Fig. S3D) DDX20 proteins. Moreover, the stability of DDX20 protein dropped significantly when MYC-TRIM25 was co-overexpressed (Fig. 2c). As for other potential E3 ubiquitin ligases, we searched the Biogrid database and also detected their impact on DDX20’s ubiquitination and protein levels in 293T cells (Additional file 1: Fig. S3E–G). Although most of them appeared to alter DDX20 ubiquitination, DDX20 protein level was not significantly increased when these E3 ubiquitin ligases were knocked down.

Fig. 2
figure 2

TRIM25 regulates DDX20 via its 1–244 domain. A, B Western blot analysis of the levels of DDX20 in 293T cells with control (SCR shRNA) or knockdown of TRIM25. The relative DDX20 protein level is illustrated graphically. C 293T cells co-transfected with the mentioned plasmids for 48 h were incubated with CHX (150 μM) for the indicated times, and the cell lysates were subjected to Western blot analysis of DDX20-FALG. Relative levels of DDX20-FLAG proteins are depicted graphically (right side). D Truncated forms of DDX20-FLAG were constructed based on its functional domains. Immunofluorescence experiments were performed to determine the sub-cellular localization of each truncated protein in the cells, and the superscript letters a-d, represent DDX20-FLAG(1–244), DDX20-FLAG(245–825), DDX20-FLAG(1–406), and DDX20-FLAG(407–825), respectively. A FLAG tag specific antibody was used to follow these proteins. E 293T cells were co-transfected with HIS-Ubiquitin and full-length or truncated forms of DDX20-FLAG, precipitates were pull down by nickel beads, and ubiquitylated target protein was detected using an anti-FLAG antibody. F 293T cells were co-transfected with truncated forms of DDX20-FLAG and MYC-TRIM25, and immunoprecipitation was performed with an anti-FLAG antibody. TRIM25, DDX20 and GAPDH proteins were detected by Western blotting using the respective antibodies. G Effect of MYC-TRIM25 overexpression on the ubiquitination of the DDX20-FLAG 1–244 domain in 293T cells. Right: Quantitative analysis of relative ubiquitinated DDX20-FLAG (1–244). INPUT: The whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. *p < 0.05, between the indicated groups. Data are shown as the mean ± SD of three independent experiments. Relative ubiquitination level of target protein = gray value of ubiquitination band in each lane/(gray value of target protein/gray value of internal reference gene)

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After constructing expression plasmids containing sequential structural domains of DDX20 including: DDX20-FLAG (1–244), DDX20-FLAG (245–825), DDX20-FLAG (1–406) and DDX20-FLAG (407–825) (Fig. 2d), we examined ubiquitination events that may occur in various structural domains of DDX20 (Fig. 2e; Additional file 1: Fig. S4, A and B). To determine which domain of DDX20 is regulated by TRIM25, we examined the interactions between different DDX20 truncation mutants and TRIM25. The region spanning amino acids 1 to 244 of DDX20 was found to be sufficient to form an intracellular complex with TRIM25 (Fig. 2f) and overexpression of MYC-TRIM25 enhanced the ubiquitination of DDX20-FLAG (1–244) (Fig. 2g).

DAPK suppresses the ubiquitination on the 1–244 fragment of DDX20

In our previous investigation, we found that DAPK did not exert an impact on the mRNA level of DDX20, but enhanced the stability of DDX20 protein [19]. Given that DDX20 undergoes degradation via the proteasome pathway, we postulated that DAPK positively regulates DDX20 stability by disrupting its ubiquitination. To corroborate this hypothesis, an HA-DAPK expression plasmid was co-transfected with DDX20-FLAG; the ubiquitination levels of DDX20 were significantly reduced upon DAPK overexpression (Fig. 3a; Additional file 1: Fig. S4D). Similar results were obtained for the endogenous DDX20 protein (Fig. 3b; Additional file 1: Fig. S4C). Conversely, when DAPK was knocked down, the relative ubiquitination level of endogenous DDX20 was significantly increased (Additional file 1: Fig. S4E).

Fig. 3
figure 3

DAPK suppresses the ubiquitination on the 1–244 domain of DDX20. A Effect of HA-DAPK overexpression on DDX20-FLAG ubiquitination in 293T cells. Precipitates were pulled down by nickel beads, and ubiquitylated target protein was detected using an anti-FLAG antibody. The proteins of HA-DAPK, DDX20-FLAG and GAPDH were detected by Western blotting with respective antibodies. Right: Quantitative analysis of relative ubiquitinated DDX20-FLAG in cells expressing HA-DAPK or not. B Effect of HA-DAPK overexpression on the ubiquitination of endogenous DDX20 in 293T cells. Whole cell lysates (containing HA-DAPK) were immunoprecipitated using an anti-DDX20 antibody and blotted with an anti-Ubiquitin antibody. The working concentration of MG132 was 20 μM. C The co-localization of GFP-DAPK (green) and DDX20-mcherry (red) were detected. Scale bar denotes 10 μm. D, E HA-DAPK and DDX20-FLAG plasmids were transfected into 293T cells. Protein complexes were co-immunoprecipitated with anti-HA (D)or Flag antibodies (E), and DDX20 and DAPK were detected, respectively. F Co-immunoprecipitation assay of HA-DAPK and different DDX20 domains in the whole cell lysates of 293T cells expressing indicated proteins. G Effect of HA-DAPK overexpression on the different domains of DDX20 in 293T cells. H Effect of HA-DAPK overexpression on the ubiquitination of truncated domains DDX20 in 293T cells. INPUT: Whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. ***p < 0.001, between the indicated groups

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We then set out to investigate the mechanisms underlying the mode by which DAPK regulates DDX20 ubiquitination. Co-localization of GFP-DAPK with DDX20-mCherry was observed using confocal microscopy (Fig. 3c). Moreover, irrespective of which tag was actually used for co-immunoprecipitation and despite the use of exogenous protein co-precipitation DAPK and DDX20 formed protein complexes (Fig. 3d, e). The truncated forms of DDX20 were employed to identify the regulatory regions of DAPK interacting with DDX20. Amino acids 1–244 at the N-terminal region of DDX20 constituted the region capable of forming a complex with DAPK in cells (Fig. 3f; Additional file 1: Fig. S4F and G). Furthermore, overexpression of DAPK increased the protein levels of both the DDX20 1–244 and the 1–406 fragments but had no effect on the DDX20 245–825 or the DDX20 407–825 regions (Fig. 3g; Additional file 1: Fig. S4H, I). A ubiquitination assay indicated that increased levels of DAPK could diminish the ubiquitination of DDX20-FLAG (1–244) (Fig. 3h; Additional file 1: Fig. S4J). Collectively, these findings suggest that amino acids 1–244 at the N-terminal region of DDX20 constitute the domain capable of forming a complex with DAPK in cells; furthermore, DAPK enhanced DDX20 levels by markedly reducing the ubiquitination of the latter.

The kinase activity of DAPK is required for the regulation of DDX20.

To identify the specific region of DAPK that regulates DDX20, we generated various expression plasmids harboring different DAPK truncations based on the different functional regions of DAPK, including HA-DAPK (1–364) containing the DAPK kinase region and calmodulin-binding region, HA-DAPK lacking the ROC-COR region, the Death domain, and the Ser tail-enriched HA-DAPK (1–666), as well as HA-DAPK lacking the Death domain and Ser-rich tail (1–1313) (Fig. 4a) [26]. When co-expressed with DDX20-FLAG, each truncation mutant was pulled down from cell lysates using an anti-HA antibody, suggesting that all of these HA-DAPK fragments could form a complex with DDX20-FLAG in cells (Fig. 4b). Furthermore, DDX20 retained its binding ability to each truncation mutant (Fig. 4c). Our findings indicated that DAPK can form a complex with DDX20 via its N-terminal domain encompassing amino acids 1–364 of DAPK in cells.

Fig. 4
figure 4

The kinase domain of DAPK is required for its regulation on DDX20. A Schematic representation of five HA-fused DAPK constructs containing amino acids 1–364, 1–666, 1–1313 and 1–1430 (full length). B–D Co-immunoprecipitation of DDX20-FLAG and the different DAPK domains in the whole cell lysates of 293T cells expressing the indicated proteins. Anti-HA or FLAG antibody were used for immunoprecipitated and the proteins in the precipitates were quantified using Western blotting. E Effect of overexpression of HA-DAPK or HA-DAPK(K42A), a kinase dead mutant of DAPK, on the ubiquitination of DDX20-FLAG in 293T cells. F, G Effect of DAPK kinase inhibitor (TC-DAPK6, 100 nM) on the expression of DDX20 in HCC cells. The relative DDX20 protein level is illustrated graphically(G). INPUT: The whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. *p < 0.05, between the indicated groups. Data are shown as the mean ± SD of three independent experiments

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To further corroborate this conclusion, we co-expressed a deletion mutant of HA-DAPK consisting of amino acids 365–1430 along with FLAG-tagged DDX20 in cells. Neither IP assays using anti-HA nor anti-FLAG antibodies detected any interaction between these two proteins (Fig. 4d), suggesting that the kinase region of DAPK is required for complex formation with DDX20. We then investigated the impact of DAPK kinase activity on DDX20 ubiquitination. Compared to the wild-type HA-DAPK, overexpression of the kinase-dead K42A HA-DAPK mutant in cells resulted in decreased levels of DDX20-FLAG protein and increased levels of its ubiquitinated form (Fig. 4e; Additional file 1: Fig. S5A). Treatment with TC-DAPK6, an inhibitor of DAPK kinase activity, significantly reduced DDX20 levels in four hepatocellular carcinoma cells lines (Fig. 4f, g). Taken together, these findings demonstrate that the kinase activity of DAPK is critical for enhancing the stability of the DDX20 protein.

DAPK regulates DDX20 via TRIM25

To investigate the direct binding between DAPK and DDX20 in cells, we employed an E. coli in vitro protein expression system to express and purify the crucial regions of both proteins (Fig. 5a, b). Following removal of the HIS-sumo tag of HIS-sumo-DDX20-FLAG (1–244) by Ulp1 enzyme, we performed IP using purified DDX20-FLAG (1–244) and HIS-sumo DAPK (1–364) proteins. While a slight binding of DDX20-FLAG (1–244) to His beads was observed, no detectable DDX20-FLAG (1–244) was found in the eluate of HIS-sumo-DAPK (1–364) and DDX20 (1–244) mixture (Fig. 5c). Thus, the DAPK (1–364) region does not bind directly to the DDX20 (1–244) region in vitro, which can suggest that the DAPK may suppress DDX20 protein degradation via regulation of an intermediate protein.

Fig. 5
figure 5

DAPK and DDX20 form a protein complex via TRIM25 in cells. A, B The Coomassie blue staining gel of purified protein. Red asterisks indicate the HIS-sumo DAPK 364 which is a HIS-sumo fused DAPK domain containing 1–364 amino acids (A), 0: bacteriophage lysis solution, 1: supernatant after sonication lysis and centrifugation, 2: molecular weight marker, 3–4: protein solution collected after 20 mM imidazole elution, 5–7: protein solution collected after 50 mM imidazole elution, 8: protein solution collected after 500 mM imidazole elution. Yellow asterisks indicate the HIS-sumo DDX20 244 which is a HIS-sumo fused DDX20 domain containing 1–244 amino acids. B. 0: molecular weight marker, 1: bacteriophage lysis solution, 2: supernatant after high-speed centrifugation of bacteriophage ultrasonically lysed, 3–4: protein solution collected after 20 mM imidazole elution, 5–8: protein solution collected after 50 mM imidazole elution, 9: protein solution collected after 500 mM imidazole elution. C In vitro HIS pulldown assay analysis of the interaction of purified protein DAPK and DDX20. D Co-immunoprecipitation assay of DDX20-FLAG and HA-DAPK in whole cell lysates of 293T cells transfected with an shRNA targeting TRIM25. E Co-immunoprecipitated exogenous HA-DAPK and MYC-TRIM25 proteins were detected by Western blotting with anti-HA and anti-Myc antibodies, respectively. F Co-IP assay detecting the association of HA-DAPK, MYC-TRIM25 and DDX20-FLAG in 293T cells expressing the indicated plasmids. Whole cell lysates were immunoprecipitated with anti-FLAG antibody. G Effect of overexpression of HA-DAPK or HA-DAPK(K42A), a kinase dead mutant of DAPK, on the ubiquitination of DDX20-FLAG in 293T cells. HIS ubiquitylation assays were performed as described in Materials and Methods. H Western blotting of TRIM25 and GAPDH proteins in 293T cells transfected with HA-DAPK or HA-DAPK(K42A). Whole cell lysates were treated with phosphorylase inhibitor and N-Ethylmaleimide. INPUT: Whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. *p < 0.05, between the indicated groups. Data are shown as the mean ± SD of three independent experiments

Full size image

The observation that DAPK and TRIM25 form a complex with, and regulate DDX20 on the same region in cells, suggested that TRIM25 may possibly be the intermediate protein “linker” between DDX20 and DAPK. Consistently, knockdown of TRIM25 in cells co-expressing HA-DAPK and DDX20-FLAG resulted in a marked decrease in the formation of the DAPK-DDX20 complex (Fig. 5d). Moreover, HA-DAPK formed a complex with MYC-TRIM25 in cells (Fig. 5e); importantly, the protein complexes containing DAPK, TRIM25 and DDX20 also existed (Fig. 5f), hence supporting our hypothesis that TRIM25 acts as a possible intermediate protein “linker” of DAPK and DDX20.

Surprisingly, however, we found that overexpression of DAPK upregulated TRIM25 protein level (Fig. 5f). Considering that the self-ubiquitination of TRIM25 may contribute to its degradation, we investigated the impact of DAPK overexpression on the ubiquitination and TRIM25 protein levels. The ubiquitination of TRIM25 was downregulated by overexpression of the wild type DAPK, but not by its kinase dead mutant DAPK (K42A) (Fig. 5g; Additional file 1: Fig. S5B). Conversely, the protein level of TRIM25 was upregulated by DAPK, but not by the kinase-dead DAPK (K42A) (Fig. 5h). This finding was similar to the effect of DAPK on DDX20, suggesting that DAPK is likely to inhibit the E3 ubiquitin ligase activity of TRIM25, thereby enhancing the stability of these two proteins.

Discussion

DEAD/H-box proteins constitute the largest RNA helicase family in the mammalian genome and have been a major focus of research since their discovery in the late 1980s [27]. These important proteins play a central role in RNA metabolism, gene expression, signal transduction, programmed cell death, and immune responses to bacterial and viral pathogens [27]. Their ubiquitination plays a regulatory role in the activity, localization, and interaction with other protein factors. In recent years, ubiquitination processes and ubiquitin ligases corresponding to several members of RNA helicases have been reported [28,29,30,31,32,33]. Among them, the most well-known is the RIG-I-like receptor, a viral RNA receptor with a helicase domain that triggers a series of antiviral immune responses by interacting with homologous TRIM/TRIM-like E3 ligases [28, 34, 35]. KATO K’s research found that this interaction role may apply to diverse RNA helicases including RNA dead box helicase and TRIM/TRIM-like proteins [35].

To date, the degradation of the DDX20 protein has not been reported. In this respect, in our previous study, we found that DDX20 is degraded via the ubiquitin proteasomal system [19]. In the current study, we identified TRIM25 as the E3 ubiquitin ligase for DDX20. TRIM25 is a classic TRIM protein that consists of a RING domain, two B-box domains, a CC dimerization domain as well as a C-terminal SPRY domain [36]. Consequently, our results further confirm the conserved binding roles between TRIM proteins and deconjugating enzymes.

TRIM25 is an important member of the TRIM family and is critical for defending cells from infection by RNA viruses [28, 37]. Studies have demonstrated that TRIM25 plays a significant role in the regulation of melanoma differentiation-associated gene 5 (MDA5)-mediated signaling through tumor necrosis factor receptor-associated factor 6 (TRAF6). The latter is an E3 ubiquitin-protein ligase, in the context of innate immunity, resulting in the activation of NF-κB [38]. Furthermore, TRIM25 has been found to augment the functionality of zinc finger antiviral protein (ZAP) [39, 40]. Conversely, TRIM25 can also exert inhibitory effects on RIG-I signaling [41, 42]. TRIM25 overexpression inhibited HBV replication by promoting HBx degradation and enhanced the recognition of pregenomic RNA (pgRNA) by the retinoic acid-inducible gene I (RIG-I) [43]. DDX20 has been reported to suppress the activity of NF-κB, thereby inducing type I IFN expression [44, 45]. As for DDX20, this protein was overexpressed in HBV-related HCC patients as shown in our previous study as well as in patient samples in PDC database (https://proteomic.datacommons.cancer.gov/pdc/).

Using proteomic profiles of HBV-related HCC tissues and normal tissues from PDC database, we found that TRIM25 was expressed at low levels in HBV-infected cancer patients, wheeras DDX20 was highly expressed. In addition, the role of DDX20 in viral infection has been revealed [46], so we hypothesized that DDX20 may be an alternative pathway for TRIM25 to accelerate the innate immune response.

Herein we have further shown that both DAPK and DDX20 act as tumor suppressors in HCC. The fact that TRIM25 negatively regulates DDX20 indicates that the former may act as an oncogene in HCC. Indeed, several studies have reported that TRIM25 promotes HCC cell survival via the Keap-1-Nrf2 or GRP78 pathways [47, 48]. Considering that DDX20 is able to suppress HCC cell migration and invasion [19, 49], it is possible that TRIM25 also participates in HCC metastasis via downregulation of DDX20. In addition, we have shown that DAPK exerts its tumor suppressive function in HCC cells via DDX20 [19, 50]. The formation of transitional ternary protein complex with ubiquitin E3 ligase as the core protein plays an important role in the process of protein regulation. Regulatory proteins can modulate the stability of substrate proteins by altering the binding of E3 ligases to their substrate proteins, thereby modulating substrate protein stability [51, 52] Therefore, we hypothesized that DAPK could enhance DDX20 protein stability and increase DDX20 protein expression by reducing the recruitment of TRIM25 to DDX20. However, it is unclear whether this functional dependence on DDX20 is completely mediated by the ability of DAPK to inhibit the E3 ubiquitin ligase activity of TRIM25. Further studies are warranted to explore the possible impact of this DAPK-TRIM25-DDX20 axis in the development of HCC. Our discovery of this DAPK-TRIM25-DDX20 pathway may possibly enhance our knowledge of the innate immune network and the complex hepatocarcinogenesis process. As DDX20 is involved in various cancers and regulates multiple cellular processes [44, 53, 54], the intervention of its degradation process by TRIM25 induced by DAPK might be a potential therapeutic target in the future.

Conclusion

In summary, our current study revealed for the first time that TRIM25 is an E3 ubiquitin ligase for DDX20. Furthermore, DAPK upregulates DDX20 protein stability via suppression of TRIM25-mediated DDX20 ubiquitination and degradation.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

DAPK:

Death-associated protein kinase

DDX20:

DEAD-box helicase 20 (DDX20)

HCC:

Hepatocellular carcinoma

HCV:

Hepatitis C virus

MDA5:

Melanoma differentiation-associated gene 5

MLC:

Myosin light chain

NLRP3:

NACHT, LRR, and PYD domains-containing protein 3

TRAF6:

Tumor necrosis factor receptor-associated factor 6

TRIM25:

Tripartite motif containing 25

TSC2:

Tuberous sclerosis 2

ZAP:

Zinc finger antiviral protein

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Acknowledgements

We thank Peng Lv for technical help with ubiquitination studies and for sharing the relevant expression plasmids. We appreciate the great help from Innovation and Transformation Center, Fujian University of Traditional Chinese Medicine.

Funding

This work was supported by funds from the National Science Foundation for Young Scientists of China (82003095 to Q.W.), the Natural Science Foundation of Fujian Province (82023N5012 to Y.L.), and Youth Science and Technology Innovation Talent Cultivation Program of FJTCM (XQC2023007 to Y.L.).

Author information

Author notes

  1. Yan Ye, Xiuli Zhang and Chenyi Wang have contributed equally to this work.

Authors and Affiliations

  1. College of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, Fujian, China

    Yan Ye, Xiuli Zhang, Qingshui Wang & Yao Lin

  2. The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, 350000, Fujian, China

    Yan Ye, Xiuli Zhang, Qingshui Wang & Yao Lin

  3. Ganzhou Key Laboratory of Molecular Medicine, the Affiliated Ganzhou Hospital of Nanchang University, Ganzhou, 341000, Jiangxi, China

    Yan Ye

  4. College of Life Sciences, Fujian Normal University, Fuzhou, 350117, Fujian, China

    Chenyi Wang, Yide Huang, Luyun Xu, Hongxia Liu & Ke Li

  5. Department of Central Laboratory, Fuzhou Second Hospital, Fuzhou, 350007, Fujian, China

    Nannan Liu & Tao Zhang

  6. The Fred Wyszkowski Cancer Research Laboratory, Faculty of Biology, Technion-Israel Institute of Technology, 3200003, Haifa, Israel

    Yehuda G. Assaraf

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Contributions

YY, XZ, TZ, and YL designed the experiments. YY, XZ, CW, and QW performed most of the experiments and analyzed the data. LX, HL, KL, and NL performed part of the Co-IP experiments. YY, YH, and XZ wrote the manuscript with input from QW, YGA, and YL. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Qingshui Wang, Tao Zhang, Yehuda G. Assaraf or Yao Lin.

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Ye, Y., Zhang, X., Wang, C. et al. DAPK enhances DDX20 protein stability via suppression of TRIM25-mediated ubiquitination-based DDX20 degradation. Cancer Cell Int 24, 382 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-024-03567-z

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-024-03567-z

Keywords

Cancer Cell International

ISSN: 1475-2867

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