The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells
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CFLARL, also known as c-FLIPL, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLARL can be mono-methylated. However, the precise role of arginine methyltransferase of CFLARL remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.
Lung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLARL ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.
We show that PRMT5 up-regulated the protein levels of CFLARL by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLARL by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLARL and ITCH, which has been identified as an E3 ubiquitin ligase of CFLARL, while overexpressed PRMT1 enhances the interaction between CFLARL and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLARL as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLARL in NSCLC cells.
PRMT5 and PRMT1 mediate the distinct effects on CFLARL degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLARL degradation in human NSCLC cells.
KeywordsCFLAR PRMT1 PRMT5 ITCH Apoptosis
CASP8 and FADD like apoptosis regulator
death-inducing signaling complex
Itchy E3 ubiquitin protain ligase
non-small cell lung cancer
poly ADP-ribose polymerase
Protein arginine methyltransferase 1
Protein arginine methyltransferase 5
CFLAR, which is a CASP8 and FADD-like apoptosis regulator, also known as c-FLIP, is an important regulatory protein in the extrinsic apoptotic pathway in mammalian cells. Several transcript variants encoding different isoforms have been reported. The short form, i.e., CFLARs (c-FLIPS), contains two N-terminal death effector domains (DED), whereas the long form, i.e., CFLARL (c-FLIPL), contains an additional pseudo-caspase domain in which the active center cysteine residue that confers the proteolytic activity of caspases is substituted by a tyrosine residue . CFLARL can inhibit and prevent apoptosis by interfering with procaspase 8/10 for binding to the FADD domain to decrease caspase 8 activation. This binding prevents further death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade . High levels of CFLARL have been found in many different types of human cancers, and the excessive expression of the protein indicates a high degree of tumor malignancy . In the clinic, CFLARL can be used as an independent adverse prognostic biomarker of colorectal cancer (CRC) . Many chemotherapeutic agents have been shown to down-regulate CFLARL at the protein and mRNA level. Silencing its expression has been shown to facilitate apoptosis in chemotherapeutic agent-induced apoptosis. Therefore, CFLARL is a promising therapeutic target in some cancer treatments.
CFLARL can be regulated at both the transcriptional and post-translational level. NF-κB can induce the up-regulation of CFLARL at the mRNA and protein level and inhibit Fas, TNFR1 and TRAIL receptor-induced apoptosis . c-MYC, FOXO3a, and c-Fos inhibit the transcription of CFLARL [6, 7]. Additionally, CFLARL has been shown to be down-regulated following treatment with compounds such as cycloheximide (CHX) and anisomycin [8, 9]. As the half-life of CFLARL is short, the ubiquitin-proteasome system plays an important role in regulating CFLARL degradation and stability. CFLARs is highly prone to ubiquitination and degradation likely due to its unique C-terminal tail . The E3 ubiquitin ligase ITCH is thought to be responsible for CFLARL ubiquitination and degradation . ITCH has also been shown to be an important regulator of CFLARs ubiquitination and stability [12, 13]. Furthermore, phosphorylation events play a vital role in the regulation of CFLARL protein levels; for example, the serine residue 273 of CFLARL is phosphorylated by AKT, which is important for the reduction of CFLARL via an ITCH-dependent mechanism . The proteins that interact with CFLARL can also affect its stability. Recently, XRCC6 was shown to interact with, stabilize, and protect CFLARL from ubiquitin-proteasomal degradation . XRCC6 usually forms a stable heterodimer consisting of two subunits (XRCC6 and XRCC5) . Evidence suggests that XRCC proteins modulate ATM activity following DNA damage . XRCC6 acts as an ATP-dependent single strand DNA helicase and has been found to play an important role in immune system disorders, aging and carcinogenesis .
Post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, ADP-methylation, and SUMOylation, are highly important for the regulation of protein functioning in eukaryotic cells. Among these post-translational modifications, protein arginine methylation governs many cellular processes, such as cell growth, proliferation, differentiation and development . The PRMT family members play a pivotal role in the regulation of the arginine methylation of both histones and other cellular proteins . Three distinct types of methylated arginine residues, namely, omega-NG-mono-methylarginine (MMA), symmetric omega-NG,NG-dimethylarginine (sDMA), and asymmetric omega-NG,NG-dimethylarginine (aDMA), have been identified in mammalian cells . The PRMT enzymes are classified into two groups depending on the type of modification they catalyze. Type I PRMT enzymes (PRMT1–4, PRMT6 and PRMT8) generate MMA and aDMA, whereas type II PRMT enzymes (PRMT5, PRMT7 and PRMT9) catalyze MMA and sDMA . PRMT5 is a type II methyltransferase that modulates cell growth and transformation. PRMT5 hypermethylates histones H3R8 and H4R3 in promoters and restrains the cell cycle and tumor suppressor genes [23, 24, 25]. PRMT5 interacts with and methylates P53 at R333, R335, and R337 when DNA is damaged, inhibiting oligomerization between MDM2 and P53 [26, 27]. In particular, PRMT5 can co-localize with EGFR and regulate its monomethylation. R1175 methylation modulates EGF-induced EGFR trans- autophosphorylation at Y1173 .
PRMT1 was the first mammalian protein arginine methyltransferase identified . Most protein arginine methylation is catalyzed by PRMT1 . PRMT1 preferentially methylates arginine residues flanked by one or more glycine residues . According to its three-dimensional structure, PRMT1 is active as a homodimer . Moreover, PRMT1 has been reported to be a negative protein in Wnt/beta-catenin signaling by the methylation of Axin . However, the mechanism by which the members of the protein arginine methyltransferase family, including PRMT5 and PRMT1, modulate apoptosis remains to be elucidated. Our work suggests that PRMT5 and PRMT1 regulate apoptosis by affecting CFLARL turnover.
Cell lines and cell culture
The lung cancer cell lines A549, H157, H460 and H1299 were cultured in RPMI 1640 supplemented with 5% (v/v) NBCS. The HEK293FT cell line was cultured in DMEM medium supplemented with 5% (v/v) newborn calf serum. All cell lines were originally obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air. The cells we used are routinely authenticated and tested for mycoplasma contamination.
Antibodies and reagents
Doxorubicin and pemetrexed powder was purchased from Sigma Aldrich (Merck, Darmstadt, Germany) and diluted in dimethyl sulfoxide (DMSO). Stock solutions were stored at − 20 °C and diluted to the desired concentrations with growth medium before use. The PRMT5 (P4847), PRMT1 (G1544) and FLAG (F7425) antibodies were purchased from Sigma Aldrich (America). The CFLARL (ALX-804-961-0100) antibody was purchased from Enzo Biochem. The monomethyl arginine (8015S), CASP8 (9746 L) and PARP-1 (#9542) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts, US). The symmetric dimethyl arginine and asymmetric dimethyl arginine antibodies were purchased from Millipore.
Western blot analysis
The preparation of whole-cell protein lysates and procedures used for the western blot analysis have been previously described . The cells were harvested and rinsed with pre-chilled PBS. Then, the cells were lysed and centrifuged at 4 °C for 15 min. Samples of the whole-cell protein lysates (35 μg) were electrophoresed on a 12% denaturing polyacrylamide slab gel and then transferred to a polyvinylidene fluoride (PVDF) membrane by electroblotting. The proteins were probed with the appropriate primary antibodies and subsequently the secondary antibodies. Antibody binding was detected by an HRP system according to the manufacturer’s protocol.
The siRNAs were synthesized by Boshang. The small interfering RNA (siRNA) duplexes used for the controls have been previously described . The PRMT5 siRNA duplexes target the sequences 5’-GCCCAGUUUGAGAUGCCUU-3′ (#1) and 5’-CCGCUAUUGCACCUUGGAA-3′ (#2). The PRMT1 siRNA duplexes target the sequences 5’-CCACCAGCCCCGAGUCCCC-3′ (#1) and 5’-ACCGCAACUCCAUGUUUCA-3′ (#2). The negative control was 5’-UUCUCCGAACGUGUCACGU-3′. The siRNA transfections were carried out with the jet PRIME® siRNA Transfection Reagent (Polyplus) following the manufacturer’s instructions.
Construction of the plasmids
The PRMT5 gene was amplified by PCR from H1299 cell genomic DNA using the following primers: MYC-PRMT5 sense: 5’-CGGATCCGCCGCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGGCGGCGATGGCGGTCG-3′, PRMT5 sense: CGGATCCGCCGCCACCATGGCGGCGATGGCGGTCG antisense: 5’-CGAATTCCTAGAGGCCAATGGTATATGAG-3′; MYC-PRMT1 sense: 5’-CGGATCCGCCGCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGGCGGCAGCCGAGGCCG-3′; PRMT1 (variant 1) sense: 5’-CGGATCCGCCGCCACCATGGCGGCAGCCGAGGCCG-3′ antisense: 5’-CGAATTCTCAGCGCATCCGGTAGTCGGT-3′; CFLARL sense: 5’-GGGTACCGCCGCCACCATGTCTGCTGAAGTCATCCAT-3’ CFLARL antisense: 5’-CCGCTCGAGTTATGTGTAGGAGAGGATAAG-3′; HA-ITCH sense: 5’-CGGATCCGCCGCCACCATGTACCCCTACGACGTGCCCGACTACGCCTCTGACAGTGGATCACAACT-3′; and ITCH sense: 5’-CGGATCCGCCGCCACCATGTCTGACAGTGGATCACAACT-3′ antisense: 5’-CGGGCCCTTACTCTTGTCCAAATCCTTCTG-3′. The plasmid construction procedures have been previously described [35, 36].
Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM Na2EDTA; 1 mM EGTA; 2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na3VO4; 0.5% Triton) on ice for 30 min then purified via centrifugation for 15 min at 4 °C. The supernatants were incubated with antibody at 4 °C for 1 h. Then the mixture was incubated with protein A beads (ThermoFisher) at 4 °C for 2 h. The beads were washed twice with 1 ml of lysis buffer. 20 μl 2 × SDS buffer were added for elution (100 °C, 10 min). Samples were centrifuged for western blot analysis.
GST pull-down assay
The HEK293FT cells were collected, lysed in immunoprecipitation lysis buffer and incubated on ice for 30 min. The lysates were purified via centrifugation for 15 min at 4 °C. The supernatants were incubated with glutathione sepharose at 4 °C overnight. The beads were washed 2 times with 900 μl of IP lysis (1% PIC) buffer, followed by incubation with 2XSDS at 100 °C for 10 min. Then, the beads were centrifuged for 5 min at room temperature. Finally, the supernatants were thoroughly collected for SDS-PAGE and western blot analysis.
Flow cytometry analysis
Annexin V-FITC Apoptosis Detection Kit (Biobox Biotech, Nanjing, China) was used for cell apoptosis analysis according to the manufacture’s protocol.
GraghPad Prism version 5.00 was used for statistical analysis. All data are presented as the mean ± SD. Differences between groups were identified using Student’s t-test. P < 0.05 was considered statistically significant.
PRMT5 and PRMT1 regulate protein level of CFLARL and its sensitivity to chemotherapeutic agents
Both PRMT5 and PRMT1 can interact with CFLARL
PRMT1 and PRMT5 regulates CFLARL degradation independently of their enzymatic activity
In order to characterize whether the arginine methylation or the physical interaction of CFLARL and PRMT1/5 regulates the degradation of CFLARL, we constructed the plasmids containing PRMT1 or PRMT5 genes with dead mutations as described before [40, 41]. The plasmids are designated as pcDNA3.1-PRMT5-T139/144A and pcDNA3.1-PRMT1- G98R, respectively. As shown in Fig. 3E, CFLARL was upregulated after overexpression of both wild-type PRMT5 and its dead mutation in A549 cells. Similarly, CFLARL was downregulated after overexpression of both wild-type PRMT1 and its dead mutation in A549 cells (Fig. 3F), suggesting it is the physical interaction of CFLARL and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity.
PRMT1 and PRMT5 regulate poly-ubiquitination of CFLARL by affecting the interaction between CFLARL and ITCH
PRMT5 and PRMT1 regulate degradation of CFLARL
PRMT5 and PRMT1 modulate caspase 8 cleavage and apoptosis induced by the anti-cancer drugs in NSCLC cells
PRMT5 and PRMT1 modulate apoptosis in NSCLC cells by regulating protein levels of CFLARL
PRMT5 competes with PRMT1 for binding to CFLARL
In the present study, we report a new finding that PRMT5 and PRMT1, which are both arginine methyltransferases, distinctly regulate the turnover of CFLARL in human NSCLS cells independently of its enzymatic activity. CFLARL is a critical regulator of apoptosis induction and drug resistance in multiple cancers, such as colon, rectum and lung cancer, and has been considered a potential anti-cancer molecular target. Thus, investigating its molecular mechanisms is particularly meaningful. Our present work verified that PRMT5 and PRMT1 could affect its ubiquitination, degradation. Although overexpression of PRMT5 catalyzed CFLARL to produce arginine symmetric dimethylation, and PRMT1 catalyzed CFLARL to produce arginine asymmetric demethylation, the dead mutations of PRMT1 and PRMT2 had the same effect on the CFLARL level as the wild-type ones had, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity.
Several reports have shown that PRMT5 overexpression is associated with hyper-proliferation and apoptosis resistance in cancer cells, and the inhibition of its activity leads to the repression of cancer genes and slows growth [46, 47]. PRMT5 plays essential roles in the regulation of protein function, quality control and signal transduction . Our results show that PRMT5 enhance CFLARL stability. The link between PRMT5 and CFLARL protein longevity is novel and reasonable.
In our study, we found that PRMT5 and PRMT1 regulate the protein level of CFLARL and the sensitivity to chemotherapy drugs. The experiments showed that PRMT5 knockdown or PRMT1 overexpression promoted cell apoptosis induced by doxorubicin and pemetrexed. The PRMT5 overexpression and PRMT1 knockdown inhibited apoptosis induced by chemotherapeutic agents. Furthermore, cellular apoptosis induced by doxorubicin along with the PRMT5 knockdown or PRMT1 overexpression could be inhibited by the exogenous replenishment of CFLARL, suggesting that PRMT5 and PRMT1 affect apoptosis by regulating the protein level of CFLARL in NSCLC cells. We speculated how these two arginine methyltransferases contributed to apoptosis induced by chemotherapeutic agents. Therefore, we verified that both PRMT5 and PRMT1 bound CFLARL, regulating CFLARL by affecting its ubiquitin-proteasome degradation. The level of CFLARL polyubiquitination is increased after overexpressing PRMT1 or knocking down PRMT5 in HEK293FT cells. Our data showed that PRMT5 and PRMT1 were involved in the interaction between CFLARL and the E3 ligase ITCH. The PRMT5 silencing and PRMT1 overexpression enhanced the interaction between CFLARL and ITCH, leading to an altered ubiquitination level and, eventually, the degradation of CFLARL.
In summary, in the present study, we explored the role of the interaction between CFLARL and PRMT5/PRMT1 in apoptosis in NSCLC cells. We also demonstrated that PRMT1 and PRMT5 had opposing effects on chemotherapeutic agent-mediated apoptosis in lung cancer cells. The identification of PRMT5 and PRMT1 as CFLARL regulators involved in cellular apoptosis may help in developing new strategies to increase the sensitivity of cancer cells to chemotherapy, which may eventually benefit lung cancer treatments.
Special thanks to Dr. Austin Cape for his helpful editing advances.
This work was supported by the grants from National Natural Science Foundation of China (31571422, 31771526, 31371402 and 81672855) and Science and technology development plan of Shandong Province (2016GSF201153).
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
ML, WA, LX and LS conducted experiments; XL, ML, LS and YL designed experiments and analyzed data; ML, XL and LS prepared the manuscript. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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