Background
Transcriptome dynamics are governed by RNA synthesis and degradation. Regulation of mRNA stability plays a central role in controlling gene expression in the vast majority of eukaryotic cells [
1‐
4]. The mRNA stability is modulated by
cis-acting elements in the 3′ untranslated region
(UTR) as well as
trans-acting factors [
5,
6]. The AU-rich element (ARE) is the best-studied
cis-acting element in short-lived mRNA [
7]. Several ARE-binding proteins have been shown to regulate mRNA turnover/decay [
8], which is initiated by deadenylation via deadenylases including poly(A)-specific ribonuclease (PARN) and the polyA nuclease 2 (PAN2)-PAN3 and carbon catabolite repression (CCR4)-negative on TATA-less (NOT) complexes [
9]. CCR4-NOT is a highly conserved multisubunits molecular machine with an approximate molecular mass of 1 MDa that contains two deadenylases, CCR4 (also named CNOT6) and CAF1 (also named CNOT7) [
10,
11]. The largest subunit of CCR4-NOT, namely CCR4-NOT complex subunit 1 (CNOT1), serves as a hub of protein-protein interactions [
11]. Specific mRNAs can be targeted by RNA-binding proteins, such as ARE-containing mRNAs can be recognized by tristetraprolin (TTP), to recruit deadenylase complexes for mRNA degradation [
12,
13].
TTP is an extensively studied ARE-binding protein. There are four TTP members in rodents, including TTP, Zfp36l1, Zfp36l2, and Zfp36l3, and they all contain conserved tandem CCCH zinc finger RNA–binding domains and a conserved C-terminal NOT1-binding domain [
14]. TTP contains intrinsically unstructured regions outside these two conserved regions [
15‐
17]. The metazoa type of NOT1-binding domain is missing in most fungi; for examples, the TTP homologs CTH1 and CTH2 in
Saccharomyces cerevisiae, and Zfs1p in
S. pombe, are no containing NOT1-binding domain [
14]. TTP was induced by lipopolysaccharide (LPS) and served as an anti-inflammatory factor to inhibit cytokine expression such as TNF-α [
18]. We demonstrated that Zfp36l1 and Zfp36l2 proteins were maintained at a constant level and were phosphorylated under LPS stimulation [
19]. Zfp36l3 expression is limited to the placenta and yolk sac, and is important for overall fecundity [
20]. TTP family proteins are serine/threonine-rich, and they appear as multiple bands in SDS-PAGE, indicating that they are highly phosphorylated [
21,
22]. TTP can be phosphorylated by mitogen-activated protein kinase (MAPK) p38-activated protein kinase 2 (MK2) at serines 52 and 178 in mouse macrophages to allow binding of 14–3-3 adaptor proteins, which inhibits the mRNA destabilizing activity of TTP [
23,
24]. In contrast, PP2A can compete with 14–3-3 proteins to dephosphorylate TTP at Ser178 and thereby activate decay of cellular mRNAs [
25,
26]. The substitution of these two serines (Ser52 and Ser178) to non-phosphorylated alanines in the endogenous murine locus encoding TTP gave rise to a strong and dominant anti-inflammatory phenotype [
27]. One molecular mechanism for TTP-mediated mRNA decay is the recruitment of the CCR4-NOT deadenylase complex through direct interaction with CNOT1, resulting in the decay of ARE-containing mRNAs [
28,
29]. TTP phosphorylated by MK2 inhibits the deadenylase recruitment due to association with 14–3-3 [
30,
31]. In addition to the p38-MK2 pathway, ERK signaling has been reported to regulate protein stability, subcellular localization, and function of TTP [
32,
33].
Previously, Ser316 phosphorylation was identified by mass spectrometry analysis in both in vitro MK2 phosphorylation and LPS-stimulated RAW264.7 macrophages [
27]. Ser316 is located in the C-terminal conserved region of TTP family proteins, and this region is critical for CCR4-NOT complex recruitment [
14,
28,
34]. In a SILAC (stable isotope labeling by amino acids) analysis, Ser316 phosphorylation might be an additional residue responding to MK2/3 in addition to the Ser52/Ser178 [
35]. In this study, we generated a specific antibody against phospho- Ser316 of TTP to examine the Ser316 phosphorylation under LPS stimulation in mouse RAW264.7 macrophages and find out the possible kinases and phosphatases. We also created phosphomimetic mutant (S316D) and non-phosphorylated mutant (S316A) to demonstrate that the Ser316 phosphorylation would inhibit CCR4-NOT complex recruitment. The TTP knockout RAW264.7 cells were created by CRISPR/Cas9 gene editing, after transfected with TTP S316A mutant would lead to downregulation of TTP –targeted mRNA. The results confirmed that Ser316 phosphorylation of TTP plays an important function in TTP-mediated mRNA destabilization in LPS-stimulated RAW264.7 cells.
Materials and methods
Cell culture
Human embryonic kidney (HEK) 293T cells (CRL-3216) and mouse NIH3T3 cells (CRL-1658) were purchased from American Type Culture Collection (ATCC) and were cultured in DMEM containing 3.7 g/l sodium bicarbonate and supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a 5% CO2 humidified atmosphere (37 °C). Mouse BALB/c macrophage RAW264.7 cells (TIB-71) were purchased from ATCC and were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a 5% CO2 humidified atmosphere (37 °C). The RAW264.7 cells were treated with 100 ng/ml of LPS from E. coli O111:B4 (Sigma-Aldrich) for different time intervals, combined with the 2 or 5 μM of MK2 inhibitor PF3644022 (Sigma-Aldrich) and 25 or 50 μM of RSK1 inhibitor BI-D1870 (Abcam).
Plasmid constructs
The plasmids for TTP, Zfp36l1, Zfp36l2 and 14–3-3ζ expression were constructed as described [
19,
21,
22,
36]. The S316 and S318, S52, and S178 mutants in TTP were created by PCR (for S316 and S318) or Q5 site-directed mutagenesis kit (New England Biolabs) (for S52 and S178) using the primers indicated in Table
S1. The PCR products were ligated to pCMV-Tag2 (Stratagene) and pEGFP-C2 (Clontech) for mammalian cell expression. Mouse Cnot1@800–1310 was PCR amplified from a full- length Cnot1 cDNA template (OriGene) with the primers: 5′-CAGGCTCAGGCCCAGGTT-3′ and 5′-TTATTAGGCCTGAGCCAGTGCAATAC-3′. The PCR products were cloned into pGEX4T-1 to express glutathione S-transferase (GST)-fused proteins in bacteria.
RNA isolation, reverse transcription, and quantitative PCR
Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen). For mRNA stability analysis, the cells were treated with 10 μg/ml actinomycin D (transcription inhibitor) for various times to inhibit new transcription. After DNase I digestion, 2 μg total RNA was reverse-transcribed to produce cDNA using M-MLV reverse transcriptase and oligo dT primer (Promega). Real-time PCR was performed with the 7300 Real-Time PCR System (Applied Biosystems) in a total volume of 20 μl. Expression of genes encoding TTP, TNFα and actin was assessed using SYBR Green PCR Master Mix (Applied Biosystems) with 50 ng of cDNA and 160 nM of each primer: 5′-GGATCTCTCTGCCATCTACGA-3′ and 5′-CAGTCAGGCGAGAGGTGAC-3′ for TTP; 5′-GACCCTCACACTCAGATCATCTTCT-3′ and 5′-CCTCCACTTGGTGGTTTGCT-3′ for TNFα; 5′-TCCTTCCTGGGCATGGAGTC-3′ and 5′-ACTCATCATACTCCTGCTTG-3′ for β-actin. The PCR amplification conditions were 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Real-time PCR data were analyzed using the 2–∆∆CT relative quantitation method.
HEK293T cells were transfected with the plasmids using Turbofect reagent (Thermo). Cells harvested 24 h after transfection were lysed with NET buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (10 mM β-glycerol phosphate, 0.1 mM Na2MoO4, 0.1 mM Na3VO4, pH 10.0, 10 mM NaF) and centrifuged at 15,000×g for 10 min. The supernatants were immunoprecipitated using anti-Flag M2 agarose (Sigma-Aldrich) at 4 °C for 2 h. After the IP mixture was washed three times with NET buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Millipore), and western blotting was performed using anti-Flag (1:2000)(Sigma-Aldrich), anti-HA (1:2000) (Bethyl Laboratories), anti-TTP and anti-p-S316 (1:1000) (produced in our lab), anti-α-tubulin (1:1000), anti-β-actin (1:1000), anti-phospho-ERK1/2 (1:1000), anti-p38 (1:1000), anti-phosph-p38 (1: 1000), anti-MK2 (1:1000), anti-phospho-MK2 (1:200), anti-phospho-RSK1(1:200), anti-CNOT3 (1:1000) and anti-CNOT6 (1:1000) (all from Cell Signaling Technology), anti-GAPDH (1:5000), anti-CNOT1 (1:1000), and anti-CNOT7 (1:500) (all from Proteintech Group), anti-ERK1/2 (1:2000) and anti-RSK1(1:2000) (both from Santa Cruz Biotechnology), and anti-DDX6 (1:1000)(Abcam). All experiments were carried out at least three times, and represented results were displayed.
Generation of rabbit anti-phospho-S316 of TTP
A peptide containing the sequence surrounding phospho-S316 of TTP (RLPIFNRIpSVSE) was synthesized and purified by Kelowna International Scientific Inc. (Taiwan). A specific rabbit antiserum was produced by LTK BioLaboratories (Taiwan). The antiserum was affinity purified using the immunizing peptide (LTK BioLaboratories).
In vitro kinase assay
Each reaction mixture contained 2 μg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 μl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha (SignalChem), RSK1 (SignalChem), and MK2 (SignalChem) in a final volume of 30 μl. The kinases can be from cell extracts. 300 μg of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 μg of GST-TTP in the buffer containing 20 mM HEPES, pH 7.7, 75 mM NaCl, 0.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 100 μg/ml PMSF. The mixture was rotated at 4 °C for 3 h and pelleted by centrifugation at 10,000×g for 20 s. After 4 × 1-ml washes in HEPES binding buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100), the beads were resuspended in 30 μl for kinase assay. The reaction mixtures were incubated at 30 °C for 30 min and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.
GST pull-down assays
Glutathione-Sepharose 4B (~ 8 μl, GE Healthcare Life Sciences) were incubated with 2 μg of bacterially expressed GST, or GST-Cnot1@800–1015 or GST-14-3-3 in phosphate-buffered saline containing 1% (v/v) Triton X-100 on a rotary shaker for 20 min at room temperature. After washing three times with the same buffer, the Sepharose was combined with lysates (300 μg protein) of RAW264.7 cells that had undergone various treatments in a final volume of 200 μl of buffer containing 20 mM HEPES, pH 7.9, 100 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% (v/v) NP-40, 1% (v/v) Triton X-100, 1 mM DTT, and 1 mM PMSF. The mixtures were incubated at 4 °C for 2 h on a rotary shaker, and then the Sepharose was washed four times with the same buffer lacking DTT and PMSF but containing 0.2 M NaCl and once with 50 mM Tris, pH 6.8. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by western blotting.
RNA pull-down assays
Cytoplasmic extracts from LPS-stimulated RAW264.7 cells were prepared by hypotonic buffer (10 mM HEPES, pH 7.5, 10 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 mM DTT, 0.05% NP-40, and protease inhibitor cocktails). Potassium acetate was adjusted to 90 mM, and 0.1 U·μL
− 1 RNasin (Promega) and 20 μg·μL
− 1 yeast tRNA was added to each lysate. To prevent non-specific binding, heparin-agarose (Sigma-Aldrich) was incubated with each lysate for 15 min at 4 °C and then centrifuged for 1 min at 8000 rpm, 4 °C. Each supernatant was further cleaned with streptavidin-Sepharose (8 μL
; Invitrogen) for 1 h at 4 °C and then centrifuged for 1 min at 8000 rpm, 4 °C. The biotin-labeled
TNFα ARE was added as described [
19]. The pulled-down RNA-protein complexes were washed four times with binding buffer (hypotonic buffer containing 90 mM potassium acetate) and separated by SDS-PAGE (10% acrylamide) for western blotting analysis.
HEK293T cells (1 × 105) were seeded in each well of a 12-well plastic culture plate. For gene knockdown, the cells were transfected with 5 nM of a small interfering RNA (siRNA) for CNOT1, CNOT6, CNOT7, RSK1, or MK2 (Invitrogen) using Lipofectamine 3000 (Invitrogen). After 24 h, the cells were transferred into a fresh medium and transfected with 1 μg of Flag-TTP wild-type, −TTP S52,178A or –TTP S316A expression plasmids using Turbofect reagent (Thermo). After another 24 h, cells were harvested, and whole cell extracts were isolated for western blotting or immunoprecipitation.
CRISPR-based sgRNAs were designed on Benchling (
https://benchling.com) and CHOPCHOP (
https://chopchop.cbu.uib.no/) to search the specific target-sequences of Cas9 RNP complexes on mouse
Ttp gene. Based on the description of the target score [
37] (
https://crispr.mit.edu/about), we designed four sgRNAs, which contain a relatively higher on-target with a lower off-target score (Table
S2). Each DNA template of TTP sgRNA encoding for a T7 promoter, a 20 nt target sequence, and a published sgRNA scaffold [
38] were assembled by overlapping PCR. Each PCR reactions contain 20 nM premix of TTP sgRNAs and bottom scaffold (Table
S2), 1 μM premix of T7 oligo primer and sgRNA-reverse, 200 μM dNTP, and Q5 polymerase (NEB) according to the manufacturer’s protocol. The thermocycler setting consisted of 30 cycles of 95 °C for 10 s, 59 °C for 10 s and 72 °C for 10 s [
39].
The assembled PCR products were extracted once with phenol:chloroform:isoamyl alcohol and then once with chloroform, before isopropanol precipitation overnight at − 20 °C. The DNA pellet was washed three times with 70% ethanol and dissolved in DEPC-treated water. The T7 in vitro transcription reaction consisted of 30 mM Tris–HCl (pH 7.9), 20 mM MgCl
2, 0.01% Triton X-100, 2 mM spermidine, 10 mM DTT, 5 mM of ribonucleotide triphosphate, 100 μg/ml T7 polymerase and 1 μM DNA templates. The reaction was incubated at 37 °C for 4 h, and RNase-free DNase was added to digest the DNA template 37 °C for 1 h. The reaction was stopped by adding 2xSTOP solution (95% deionized formamide, 0.05% bromophenol blue and 20 mM EDTA) at 60 °C for 5 min. The RNA was purified by electrophoresis in 10% polyacrylamide gel containing 6 M urea. The RNA band was excised from the gel, ground up in a 15-ml tube, and eluted with 5 vol. of 300 mM sodium acetate (pH 5.0) overnight at 4 °C. One equivalent of isopropanol was added to precipitate the RNA at − 20 °C. The RNA pellet was centrifuged and washed three times with 70% ethanol and dried by vacuum. To refold the sgRNA, the RNA pellet was first dissolved in 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, and 1 mM TCEP. The sgRNA was heated to 70 °C for 5 min and cooled to room temperature. MgCl
2 was added to a final concentration of 1 mM. The sgRNA was again heated to 50 °C for 5 min, cooled to room temperature and kept on ice. The sgRNA concentration was determined by OD
260nm and adjusted to 100 μM using 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, 1 mM TCEP, and 1 mM MgCl
2. The sgRNA was store at − 80 °C [
39].
Transfection of RAW264.7 cells was performed according to the instructions of Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Invitrogen). The Cas9 RNP were prepared by incubating the purified Cas9 protein with sgRNA at 1:4 molar ratios. After transfection, the cells were incubated at 37 °C for 48 h, and followed by single-cell sorting using BD, FACSJazz automated cell sorter to perform the single isolation (TechComm at NTU).
Genomic DNA isolation and PCR analysis
Genomic DNA was extracted by QuickExtract™ DNA Extraction Solution (Epicentre). Cells were mixed by 200 μl of QuickExtract Solution and vortex for 15 s. The tubes were transferred to 65 °C and incubate for 6 min. After 15 s vortex, samples were incubated at 98 °C for 2 min. Store the DNA at − 20 °C or − 80 °C for long-term storage according to the manufacturer’s protocol. Genomic DNA was PCR amplified using primer-1 and primer-4 (Table
S2). Theoretically, sgRNA mTTP KO-1 and sgRNA mTTP KO-4 will generate double-strand DNA breaks on each target site, and cause a 793 nt deletion between exon 1 and exon 2 on the
Ttp gene in RAW264.7 cells. Therefore, the size of PCR product generated by primer-1 and primer-4 will be reduced from 2001 nt to 1208 nt. Besides, primer-2 and primer-3 locate on the predicted cutting fragment. If the TTP knock-out happened, it would not have PCR products between primer-1 and primer-2 or primer-3 and primer-4 theoretically (Table
S2).
Indirect immunofluorescence staining
NIH3T3 cells were typically seeded at 50% on sterile glass coverslips before 16–18 h. After transfected with GFP-fused TTP expression vector using Lipofectamine 2000 (Thermo) for 24 h and treated with 20 ng/ml of leptomycin B (LMB) for 6 h, cells were washed briefly with PBS and then fixed in 4% paraformaldehyde/PBS for 30 min at room temperature. After gently washed twice with PBS (5 min for each), cells were permeabilized with 0.1% Triton X-100/PBS for 10 min at room temperature. Following another two times washed with PBS (5 min for each), blocked the cells for 30 min with PBS containing 1% BSA. Then the cells were incubated with appropriately diluted DDX6 antibody overnight at 4 °C, washed with PBS three times (10 min for each), further incubated with secondary antibody (Alexa Fluor 594 goat anti-rabbit Ig, molecular probe) and DAPI (Sigma-Aldrich) for 1.5 h in the dark, and washed with PBS three times (10 min for each) in the dark. Mount in mounting fluid and store at − 20 °C until detected by Leica SP5 confocal Microscopy system.
Statistical analysis
All data are presented as the mean ± SD of at least three independent experiments. Statistical significance (*P < 0.05, **P < 0.01 or ***P < 0.001) was determined by one-tailed Student’s t-test.
Discussion
TTP is a highly phosphorylated protein, and the functional regulation by phosphorylation is an important subject in TTP study [
46‐
48]. We generated a specific antibody against phospho-Ser316 and demonstrated that ERK-RSK1 and p38-MK2 signaling pathways phosphorylate TTP at Ser316 in LPS-stimulated RAW264.7 cells (Figs.
1 and
3). The previous results showing that the TTP-mediated turnover of
TNFα mRNA is inhibited by the combined activation of ERKs and p38 [
33]. Our finding suggests that Ser316 might be involved in this effect through phosphorylated by RSK1 and MK2 in response to differential MAPK signals. The IP, GST pull-down, and RNA pull-down assays (Fig.
2, Fig.
6, and Fig.
S5) demonstrated that Ser316 phosphorylation weakens the interaction with CNOT1 in the CCR4-NOT deadenylase complex. It was consistent with a report that described the human TTP peptide (residues 312–326, mouse TTP residues 305–319) containing phospho-S323 (like mouse S316) showed lower CNOT1@800–999 binding affinity than the wild-type peptide [
28]. Additionally, the ERK–RSK pathway also phosphorylates one of the TTP family proteins, ZFP36L1, at S334 in the conserved C-terminal NOT1-binding domain and inhibits its interaction with CNOT7 [
34]. We also provide evidence to prove the Ser316 phosphorylation was removed by PP2A (Fig.
S1B).
CNOT1 is a scaffold protein that interacts with the deadenylase CNOT7 via its central MIF4G domain [
49], with CNOT9 via a DUF3819 domain [
50,
51], with the CNOT2-CNOT3 heterodimer via a C-terminal SH domain [
52,
53], and with CNOT10-CNOT11 via its N-terminus [
54]. CNOT6 interacts with CNOT7 via its N-terminal leucine-rich repeat domain, but it does not interact directly with CNOT1 [
55]. IP showed that TTP forms a complex with CNOT1, CNOT3, CNOT6, and CNOT7 (Fig.
2 A). When we used tandem mass spectrometry to analyze TTP-associated proteins, several CCR4-NOT complex subunits were detected, including CNOT1, CNOT2, CNOT6, CNOT6L, CNOT10, and CNOT11 (data not shown). Knockdown analyses demonstrated that CNOT1, CNOT6, and CNOT7 play roles in TTP mRNA destabilization activity (Fig.
2 B and Fig.
S2). When CNOT1 was knocked down, CNOT6 and CNOT7 could not be co-precipitated with TTP (Fig.
2 B). Therefore, knockdown of CNOT1 had a greater effect on TTP activity than knockdown of CNOT6 or CNOT7 (Fig.
S2C). Through the interaction with CNOT1, TTP might communicate with other components of the mRNA decay machinery. The MIF4G domain of CNOT1 interacts with the translation repressor DDX6 to bridge deadenylation and decapping [
50,
51,
56]. Recent studies have shown that CNOT9 interacts with GW182/TNRC6C and involves microRNA-mediated repression [
50,
51]. However, in our immunofluorescence staining and IP results (Fig.
2 C), the interaction between TTP and DDX6 is phosphorylation independent. TTP might associate with DDX6-containing P-body in RAW264.7 cells (Fig.
6 C) [
57].
In response to LPS stimulation, the
TNFα mRNA was dramatically induced at 30 min and decreased at 1 h (Fig.
5 A), while the decrease was not observed in TTP KO cells, indicating TTP plays a role in this response. When ectopic expression of TTP S316A or TTP S316D in TTO KO cells, the decrease at 1 h was not recovered (Fig.
5 C). We suggest that the dynamic TTP phosphorylation is required for the bi-phasic
TNFα expression [
17,
58], and the lower amount and hypo-phosphorylated TTP at 1 h induction (Fig.
4 A) exhibits higher mRNA destabilization activity. Like phosphorylation at serines 52 and 178 by p38-MK2 signaling [
32], Ser316 phosphorylation of TTP also displayed cytoplasmic localization (Fig.
6 C). It might be due to the interaction between hyper-phosphorylated TTP and 14–3-3 protein (Fig.
6 A). We observed serines 52 /178 and Ser316 played functions independently in the recruitment of the CCR4-NOT deadenylase complex. That is TTP phosphorylation on either Ser52/178 or Ser316 would decrease association with CCR4-NOT complex (Fig.
2 D). It is consistent with a recent report in knockout mice study showing that Ser316 is another residue phosphorylated by MK2/3 in addition to serines 52 and 178, and they regulate TNF biosynthesis independently [
35]. However, the Ser316 phosphorylation seems weaker in TTP Ser52,178A mutant than wild-type (Fig.
1 A and D). Whether phosphorylation at serines 52 and 178 affects Ser316 phosphorylation will be further clarified and investigated. TTP contains intrinsically disordered regions (IDRs) which facilitate rapid degradation of TTP protein [
15]. The serines 52 and 178 are located in IDR, and those phosphorylations can inhibit TTP protein degradation [
32,
58]. Ross and his colleagues generated the mouse strain expressed TTP-S52,178A, and the mutant protein was unstable and expressed low levels in mice, but it functioned higher mRNA destabilization activity than wild-type [
27]. TTP-S316A or -S316D mutants did not alter their protein half-lives in our preliminary examination. Our results imply the complex regulation of TTP phosphorylation, which might control a network of protein-protein interaction to modulate target mRNA stability.
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