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. 2011 Jun 17;286(24):21577-87.
doi: 10.1074/jbc.M110.201137. Epub 2011 Apr 20.

Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin

Won Hyeok Lee  1 , Hyun Hee Lee, Mai-Tram Vo, Hyo Jeong Kim, Myoung Seok Ko, Yeong-Cheol Im, Young Joo Min, Byung Ju Lee, Wha Ja Cho, Jeong Woo Park
Affiliations

Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin

Won Hyeok Lee et al. J Biol Chem. .

Abstract

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

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Figures

FIGURE 1.
FIGURE 1.
Inhibition of CK2 decreases the VEGF mRNA decaying activity of TTP. A, CK2 inhibitor DRB decreases the mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR and pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with CK2 inhibitor DRB or p38 MAPK inhibitor SB203580 at indicated concentrations for 12 h (A), and luciferase (Luc.) activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3′UTR-transfected cells was set to 1. Each bar represents the mean ± S.D. of three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001). ns, not significant. B and C, inhibition of CK2α by siRNA (siCK2α) decreases the mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR, pcDNA6/V5-TTP, and siCK2α or scRNA. At 24 h post-transfection, expression level of CK2α was determined by immunoblotting (IB) (B) and luciferase activity was determined (C). Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3′UTR-transfected cells was set to 1. Each bar represents the mean ± S.D. of three independent experiments (**, p < 0.01; ***, p < 0.001). D, TTP-mediated down-regulation of endogenous VEGF mRNA is attenuated by siCK2α. Colo320 cells were cotransfected with pcDNA6/V5-TTP and siCK2α or scRNA. At 24 h post-transfection, VEGF mRNA was determined by quantitative real time PCR. The expression level obtained from mock-transfected cells was set to 1. Each bar represents the mean ± S.D. of three independent experiments (**,p < 0.01; ***, p < 0.001).
FIGURE 2.
FIGURE 2.
Overexpression of CK2α increases VEGF mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR and pcDNA6/V5-TTP pcDNA3/HA-CK2α. At 24 h post-transfection, the expression levels of CK2α and TTP were determined by immune blots (IB) (A) and luciferase activity was determined (B). Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells cotransfected with psiCHECK2-VEGF 3′UTR and pcDNA6/V5-TTP was set to 1. Each bar represents the mean ± S.D. of three independent experiments (**, p < 0.01; ***, p < 0.001). ns, not significant.
FIGURE 3.
FIGURE 3.
Inhibition of CK2 by DRB decreases the instability of TTP protein. A–C, inhibition of CK2α by CK2 inhibitors or siRNA decreases the expression level of the TTP protein but does not affect the expression level of the TTP transcript. Colo320 cells were treated with CK2 inhibitor DRB or TBB at the indicated concentrations for 12 h or transfected with siCK2α or scRNA. TTP protein levels were determined by immunoblotting (IB) (A, top panel, and B) and TTP mRNA levels were determined by (A, bottom panel) semi-quantitative PCR, and real time PCR (C). D and E, DRB treatment decreases the stability of TTP protein. Colo320 cells were transfected with pcDNA6/V5-TTP, and at 24 h post-transfection cells were incubated with 10 μg/ml cycloheximide (CHX) in the presence or absence of DRB. D, cells were harvested at indicated times. and TTP protein levels were determined by immunoblotting with anti-V5 antibody. E, graph showing the expression levels of TTP protein. The expression levels of TTP protein in CHX-treated cells at 0 h were set to 1. The results are presented as the means ± S.D. of three independent experiments. (**, p < 0.01; ***, p < 0.001). ns, not significant.
FIGURE 4.
FIGURE 4.
CK2 inhibitor DRB enhances phosphorylation, ubiquitination, and proteasomal degradation of the TTP protein. Colo320 cells were transfected with pcDNA6/V5-TTP, and at 24 h post-transfection cells were treated with DRB in the presence or absence of MG132 at the indicated concentrations. A, proteasome inhibitor MG132 abolishes the decrease of TTP protein induced by DRB. TTP protein levels were determined by immunoblotting (IB) with anti-V5 antibody. B, treatment of calf intestinal alkaline phosphatase (CIP) converts slow migrating TTP to fast migrating TTP. Cell lysates were subjected to in vitro calf intestinal alkaline phosphatase treatment (20 units) for 30 min at 35 °C (5th lane). The samples were then analyzed for TTP protein by immunoblotting with anti-V5 antibody. C, CK2 inhibitor DRB increases the ubiquitination of TTP. Colo320 cells were cotransfected with pcDNA6/V5-TTP and pcDNA3/FLAG-UB. At 24 h post-transfection, cells were treated with DRB in the presence or absence of MG132. Immunoprecipitation (IP) was performed by incubating the cell lysates with anti-V5 antibody and then blotting with anti-FLAG antibody.
FIGURE 5.
FIGURE 5.
Mutations of putative CK2 phosphorylation sites of TTP do not disrupt the effects of CK2 on TTP. A, mutation at the four CK2 phosphorylation sites does not affect the decrease of TTP protein levels induced by CK2 inhibitor DRB. Colo320 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5-TTP(mut all) and were treated with DRB for 12 h. The expression level of the TTP protein was determined by immunoblotting (IB) with anti-V5 antibody. B, mutation at the four CK2 phosphorylation sites does not change the phosphorylation of TTP induced by DRB. Colo320 cells transfected with pcDNA6/V5-TTP or pcDNA6/V5-TTP(mut all) were treated with MG132 in the presence or absence of DRB for 12 h. The expression level of TTP was determined by immunoblotting with anti-V5 antibody. C, mutation of the CK2 phosphorylation sites does not affect the decrease of mRNA decaying activity of TTP induced by DRB. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR and wild-type TTP (pcDNA6/V5-TTP) or point mutant TTP: pcDNA6/V5-TTP(S26A), pcDNA6/V5-TTP(S169A), pcDNA6/V5-TTP(S279A), pcDNA6/V5-TTP(S325A), or pcDNA6/V5-TTP(mut all). At 24 h post-transfection, cells were treated with DRB for 12 h, and luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells transfected with psiCHECK2-VEGF 3′UTR was set to 1. Each bar represents the mean ± S.D. of three independent experiments (***, p < 0.001).
FIGURE 6.
FIGURE 6.
Effects of CK2 on TTP is mediated by the MKP-1/p38 MAPK pathways. A, CK2 inhibition by DRB increases phosphorylation of p38 MAPK and MK2. Colo320 cells were transfected with pcDNA6/V5-TTP, and the cells were treated with 30 μm DRB at 24 h post-transfection. Cells were harvested at indicated times after DRB treatment, and cell lysates were analyzed for TTP, p38 MAPK, phosphorylated p38 MAPK, MK2, and phosphorylated MK2 by immunoblotting (IB). B, mutation of MK2 phosphorylation sites of TTP blocks the DRB-induced degradation of TTP. Colo320 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5-TTP(S60A and S186A) and were treated with DRB for 12 h. The expression level of the TTP protein was determined by immunoblotting with anti-V5 antibody. C, p38 MAPK inhibitor SB203580 attenuates the decrease mRNA decaying activity of TTP induced by siRNA against CK2α. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR, pcDNA6/V5-TTP, and siCK2α or scRNA. Cells were treated with SB203580 for 12 h, and luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells transfected with psiCHECK2-VEGF 3′UTR was set to 1. Each bar represents the mean ± S.D. of three independent experiments (***, p < 0.001). D, inhibition of CK2α by DRB decreases the expression of MKP-1. Colo320 cells were treated with DRB, and cells were harvested at the indicated times after DRB treatment. Cell lysates were analyzed for MKP-1 by immunoblotting with anti-MKP-1 antibody. E, overexpression of CK2α increases the expression of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP and pcDNA3/HA-CK2α. At 24 h post-transfection, expression of CK2α and MKP-1 was determined by immunoblotting. F, mutation of CK2 phosphorylation sites of MKP-1 blocks the CK2α-induced phosphorylation of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP, pcDNA3/HA-CK2α, and pcDNA3.1/FLAG-MKP-1 or pcDNA3.1/FLAG-MKP-1 (S131A and S235A). At 24 h post-transfection, cell lysates were analyzed for CK2α, MKP-1, and phosphorylated MKP-1 by immunoblotting. G, inhibition of MKP-1 by siRNA abolishes the effects of CK2 on TTP expression and p38 MAPK phosphorylation. Colo320 cells were cotransfected with pcDNA6/V5-TTP, pcDNA3/HA-CK2α, and siRNA against MKP-1 (siMKP-1) or scRNA. At 24 h post-transfection, cell lysates were analyzed for CK2α, TTP, MKP-1, p38 MAPK, and phosphorylated p38 MAPK by immunoblotting. H, inhibition of MKP-1 by MKP-1 inhibitor triptolide or siRNA abolishes the effects of CK2α on mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3′UTR, pcDNA6/V5-TTP, pcDNA3/HA-CK2α, and siMKP-1 or scRNA. Cells were treated with triptolide for 12 h, and the luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells cotransfected with psiCHECK2-VEGF 3′UTR and pcDNA6/V5-TTP was set to 1. Each bar represents the mean ± S.D. of three independent experiments (*, p < 0.05; ***, p < 0.001). ns, not significant.
FIGURE 7.
FIGURE 7.
TGF-β increases mRNA decaying activity of TTP through activation of CK2/MKP-1 pathway. A, TGF-β increases CK2 activity. Colo320 cells were treated with TGF-β at the indicated concentrations in the presence or absence of DRB for 4 h. CK2 activity in the cell lysates was determined as described under “Experimental Procedures.” The CK2 activity obtained from untreated cells was set to 1. Each bar represents the mean ± S.D. of three independent experiments (**, < 0.01; ***, p < 0.001). B, TTP-mediated down-regulation of endogenous VEGF mRNA is enhanced by TGF-β, and this effect is mediated by CK2. Colo320 cells were transfected with pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with 2 ng/ml of TGF-β in the presence or absence of DRB. VEGF mRNA was determined by quantitative real time PCR. The expression level obtained from mock-transfected and untreated cells was set to 1. Each bar represents the mean ± S.D. of three independent experiments (**, < 0.01; ***, p < 0.001). C, TTP-mediated destabilization of VEGF mRNA is enhanced by TGF-β, and this effect is mediated by CK2. Colo320 cells were transfected with pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with 2 ng/ml of TGF-β in the presence or absence of DRB for 6 h. Expression of VEGF mRNA was determined by quantitative real time PCR at indicated times after the addition of 5 μg/ml actinomycin D. Results shown on the graph represent means ± S.E. of three independent experiments (*, < 0.05; **, p < 0.01). D, TGF-β significantly enhances the growth inhibitory effect of TTP, and DRB attenuates the TGF-β effect. Colo320 cells were transfected with pcDNA6/V5-TTP or empty vector. At 24 h post-transfection, cells were treated with 2 ng/ml of TGF-β in the presence or absence of DRB. Cell viability was determined at 48 h using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell proliferation assay. The data represent means ± S.D. of three independent experiments (*, p < 0.05; ***, p < 0.001).
FIGURE 8.
FIGURE 8.
Model of regulation of TTP expression by CK2. The p38 MAPK/MK2 signaling pathway can induce phosphorylation (P) of TTP, which leads to proteasomal degradation of TTP protein. However, when CK2 is activated by TGF-β stimulation, it can activate MKP-1, which in turn inhibits the p38 MAPK/MK2 pathway by dephosphorylation of p38 MAPK, thereby protecting the TTP protein from proteasomal degradation.
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