Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: 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 3UTR and pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with CK2 inhib- itor DRB or p38 MAPK inhibitor SB203580 at indicated concentrations for 12 h (A),andluciferase(Luc.)activitywasdetermined.Renillaluciferaseactivitywas normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3UTR-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 CK2 by siRNA (siCK2) decreases the mRNA decaying activity of TTP. Colo320 cells were cotrans- fected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, and siCK2 or scRNA. At 24 h post-transfection, expression level of CK2 was determined by immu- noblotting (IB) (B) and luciferase activity was determined (C). Renilla luciferase activitywasnormalizedtofireflyactivity.Theluciferaseactivityobtainedfrom psiCHECK2-VEGF 3UTR-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 bysiCK2.Colo320cellswerecotransfectedwithpcDNA6/V5-TTPandsiCK2 or scRNA. At 24 h post-transfection, VEGF mRNA was determined by quanti- tative 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).

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Inhibition, Activity Assay, Transfection, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: 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 M 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 CK2. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, and siCK2 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 3UTR was set to 1. Each bar represents the mean S.D. of three independent experiments (***, p 0.001). D, inhibition of CK2 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 CK2 increases the expression of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP and pcDNA3/HA-CK2. At 24 h post-transfection, expression of CK2 and MKP-1 was determined by immunoblotting. F, mutation of CK2 phosphorylation sites of MKP-1 blocks the CK2-induced phosphorylation of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP, pcDNA3/HA-CK2, 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 CK2, 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-CK2, and siRNA against MKP-1 (siMKP-1) or scRNA. At 24 h post- transfection, cell lysates were analyzed for CK2, 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 CK2 on mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, pcDNA3/HA-CK2, 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 3UTR 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.

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Inhibition, Phospho-proteomics, Transfection, Western Blot, Mutagenesis, Expressing, Activity Assay, Luciferase, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: 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- 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.

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Expressing, Phospho-proteomics, De-Phosphorylation Assay