(2008) Mol

(2008) Mol. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2 overexpression. TGF-1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2 or TTP reversed TGF-1-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-1 enhances the activity of CK2. transcripts (2), indicating a possible role for TTP in angiogenesis and tumor growth (3, 4). Up-regulation of mRNA Atovaquone can be induced by 12-mRNA contains AREs, and its stability is regulated by TTP itself (8). TTP is usually highly phosphorylated both and (35) and ensures the maintenance of G2 arrest and apoptosis following spindle damage (36). We previously reported that TTP down-regulates the expression of VEGF and inhibits the growth of human colon cancer cells and (37). In this study, we demonstrate that CK2 increases the mRNA decaying activity of TTP in human colon cancer cells by protecting the TTP protein from phosphorylation and proteasomal degradation. CK2 decreased phosphorylation of p38 MAPK, and the effects of CK2 were attenuated either by treatment with an inhibitor of MAPK phosphatase 1 (MKP-1) or an siRNA against mRNA decaying activity of TTP. Finally, TGF- suppressed the growth of colon cancer cells, and this event was mediated by CK2 and TTP, indicating that TGF- functions as a tumor suppressor via the activation of CK2 and TTP in Colo320 cells. Collectively, our results show that CK2 increases the mRNA decaying activity of TTP through activation of MKP-1 and that TGF- plays a role in the activation of the CK2-MKP-1-TTP signaling pathway. EXPERIMENTAL PROCEDURES Cells Human colon cancer cells (Colo320) were purchased from Korean Cell Collection Lender (KCLB, Seoul, Korea). Colo320 cells were cultured in RPMI 1640 media, supplemented with 10% FBS (heat-inactivated fetal bovine serum) (Invitrogen), and managed at 37 C in a humidified atmosphere of 5% CO2. For the MTS cell proliferation assay, cells were plated in RPMI 1640 medium in triplicate at 1.2 104 cells/well in 96-well culture plates. At the indicated occasions, CellTiter 96? Aqueous One Answer Reagent (Promega, Madison, WI) was added to each well according to the manufacturer’s protocols, and absorbance at 490 nm (polymerase (Sun Genetics, Daejeon, Korea) and the appropriate primers as follows: mRNA expression using quantitative real time PCR, performed using the ABI Prism 7900 HT to monitor real time increases in the fluorescence of SYBR Green dye (Qiagen, Hilden, Germany). Specificities of each primer pair were confirmed via melting curve analysis and agarose gel electrophoresis. The PCR primer pairs were as follows: qVEGF, 5-ATCTTCAAGCCATCCTGTGTGC-3 and 5-TGCGCTTGTCACATTTTTTCTTG-3; qTTP, 5-CCCCAAATACAAGACGGAACTC-3 and 5-GGGCCGCCAGGTCTTC-3; and qGAPDH, 5-ACATCAAGAAGGTGGTGAAG-3 and 5-CTGTTGCTGTAGCCAAATTC-3. Plasmid, siRNAs, Transfection, and Dual-Luciferase Assay The pcDNA6/V5-TTP plasmid construct has been explained previously (37). Plasmid constructs made up of full-length cDNA of human CK2 or human ubiquitin were purchased from Addgene (Cambridge, UK). Full-length cDNA of human CK2 and human ubiquitin were amplified from these plasmids, and full-length cDNA of human MKP-1 was amplified from your cDNA of Colo320 cells using PCR. PCR products were ligated into BamHI/XhoI sites of Atovaquone pcDNA3-HA or pcDNA3.1-FLAG vectors (Invitrogen) to generate pcDNA3/HA-CK2, pcDNA3/FLAG-Ub, and pcDNA3/FLAG-MKP-1. Site-directed mutants of TTP with single (S21A, S169A, S279A, or S325A), double (S60A/S186A), or quadruple amino acid substitutions (S21A/S169A/S279A/S325A) and MKP-1 with double amino acid substitutions (S131A/S235A) were generated using pcDNA6/V5-TTP and pcDNA3/FLAG-MKP-1 as a template, respectively, using a QuikChange site-directed mutagenesis kit (Stratagene, San Diego) according to the manufacturer’s instructions. Mutagenic primers utilized for generation of site-directed mutants of TTP or MKP-1 were as follows: TTP-S21A, 5-AGTGCCCGTGCCAGCCGACCATGGAGGG-3 and 5-CCTCCATGGTCGGCTGGCACGGGCACTG-3; TTP-S60A, 5-CTGGCCGCTCCACCGCCCTAGTGGAGGGC-3 and 5-GCCCTCCACTAGGGCGGTGGAGCGGCCAG-3; TTP-S169A, 5-CATCCACAACCCTGCCGAAGACCTGGCGG-3 and 5-CCGCCAGGTCTTCGGCAGGGTTGTGGATG-3; TTP-S186A, 5-CTTCGCCAGAGCATCGCCTTCTCCGGCCTGC-3 and 5-GCAGGCCGGAGAAGGCGATGCTCTGGCGAAG-3; TTP-S279A, 5-GTACAGTCCCTGGGAGCCGACCCTGATGAATATG-3 and 5-CATATTCATCAGGGTCGGCTCCCAGGGACTGTAC-3;TTP-S325A, 5-CGCATCTCTGTTGCCGAGCTCGAGTCTAG-3 and Atovaquone 5-CTAGACTCGAGCTCGGCAACAGAGATGCG-3; MKP-1-S131A, 5-GAAGCGTTTTCGGCTGCCTGCCCGGAGCTG-3 and 5-CAGCTCCGGGCAGGCAGCCGAAAACGCTTC-3; and MKP-1-S235A, 5-GGCAGACATCAGCGCCTGGTTCAACGAGGC-3 and 5-GCCTCGTTGAACCAGGCGCTGATGTCTGCC-3. Cells were transfected with plasmid constructs.Chem. CK2 and TTP and the TTP function. The siRNA against CK2 or TTP reversed TGF-1-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-1 enhances the activity of CK2. transcripts (2), indicating a possible role for TTP in angiogenesis and tumor growth (3, 4). Up-regulation of mRNA can be induced by 12-mRNA contains AREs, and its stability is regulated by TTP itself (8). TTP is usually highly phosphorylated both and (35) and ensures the maintenance of G2 arrest and apoptosis following spindle damage (36). We previously reported that TTP down-regulates the expression of VEGF and inhibits the growth of human colon cancer cells and (37). In this study, we demonstrate that CK2 increases the mRNA decaying activity of TTP in human colon cancer cells by protecting the TTP protein from phosphorylation and proteasomal degradation. CK2 decreased phosphorylation of p38 MAPK, and the effects of CK2 were attenuated either by treatment with an inhibitor of MAPK phosphatase 1 (MKP-1) or an siRNA against mRNA decaying activity of TTP. Finally, TGF- suppressed the growth of colon cancer cells, and this event was mediated by CK2 and TTP, indicating that TGF- functions as a tumor suppressor via the activation of CK2 and TTP in Colo320 cells. Collectively, our results show that CK2 increases the mRNA decaying activity of TTP through activation of MKP-1 and that TGF- plays a role in the activation of the CK2-MKP-1-TTP signaling pathway. EXPERIMENTAL PROCEDURES Cells Human colon cancer cells (Colo320) were purchased from Korean Cell Collection Lender (KCLB, Seoul, Korea). Colo320 cells were cultured in RPMI 1640 media, supplemented Atovaquone with 10% FBS (heat-inactivated fetal bovine serum) (Invitrogen), and managed at 37 C in a humidified atmosphere of 5% CO2. For the MTS cell proliferation assay, cells were plated in RPMI 1640 medium in triplicate at 1.2 104 cells/well in 96-well culture plates. At the indicated occasions, CellTiter 96? Aqueous One Answer Reagent (Promega, Madison, WI) was added to each well according to the manufacturer’s protocols, and absorbance Atovaquone at 490 nm (polymerase (Sun Genetics, Daejeon, Korea) and the appropriate primers as follows: mRNA expression using Angiotensin Acetate quantitative real time PCR, performed using the ABI Prism 7900 HT to monitor real time increases in the fluorescence of SYBR Green dye (Qiagen, Hilden, Germany). Specificities of each primer pair were confirmed via melting curve analysis and agarose gel electrophoresis. The PCR primer pairs were as follows: qVEGF, 5-ATCTTCAAGCCATCCTGTGTGC-3 and 5-TGCGCTTGTCACATTTTTTCTTG-3; qTTP, 5-CCCCAAATACAAGACGGAACTC-3 and 5-GGGCCGCCAGGTCTTC-3; and qGAPDH, 5-ACATCAAGAAGGTGGTGAAG-3 and 5-CTGTTGCTGTAGCCAAATTC-3. Plasmid, siRNAs, Transfection, and Dual-Luciferase Assay The pcDNA6/V5-TTP plasmid construct has been explained previously (37). Plasmid constructs made up of full-length cDNA of human CK2 or human ubiquitin were purchased from Addgene (Cambridge, UK). Full-length cDNA of human CK2 and human ubiquitin were amplified from these plasmids, and full-length cDNA of human MKP-1 was amplified from your cDNA of Colo320 cells using PCR. PCR products were ligated into BamHI/XhoI sites of pcDNA3-HA or pcDNA3.1-FLAG vectors (Invitrogen) to generate pcDNA3/HA-CK2, pcDNA3/FLAG-Ub, and pcDNA3/FLAG-MKP-1. Site-directed mutants of TTP with single (S21A, S169A, S279A, or S325A), double (S60A/S186A), or quadruple amino acid substitutions (S21A/S169A/S279A/S325A) and MKP-1 with double amino acid substitutions (S131A/S235A) were generated using pcDNA6/V5-TTP and pcDNA3/FLAG-MKP-1 as a template, respectively, using a QuikChange site-directed mutagenesis kit (Stratagene, San Diego) according to the manufacturer’s instructions. Mutagenic primers utilized for generation of site-directed mutants of TTP or MKP-1 were as follows: TTP-S21A, 5-AGTGCCCGTGCCAGCCGACCATGGAGGG-3 and 5-CCTCCATGGTCGGCTGGCACGGGCACTG-3; TTP-S60A, 5-CTGGCCGCTCCACCGCCCTAGTGGAGGGC-3 and 5-GCCCTCCACTAGGGCGGTGGAGCGGCCAG-3; TTP-S169A, 5-CATCCACAACCCTGCCGAAGACCTGGCGG-3 and 5-CCGCCAGGTCTTCGGCAGGGTTGTGGATG-3; TTP-S186A, 5-CTTCGCCAGAGCATCGCCTTCTCCGGCCTGC-3 and 5-GCAGGCCGGAGAAGGCGATGCTCTGGCGAAG-3; TTP-S279A, 5-GTACAGTCCCTGGGAGCCGACCCTGATGAATATG-3 and 5-CATATTCATCAGGGTCGGCTCCCAGGGACTGTAC-3;TTP-S325A, 5-CGCATCTCTGTTGCCGAGCTCGAGTCTAG-3 and 5-CTAGACTCGAGCTCGGCAACAGAGATGCG-3; MKP-1-S131A, 5-GAAGCGTTTTCGGCTGCCTGCCCGGAGCTG-3 and 5-CAGCTCCGGGCAGGCAGCCGAAAACGCTTC-3; and MKP-1-S235A, 5-GGCAGACATCAGCGCCTGGTTCAACGAGGC-3 and 5-GCCTCGTTGAACCAGGCGCTGATGTCTGCC-3. Cells were transfected with plasmid constructs using TurboFectTM transfection reagent (Fermentas, Hanover, Germany). Small interfering RNAs (siRNA) against human (siMKP-1) (sc-35937), human (siTTP) (sc-36761), and control siRNA (scRNA) (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Colo320 cells (1.5 or 3 105).