CK-II activated by upstream signals triggers dual phosphorylation of Ser113 and Ser118 on cPGES, which in turn promotes the recruitment of cPGES into the Hsp90 complex, leading to its full activation. [10?mM Tris/HCl (pH?7.4) containing 150?mM NaCl] and suspended in 20?l of sample buffer for SDS/PAGE. SDS/PAGE and immunoblotting were performed as described previously [10,18]. phosphorylation of cPGES Recombinant cPGES or its mutants were incubated for 30?min at 30?C with recombinant CK-II in the presence or absence of Hsp90 in 20?mM Tris/HCl (pH?7.5) containing 50?mM KCl, 10?mM MgCl2, 40?M ATP and 10?Ci/ml of [32P]ATP (Perkin-Elmer). Aliquots of samples were taken for PGES enzyme assay, and after brief boiling, samples were applied to SDS/PAGE followed by autoradiography. Phospho-amino acid analysis Replicate immunoprecipitates resolved by SDS/PAGE were transferred on to PVDF membranes (Millipore) and the positions of cPGES Biotin Hydrazide were precisely excised. The pieces were incubated with 100?l of 6?M HCl for 1?h at 110?C. The supernatants Biotin Hydrazide were collected, evaporated and mixed with trace amounts of L-phosphoserine, L-phosphothreonine and L-phosphotyrosine (Sigma). Then the samples were spotted on to TLC plates (Merck) and electrophoresed at 1500?V for 25?min at pH?1.9 [2.5% (v/v) methanoic acid and 7.8% (v/v) ethanoic acid in water] in the first dimension and at 1300?V for 20?min at pH?3.5 [5% (v/v) ethanoic acid and 0.5% (v/v) pyridine in water] in the second dimension. After drying, separated amino acids on the plates were visualized with ninhydrin spray at 80?C for 15?min. Incorporation of 32P into each amino acid was detected by autoradiography. Construction of cPGES mutants cDNAs for cPGES point mutants were constructed by the mismatched PCR method, as described previously . The primers used were as follows: p23 sense primer, 5-ATGCAGCCTGCTTCTGCAAAGTG-3; S113A (Ser113Ala mutant) sense primer, 5-ATTAGACATGTCTTCATCTGAATCATC-3; S118A sense primer, 5-ATTAGCCATGTCTTCATCTGCATCATC-3; Y14F sense primer, 5-ATGCAGCCTGCTTCTGCAAAGTGGTACACGATCGAAGGGACTTTGTC-3; p23 FLAG antisense primer, 5-TTACTTGTCATCGTCGTCCTTGTAGTCCTCC-AGATCTGGCATTTT-3; S24G antisense primer, 5-GACGGTAAGGATGTTAATG-3; and S151G FLAG IL2RA antisense primer, 5-TTACTTGTCATCGTCGTCCTTGTAGTCCTCCAGATCT-GGCATTTTTTCATCATCACCGTC-3. To construct the S113A mutant, PCR was conducted with a set of p23 sense and S24G antisense primers (for forward strand) and with a set of S113G sense and p23 FLAG antisense primers (for reverse strand) using polymerase (Takara Biomedicals) and cPGES cDNA in pCR3.1 (Invitrogen) as a template with 25?cycles of 95?C for 30?s, 57?C for 30?s and 72?C for 30?s. The resulting forward and back strands were mixed, heated, annealed and subjected to a second PCR with p23 sense and p23 FLAG antisense primers under the same thermal conditions. The S118A mutant was constructed with a set of p23 sense and S24G antisense primers (for forward strand) and S118A sense and p23 FLAG antisense primers (for reverse strand) in a similar way. To construct the S151G mutant, PCR was carried out with a set of p23 sense and S151G FLAG antisense primers. To construct the Y14F mutant, PCR was carried out with a set of Y14F sense and p23 FLAG antisense primers. The resulting PCR products were subcloned into pCR3.1 vector with T4 DNA ligase (Invitrogen) and transformed into Top10F’ supercompetent cells (Invitrogen). The plasmids were isolated and sequenced using a cycle sequencing kit (Takara Biomedicals) and an autofluorometric DNA Biotin Hydrazide sequencer 310 Genetic Analyser (Applied Biosystems) to confirm the mutations. Bacterial manifestation of recombinant cPGES proteins The native and mutant cPGES cDNA inserts were subcloned into pET21c (Novagen) and transformed into BL21 (DE3) (Stratagene). The cells were cultured with 0.3?mM IPTG (isopropyl -D-thiogalactoside) to induce His6-tagged recombinant proteins. Bacterial cell pellets were lysed in 20?mM Tris/HCl (pH?8.0) containing 0.5?mM NaCl, 10% (v/v) Biotin Hydrazide glycerol and 6?M guanidinium chloride with stirring for 30?min at room temp (22?C). After centrifugation at 15000?for 30?min at 4?C, the resulting supernatants were applied to a nickel-nitrilotriacetic acidCagarose column (Qiagen), pre-equilibrated with 100?mM NiSO4 at a circulation rate of 10?ml/h. After washing, the bound proteins were eluted with the same buffer comprising 20C60?mM imidazole inside a stepwise manner. The purity of the recombinant proteins was verified by SDS/PAGE followed by staining with Coomassie Amazing Blue. Transfection studies Transfection of cDNAs into 3Y1?cells was performed by lipofection, as described previously [10,18]. The Biotin Hydrazide transfectants were cloned by limiting dilution in 96-well plates in tradition medium comprising 0.8?mg/ml geneticin (Invitrogen). After tradition for 2C3?weeks, appropriate clones were expanded and utilized for subsequent experiments. Building of.
CK-II activated by upstream signals triggers dual phosphorylation of Ser113 and Ser118 on cPGES, which in turn promotes the recruitment of cPGES into the Hsp90 complex, leading to its full activation
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