(A) E. coli-derived recombinant cPGES (3 μg) was incubated for 30 min at 30 °C with recombinant CK-II with (+) or without (−) Hsp90 in the presence of Mg²⁺ and [³²P]ATP. The molar proportion of cPGES, CK-II and Hsp90 was adjusted to 1:1:1. After brief boiling, samples were applied to SDS/PAGE followed by autoradiography (lower panel). Aliquots of samples were taken for PGES enzyme assay using 15 μM PGH₂ as a substrate (upper panel). Values indicate fold-increases in PGES activity relative to that of cPGES alone (n=3; means±S.D.). (B) Effects of various ratios of cPGES, CK-II and Hsp90 on cPGES activity.

(A) Time course of PGE₂ production after incubation for the indicated periods with or without 10 μM BK (lower panel). Results are means±S.D. for four independent experiments. ³²P-incorporation into cPGES (Phospho-cPGES) at each time point was assessed by immunoprecipitation and subsequent autoradiography, and equal precipitation of cPGES protein from each sample was verified by immunoblotting (upper panel). (B) Formation of the cPGES–CK-II–Hsp90 complex and cPGES phosphorylation after stimulation with BK for indicated periods were assessed by the immunoprecipitation (IP) assay (upper panel). PGES activity in cell lysates at each time point was measured (lower panel). Results are means±S.D. for seven independent experiments (*P<0.05 compared with experiments without BK stimulation). (C) Effects of DRB and geldanamycin (GA) on PGE₂ production (left-hand panel) and cPGES enzymic activity in cell lysates (right-hand panel). Cells were pre-incubated for 5 h with these agents and then treated for 1 min with (+) or without (−) BK. Results are means±S.D. for four independent experiments (*P<0.05 compared with BK stimulation without inhibitors). (D) Effects of DRB and GA on the formation of the cPGES–CK-II–Hsp90 complex, as determined by immunoprecipitation and subsequent immunoblotting. A blot representative of five independent experiments is shown.

(A) Schematic structure of cPGES mutants. (B) Expression of FLAG-tagged WT and mutant cPGES proteins in 3Y1 transfectants, as assessed by immunoblotting with anti-FLAG antibody. Equal numbers of cells (10⁵ equivalents) were applied to individual lanes. (C) cPGES enzymic activity in lysates of the transfectants. Values show relative enzymic activity, with that of parental cells regarded as 1. (D–F) PGE₂ production by the transfectants and parental cells after treatment for 30 min with 10 μM AA (D), 10 μM A23187 (E), and 10 μM bradykinin (F). Results are means±S.D. for three to five independent experiments (*P<0.05 compared with replicate parental cells). (G) Phosphorylation of WT and mutant cPGES proteins. Cells pre-labelled with ³²P were lysed and were subjected to immunoprecipitation (IP) with anti-FLAG antibody. Individual samples were resolved by SDS/PAGE and were subjected to autoradiography to detect phosphorylated cPGES or to immunoblotting with anti-FLAG antibody to verify appropriate precipitation of FLAG-tagged proteins from individual samples. (H) Recombinant WT and mutant cPGES proteins were incubated with (+) or without (−) CK-II, and their PGES activities were measured. Result indicates relative PGES activity regarding the activity of WT enzyme without incubation with CK-II as 1. (I) The mixture of cPGES (either WT or mutants), CK-II and Hsp90 was immunoprecipitated with anti-cPGES antibody, applied to SDS/PAGE, and subjected to immunoblotting with respective antibodies.

(A and B) 3Y1 cells were treated for 10 min with (+) or without (−) A23187 in the presence (+) or absence (−) of 1 μM Dex, and PGE₂ (A) and AA (B) released into the supernatants were measured. Results are means±S.D. for three independent experiments. (C) Cell lysates were subjected to immunoprecipitation (IP) with anti-cPGES antibody, followed by immunoblotting with antibodies against Hsp90 and cPGES. (D) Replicate lysates of ³²P-pre-labelled cells were subjected to immunoprecipitation with anti-cPGES antibody, followed by SDS/PAGE and autoradiography or immunoblotting with anti-cPGES antibody. Blots in (C) and (D) are representative of three independent experiments.

(A) Putative phosphorylation sites on human cPGES predicted from its primary amino acid sequence. The asterisk indicates a tyrosine residue required for enzyme catalysis [10]. TK, tyrosine kinase. (B) ³²P-pre-labelled 3Y1 cells were incubated for 1 min with or without A23187, and cPGES protein was immunoprecipitated from cell lysates. Then the pellets were resolved on SDS/PAGE and subjected to autoradiography (lower panel). Relative incorporation of ³²P into cPGES protein was determined by scanning with a BAS image analyser (Fuji film) (upper panel). (C) Phospho-amino acid analysis. ³²P-containing cPGES protein bands with or without A23187 stimulation were excised from the SDS/PAGE gel, digested, and separated by two-dimensional TLC. The positions of phosphoserine, phosphothreonine and phosphotyrosine residues are shown. (D) Effect of calf intestine alkaline phosphatase (CIAP) on cPGES activity in 3Y1 cells. After incubation of cell lysate with (+) or without (−) CIAP for 2 h at 37 °C, PGES activity was measured. (E) Effects of various protein kinase inhibitors on PGE₂ production by 3Y1 cells after incubation for 30 min with 10 μM AA. The cells were pre-treated with 10 μM chelerythrine (PKC inhibitor), 0.4 μM KN-93 (CaMK-II inhibitor), 100 μM genistein (tyrosine kinase inhibitor), 100 μM DRB (CK-II inhibitor) or vehicle (DMSO) for 5 h. PGE₂ released into the supernatants was quantified (n=4; means±S.D.; *P<0.01 compared with vehicle). (F) Effects of protein kinase inhibitors on cPGES phosphorylation. ³²P-pre-labelled 3Y1 cells were pre-incubated for 5 h with the aforementioned protein kinase inhibitors, and incorporation of ³²P into cPGES was assessed by immunoprecipitation, followed by SDS/PAGE and autoradiography. Immunoblotting with anti-cPGES antibody was performed to confirm equal sample loading into each lane. IP, immunoprecipitation; IB, immunoblotting. Representative results of three independent experiments are shown in (B–D) and (F).

(A) 3Y1 cells pre-treated for 5 h with or without 100 μM genistein (Gen) or 100 μM DRB were stimulated with (+) or without (−) 10 μM A23187 for 1 min, and PGE₂ production (middle panel) and cPGES enzymic activity (bottom panel) were assessed. Lysates of replicate ³²P-pre-labelled cells were subjected to immunoprecipitation with anti-cPGES antibody, followed by SDS/PAGE and autoradiography to detect phosphorylated cPGES (top panel). Equal precipitation of cPGES protein from each sample was verified by immunoblotting. (B) Time course of PGE₂ generation (left-hand panel) and cPGES enzymic activity (right-hand panel) after A23187 stimulation (n=4; means±S.D.). (C) A23187-induced formation of the cPGES–CK-II–Hsp90 complex. Cells were stimulated for the indicated periods with A23187, lysed, and subjected to immunoprecipitation (IP) with anti-cPGES antibody. The pellets were resolved by SDS/PAGE, followed by immunoblotting with anti-Hsp90, anti-CK-IIα and anti-cPGES antibodies. Effects of 100 μM DRB (D) or 1 μM geldanamycin (GA) (E) on cPGES–CK-II–Hsp90 complex formation. Cells were pre-treated with these compounds for 5 h and then activated for 1 min with A23187. Cell lysates were taken for immunoprecipitation (IP) with anti-cPGES antibody, followed by SDS/PAGE and immunoblotting with anti-Hsp90, anti-CK-IIα and anti-cPGES antibodies. Representative results of three or four independent experiments are shown in (A) and (C–E).

(A) Expression levels of PGE₂-biosynthetic enzymes in mock- or DN-CK-II-transfected 3Y1 cells, as assessed by immunoblotting. (B) Association of cPGES with Hsp90 (top panel) and phosphorylation of cPGES (middle panel) in mock- or DN-CK-II-transfected cells. cPGES–Hsp90 association was assessed by immunoprecipitation (IP) and subsequent SDS/PAGE and immunoblotting, and cPGES phosphorylation was assessed by immunoprecipitation from replicate ³²P-labelled cells, followed by SDS/PAGE and autoradiography. Equal sample loading into each lane was verified by immunoblotting with anti-cPGES antibody (bottom panel). Blots representative of three independent experiments are shown. (C) cPGES enzymic activity in lysates of mock- or DN-CK-II-transfected cells. Values show relative enzymic activity, with the activity of control cells regarded as 1. (D) PGE₂ production by mock- or DN-CK-II-transfected cells treated for 30 min with 10 μM AA. (E) PGE₂ production by A23187-stimulated mock- or DN-CK-II-transfected cells. Cells were treated with (+) or without (−) 10 μM A23187 for 30 min. (F) AA release from mock- or DN-CK-II-transfected 3Y1 cells. Cells were pre-incubated with [³H]AA, and [³H]AA release with (+) or without (−) stimulation with A23187 for 30 min was measured. In (C–F), results are means±S.D. of three to five independent experiments (*P<0.05 compared with replicate mock-transfected cells).

(A and B) 3Y1 cells were treated for 2 min with (+) or without (−) bradykinin (BK) in the presence or absence of the p38 inhibitor SB20358 (SB), and PGE₂ released into the supernatants (A) and PGES enzymic activity in cell lysates (B) were measured. Results are means±S.D. for three independent experiments (*P<0.05 compared with BK stimulation in the absence of SB). (C) Cell lysates were subjected to immunoprecipitation with anti-cPGES antibody, followed by SDS/PAGE and immunoblotting with antibodies for Hsp90, CK-IIα and cPGES. Replicate immunoprecipitates from ³²P-pre-labelled cells were subjected to immunoprecipitation (IP), SDS/PAGE, and then autoradiography to detect a phosphorylated form of cPGES. Blots are representative of three experiments.