Figure 1. cPGES is phosphorylated on serine residues in 3Y1 cells.

(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).