Characterization of Two Mosquito STATs, Aa STAT and Ct STAT DIFFERENTIAL REGULATION OF TYROSINE PHOSPHORYLATION AND DNA BINDING ACTIVITY BY LIPOPOLYSACCHARIDE TREATMENT AND BY JAPANESE ENCEPHALITIS VIRUS INFECTION*

Two mosquito STATs, Aa STAT and Ct STAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila , Anopheles , and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of Aa STAT and Ct STAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of Aa STAT which could be restored by sodium orthovanadate treatment. Taking all of the data to-gether, this is the first report to clone and characterize m M EDTA) m M Na 3 VO and 2 m M phenylmethylsulfonyl fluoride. The embryos, larvae, pupae, and adults homogenized with the same lysis buffer. Extracts were centrifuged at 4 °C for 10 min at 13,000 (cid:2) g , and the resulting supernatants were used for subsequent Western blotting. Nuclear extracts were prepared ac- cording to the procedures described (31). Briefly, cells were washed twice with phosphate-buffered saline and solubilized with A m M HEPES, m M KCl, m M EDTA, m M dithiothreitol, m M phenylmethylsulfonyl fluoride, 0.1 m M Na 3 VO , m M Na 4 P 2 O , m M NaF, 10% glycerol, 1 (cid:5) g/ml of aprotinin, pepstatin, and leupep-tin, pH 7.9) on ice for 30 min and then vortexed vigorously and centri- fuged at 12,000 (cid:2) g at 4 °C for 1 min. The supernatant was the cytoplasmic part at this stage. The pellets were then extracted with buffer B (20 m M HEPES, 350 m M NaCl, 10 m M KCl, 1 m M EDTA, 1 m M dithiothreitol, 1 m M phenylmethylsulfonyl fluoride, 0.1 m M Na 3 VO 4 , 0.2% Nonidet P-40, 10% glycerol, 1 (cid:5) g/ml aprotinin, pepstatin, and leupeptin, pH 7.9), rotated at 4 °C for 30 min. The extracts were cen- trifuged at 13,000 (cid:2) g at 4 °C for 5 min, and these supernatants, nuclear extracts, were quickly frozen and stored at (cid:5) 70 °C for subse- quent use by electrophoretic mobility shift assay (EMSA) or Western blotting. The