727 phosphorylation and robust activation of STAT1 (Fig. 2B). Poly(dA:dT727 phosphorylation and robust activation of

727 phosphorylation and robust activation of STAT1 (Fig. 2B). Poly(dA:dT
727 phosphorylation and robust activation of STAT1 (Fig. 2B). Poly(dA:dT) transfection induced association of STAT3 with TBK1 and IKK , whereas VACV70mer induced association of STAT3 with TBK1 but not IKK (Fig. 2B). These stimuli also induced many degrees of IKK /IKK activation and p65 phosphorylation, indicative of NF- B activation (Fig. 2B). Lastly, a time course experiment revealed that association amongst TBK1 and STAT3 was induced as early as 1.five h right after dsDNA transfection, coinciding together with the timing of Ser754 phosphorylation, whereas Tyr705 phosphorylation of STAT3 was detected at a later time point (Fig. 2C). Taken with each other, these information show that among differentVOLUME 292 sirtuininhibitorNUMBER 13 sirtuininhibitorMARCH 31,Final results TBK1 Directly Phosphorylates STAT3 at Serine 754 –Previous studies demonstrated that TBK1 and IKK , respectively, regulate the function of STAT6 and STAT1 by direct phosphorylation (14, 26). To figure out no matter whether TBK1 and IKK also phosphorylate other STATs, we examined the sequences of STATs against the optimal substrate motif for TBK1 and IKK (30, 31) and identified serine 754 within the TAD of STAT3 as a prospective TBK1/IKK phosphorylation web site (Fig. 1A). To test no matter if STAT3 is often a substrate of TBK1 or IKK , IKK and STAT3 have been overexpressed in HEK293T cells, and immunoprecipitated STAT3 was blotted with an IKK household phosphosubstrate motif antibody (see “REG-3 alpha/REG3A Protein Purity & Documentation Experimental Procedures”). Overexpression of wild-type IKK strongly induced phosphorylation of STAT3 at Tyr705 also as a internet site recognized by the IKK phosphosubstrate motif antibody (Fig. 1B). We then gen-5406 JOURNAL OF BIOLOGICAL CHEMISTRYTBK1 Regulates STAT3 Activity in Response to Cytosolic DNAFIGURE 1. IKK and TBK1 induce STAT3 phosphorylation at Ser754. A, a schematic figure displaying the domain structure of STAT3 along with the place of Ser754. Sequences of STAT3 from numerous vertebrates were aligned to examine the homology, with residues important for TBK/IKK substrate recognition marked in blue and Ser754 marked in red. Also shown would be the domain structure of STAT1 and STAT6, with previously identified IKK /TBK1 target serine residues marked in red along with the sequence alignments shown around the right. NTD, N-terminal domain. B, FLAG-tagged STAT3 (three g) was co-transfected with GST-IKK (three g) into HEK293T cells. STAT3 was immunoprecipitated (IP) and blotted with an IKK substrate motif antibody. C, FLAG-tagged wild-type, Y705F, S727A, or S754A STAT3 (3 g) had been co-transfected with wild-type or K38A (kinase-dead; KD) GST-TBK1 (3 g) into HEK293T cells. STAT3 was immunoprecipitated and blotted with phosphorylation-specific antibodies to detect Ser754, Tyr705, and Ser727 phosphorylation. D, in vitro SHH, Mouse kinase assay employing the GST-tagged C terminus of STAT3 and GST-TBK1 purified from HEK293T as described under “Experimental Procedures.” The mixture was resolved by SDS-PAGE and blotted (WB) with GST and Ser(P)754-STAT3 antibodies. Phosphorylation of STAT3 was also detected by autoradiography. Data in B are representative of 4, 3, and two independent experiments, respectively.TBK1 activators, cytosolic DNA induces probably the most robust STAT3 activation and phosphorylation at Ser754. TBK1 Is Needed for Cytosolic DNA-induced STAT3 Phosphorylation at Ser754–Given that cytosolic DNA induces robust TBK1 activation, STAT3 phosphorylation, and association amongst TBK1 and STAT3, we asked regardless of whether TBK1 is necessary for cytosolic DNA-induced Ser754 phosphorylation of STAT3. We fou.