The IB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. well as the phosphopeptide only contained phosphoserine (PS) (Physique 3E). Taken together, these results demonstrate that IKK is usually a novel BAD kinase that phosphorylates BAD at serine residue(h). IKK is usually necessary and sufficient to phosphorylate BAD at Ser26 in vitro and vivo To identify IKK-phosphorylated serine residue(s), we constructed a C-terminal truncated GST-C-BAD(1C114) and an N-terminal truncated GST-N-BAD(115C204) (Physique H3A). Immune complex kinase assays showed that GST-N-BAD(115C204) was phosphorylated by basal IKK and the phosphorylation was only slightly increased when active IKK was used (Physique H3A). By contrast, phosphorylation of GST-C-BAD(1C114) was significantly enhanced when TNF-activated IKK was Zibotentan used (Physique H3A). Two-dimensional phosphopeptide mapping analysis revealed that in comparison to GST-BAD, GST-N-BAD contained phosphopeptide (major) and (minor) (Physique H3W). These results indicate that active IKK phosphorylation site(s) is usually located in the N-terminal half of BAD. To determine the precise IKK-phosphorylation site(s) on BAD, we systemically replaced all serine residues within the N-terminal half (1C114) in the full-length GST-BAD with non-phosphorylatable alanines either individually or in different combinations, using a site-directed mutagenesis approach (Physique H3C). Immune complex kinase assays showed that TNF-activated IKK was unable to phosphorylate the GST-BAD(S26A) mutant in comparison to WT GST-BAD (Physique 4A). Comparable results were obtained with purified IKK(EE) (Physique 4B). By contrast, other GST-BAD mutants were still phosphorylated by active IKK (Physique H3Deb). Two-dimensional phosphopeptide mapping revealed that the replacement of Ser26 by Ala resulted in complete elimination of the phosphopeptide and but had no effects on phosphopeptide (Physique 4C). Analysis of IKK-phosphorylated GST-BAD proteins by tandem mass spectrometry (MS/MS) also revealed that Ser26 was phosphorylated by IKK (Physique 4D). Physique 4 IKK Is usually Necessary and Sufficient to Phosphorylate BAD at Ser26 In Vitro and In Vivo. (A and W) Phosphorylation of GST-BAD and GST-BAD(S26A) mutant proteins by active IKK (A) or purified IKK(EE) proteins (B), as described in Figure 3B. (C) Two-dimensional … To analyze the regulation of BAD Ser26 phosphorylation in vivo, we generated a Zibotentan rabbit polyclonal antibody using a synthetic BAD phosphopeptide containing phosphorylated Ser26 as an immunogen. Immunoblotting analysis revealed that the anti-phospho-Ser26 antibody specifically recognized active IKK-phosphorylated GST-BAD but not non-phosphorylated GST-BAD or GST-BAD(S26A) mutant (Figure 4E). This was not a result of the difference in the amount of GST-BAD proteins, as analyzed by immunoblotting using anti-GST antibody (Figure 4E). Thus, anti-phospho-Ser26 antibody Zibotentan specifically recognizes BAD when it is phosphorylated at Ser26 by IKK. To determine whether BAD is phosphorylated at Ser26 in response to TNF in an IKK-dependent manner, we used knockout mice that have been reconstituted with WT or [cell death was analyzed by TUNEL staining (TUNEL Apoptosis Detection kit, EMD Millipore), according to the manufacturers protocol. Statistics analysis The Zibotentan Statistic analysis was performed by either the Student test or the log-ranked (Mantel-Cox) test. ? Highlights IKK is able to inhibit TNF-induced apoptosis independently of NF-B activation Inhibition of BAD constitutes the NF-B-independent anti-apoptotic axis of IKK IKK phosphorylates BAD at Ser26 and primes it for inactivation BAD inactivation coordinates with NF-B activation to suppress TNF-induced apoptosis Supplementary Material 01Click here to view.(58K, doc) 02Click here to view.(1.4M, pdf) Acknowledgments We are grateful TNFRSF1A to Drs. David A. Brenner, Joseph A. DiDonato, Michael Karin, Stanley Korsmeyer, and Frank Mercurio for reagents that make this work possible. This work is partially supported by National Basic Research Program of China (2012CB910801), National Natural Science Foundation of China (31130035), Chinese Academy of Sciences (SIBS2010CSP001), and National Institutes of Zibotentan Health grants CA100460 (to A.L.), CA128114 (to X.L.), and GM081603 (to J.L). Footnotes Publisher’s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain..