Rev. well as double-stranded breaks that result in oncogenic chromosome translocations such as those between c-and (c-signaling pathways that impact AID phosphorylation have not been determined and no phosphatase has been reported to influence AID phosphorylation (3, 31, 36). Here we identify a novel mechanism of AID regulation by phosphorylation of serine 3, which, in contrast to serine 38 or threonine 140, acts to suppress AID activity. We show that phosphorylation of serine 3 is controlled by protein phosphatase 2 (PP2A). MATERIALS AND METHODS Protein analysis. Anti-AID antibodies were previously described (30, 31). To produce anti-pS3 antibodies, rabbits were immunized with phosphopeptide MD(pS)LLMKQC (AID 1 to 8) coupled to keyhole limpet hemocyanin. Phospho-specific antibodies were purified by negative selection on unphosphorylated peptide coupled to Sulfolink gel (Thermo Fisher Scientific), followed by positive selection on the phosphopeptide. Cells were extracted in lysis buffer (20 mM Tris, pH 8.0, 200 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 25 mM NaF, 0.1 mM vanadate, and 1 mM dithiothreitol [DTT]). For immunoprecipitation, 1 mg of extracts was incubated with anti-Flag agarose beads (Sigma-Aldrich) and AID was eluted with 0.5 g/ml of Flag peptide (Sigma-Aldrich) in lysis buffer. Western blots were performed on immunoprecipitated protein or total cell extracts with the indicated anti-AID antibody; anti-green fluorescent protein (anti-GFP) (Santa Cruz) was Rabbit polyclonal to ZNF625 used as a loading control, and anti-phosphoserine PKC substrate (Cell Signaling) was used to blot for phosphoserine. To phosphorylate AID dephosphorylation, recombinant phosphorylated AID was incubated with 1 U of purified PP2A (Upstate) for 30 min at 30C in 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.2 mg/ml bovine Asenapine maleate serum albumin (BSA). Mass spectrometry analysis of phosphorylation was performed on phosphorylated recombinant AID as previously described (30). Lymphocyte isolation, culture, and retroviral infection. Lymphocyte isolation, cultures, carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, retrovirus infection with pMX-mK-AID, and CSR to IgG1 analysis were as described previously (30, 31). Retroviral AID-Flag contained a Flag tag fused in frame to the carboxy terminus of AID. B cells were purified from mouse spleens by depletion with anti-CD43 beads (Miltenyi Biotec) and cultured in 25 g/ml lipopolysaccharide (LPS) (Sigma-Aldrich) with 5 ng/ml interleukin-4 (IL-4) (Sigma-Aldrich). Cells were stained with APC anti-mouse IgG1 (BD Biosciences). Cells were treated with the phosphatase inhibitors endothall, calyculin, and okadaic acid (Calbiochem). For the NTZ-3T3 assay, pMX-mK-AID vector with the GFP coding portion removed was used. PCR, mutation analysis, and translocation assay. The NTZ-3T3 assay and GFP gene mutational analyses were performed as previously described 9 days after retrovirus infection (29, 60). The c-value was calculated using a two-tailed Fisher’s exact test. Q-PCR analysis. RNA was Asenapine maleate extracted using Trizol (Invitrogen), cDNA prepared Asenapine maleate using Superscript II reverse transcriptase (Invitrogen) and quantitative PCR (Q-PCR) was performed using Brilliant SYBR green QPCR master mix (Stratagene) as per the manufacturer’s protocol. Reactions were performed in triplicate and analyzed with an MX3000P Q-PCR machine (Stratagene). Reactions were normalized to GAPDH. Primers used were as follows: GLT forward, 5-TAGTAAGCGAGGCTCTAAAAAGCAT; reverse, 5-AGAACAGTCCAGTGTAGGCAGTAGA; IgG1 GLT forward, 5-TATGATGGAAAGAGGGTAGCATTCACC; reverse, 5-CTCCTTCCCAATCTCCCGTG. deamination assay. The AID catalytic assay in was performed exactly as described previously (48). For the UNG cleavage assay, a 50-base oligonucleotide (5-GGAATTGAGTTGGTAGGGTAGCTAGGAGGTAAGTAGGGAAGATGGATGAT-3) was labeled with [-32P]ATP and T4 polynucleotide kinase. The single-stranded DNA (ssDNA) oligonucleotide was incubated with purified recombinant GST-AID or AID-S3A, deglycosylated with UDG (New England Biolabs), treated with.