Amino-Terminal Phosphorylation of Activation-Induced Cytidine Deaminase Suppresses c-myc/IgH Translocation

Abstract

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

“…Consistent with this view, the AID mutant G23S (47), which has compromised SHM but intact CSR activity, and the mutant S3A (48,49), which has a higher CSR activity than WT, both had intact dimerization ability and also migrated to the HMW region upon glycerol gradient centrifugation (Fig. S5).…”

Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation

“…Although no mutations at the N terminus of AID have been shown to cause CSR-specific loss of the AID function, some AID mutants with point mutations in the N-terminal region retain substantial CSR activity but severely damage SHM activity, which is most likely due to a combination of partial loss of DNA cleavage activity and less efficient cleavage of the V region compared with S regions (18)(19)(20). Conversely, a S3A mutaiton augments both CSR and SHM (21). On the other hand, the deletions and/or mutations in the nuclear export signal region (residues 183-198) result in loss of the AID function for CSR but not SHM, probably because AID with the C-terminal deletion has normal DNA cleavage activity (19,22).…”

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

“…B cell isolation and the pMX retroviral infection have been previously described (19,20). In brief, primary naive B lymphocytes from WT and UNG Ϫ/Ϫ mouse spleens were purified by negative selection with anti-CD43 beads (Miltenyi Biotec, Auburn, CA) and cultured with 25 g of lipopolysaccharide (LPS)/ml and 5 ng of interleukin-4 (IL-4; Sigma-Aldrich)/ml.…”

“…After recovery, the bacterial cells were plated on dual chloramphenicol (34 g/ml)-Kan (50 g/ml) plates, followed by incubation at 30°C for 48 h. DNA was prepared from isolated colonies, and the Kan selection marker in orf46 was PCR amplified to verify the insertion of the mutagenesis cassette into the MHV68-H2bYFP BAC. Using a protocol outlined previously (20), the kanamycin selection marker was removed, leaving behind the desired ORF46 stop mutation. The orf46 gene was PCR amplified from the putative mutant BAC, digested with DraI to confirm the presence of the stop codon and then PCR products harboring the newly introduced DraI site were sequenced (Laragen, Inc., Culver City, CA).…”

“…Supernatants were harvested 2 to 3 days after transfection and used to infect purified naive B cells after 16 h of culture in LPS and IL-4. To determine relative class switching to IgG1, naive and/or retrovirally transduced primary B cells were cultured, stained with antimouse IgG1 antibodies (Becton Dickinson), and analyzed by flow cytometry as previously described (19,20).…”