Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice

Abstract

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Fig. 1.

AID expression and RPA interaction in AID^S38A mutant mice. (A) AID protein levels in anti-CD40 plus IL-4–stimulated splenic B cells. Sixty micrograms of whole cell extracts from purified and activated splenic B cells from mice of the indicated genotypes harvested at day 2.5 of stimulation were separated by SDS-PAGE and immunoblotted with antibodies to AID or tubulin. Results from 2 independent experiments (Exp.) are shown. (B) Protein levels in LPS + anti-δ-dextran–treated splenic B cells. Whole cell extracts (60 μg) were analyzed as in A in response to LPS-IgD-dextran stimulation. (C) Interaction of AID^WT and AID^S38A mutant proteins with RPA. Nuclear extracts from AID-deficient (−/−), WT (+/+), and AID^S38/S38A mice were immunoprecipitated (IP) with AID, and the immunoprecipitate was analyzed by Western blotting (WB) for the presence of the 32-kDa subunit of RPA (Top) or AID (Bottom). Ig, Ig chains.

Fig. 2.

CSR activity in AID^S38A/S38A and AID^S38A/− mice for IgG1. B cells were isolated from the spleen of mice of the indicated genotypes and stimulated for CSR to IgG1 for 3, 3.5, and 4.5 days using anti-CD40 antibodies and IL-4. Levels of CSR in these B cells were analyzed using FACS as described in Materials and Methods. The percentage of IgG1⁺B220⁺ cells for each genotype was plotted against the days in culture. Each bar graph represents the mean of multiple experiments (Table S1), with the error bars representing SD from the mean. AID-deficient splenic B cells activated with anti-CD40 plus IL-4 were used as controls, and less than 0.1% of the cells stained for IgG1 at all 3 time points (Table S1).

Fig. 3.

CSR activity in AID^S38A/S38A and AID^S38A/− mice for IgG3. B cells isolated from mice of the indicated genotypes were stimulated for CSR to IgG3 for 3, 3.5, and 4.5 days using LPS and anti-δ-dextran. Levels of CSR in these B cells were analyzed using FACS. The percentage of IgG3⁺B220⁺ cells was plotted against time in culture. Each bar graph represents the mean of multiple experiments (Table S1), with the error bars representing SD from the mean. AID-deficient splenic B cells activated with LPS plus anti-δ-dextran were used as controls, and less than 0.4% of the cells stained for IgG1 at all 3 time points (Table S1).

Fig. 4.

SHM in Peyer's patch B cells. SHM within a 672-bp region of DNA downstream of J_H4 was detected using AID^WT/WT and AID^S38A/S38A Peyer's patch germinal center B cells. Three 12-week-old AID^S38A/S38A mice and 2 age-matched WT control mice were used. Mutation error rate attributable to PCR using Pfu (6.76 mutations/10⁻⁵ bp) was obtained by analyzing SHM in B cells obtained from AID-deficient mice.