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Regulation of class switch recombination and somatic mutation by AID phosphorylation.

McBride KM, Gazumyan A, Woo EM, Schwickert TA, Chait BT, Nussenzweig MC - J. Exp. Med. (2008)

Bottom Line: Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140).Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo.This effect is particularly pronounced in haploinsufficient mice where AID levels are limited.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

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CSR in AIDS38A and AIDT140A mice. (A) Anti-AID immunoblot of AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, wild-type (WT), AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4. Anti-tubulin immunoblot was used as a loading control. (B) Flow cytometric analysis of IgG1 expression and CFSE dye dilution by WT, AID−/−, AIDT140A, and AIDS38A (top) or AID+/−, AID−/−, AIDT140A/−, or AIDS38A/− B cells (bottom) stimulated with LPS and IL-4. The percentage of IgG1+ cells is indicated on the top right of each graph. (C) Isotype switching to IgG1 by WT, AIDT140A, and AIDS38A (top) or AIDT140A/−, AIDS38A/−, and AID+/− cells (bottom) as in B for the indicated number of independent experiments. Solid lines represent means. Bar graphs represent the mean relative efficiency and SD of IgG1 CSR compared with WT. (D) Number of mutations in the 5′ of Sμ region cloned from AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4 sorted for IgM expression and five cell divisions. Pie charts and statistical analysis, as in Fig. 2 D, represent summary of two experiments. The numbers of point mutations were as follows: 3 mutations/59,185 bp mutations for AID−/−; 18 mutations/77,875 bp for WT; 11 mutations/115,255 bp for AIDS38A; and 24 mutations/117,747 bp for AIDT140A. (E) Flow cytometric analysis of IgG3 expression in AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS or LPS and anti-dextran for 6 d. The percentage of IgG3+ cell is indicated on the top left of each graph. (F) Results of E from three independent experiments. Solid line represents the mean.
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fig3: CSR in AIDS38A and AIDT140A mice. (A) Anti-AID immunoblot of AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, wild-type (WT), AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4. Anti-tubulin immunoblot was used as a loading control. (B) Flow cytometric analysis of IgG1 expression and CFSE dye dilution by WT, AID−/−, AIDT140A, and AIDS38A (top) or AID+/−, AID−/−, AIDT140A/−, or AIDS38A/− B cells (bottom) stimulated with LPS and IL-4. The percentage of IgG1+ cells is indicated on the top right of each graph. (C) Isotype switching to IgG1 by WT, AIDT140A, and AIDS38A (top) or AIDT140A/−, AIDS38A/−, and AID+/− cells (bottom) as in B for the indicated number of independent experiments. Solid lines represent means. Bar graphs represent the mean relative efficiency and SD of IgG1 CSR compared with WT. (D) Number of mutations in the 5′ of Sμ region cloned from AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4 sorted for IgM expression and five cell divisions. Pie charts and statistical analysis, as in Fig. 2 D, represent summary of two experiments. The numbers of point mutations were as follows: 3 mutations/59,185 bp mutations for AID−/−; 18 mutations/77,875 bp for WT; 11 mutations/115,255 bp for AIDS38A; and 24 mutations/117,747 bp for AIDT140A. (E) Flow cytometric analysis of IgG3 expression in AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS or LPS and anti-dextran for 6 d. The percentage of IgG3+ cell is indicated on the top left of each graph. (F) Results of E from three independent experiments. Solid line represents the mean.

Mentions: To examine the physiological function of AID phosphorylation at positions S38 and T140, we produced mice that carry S38A or T140A mutations in AID, AIDS38A, and AIDT140A, respectively (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20081319/DC1). AIDS38A and AIDT140A mutant B cells developed normally (not depicted) and were assayed for CSR to IgG1 after stimulation with LPS and IL-4 in culture. Cell division was monitored by labeling cells with CFSE and tracking dye dilution by flow cytometry. AID protein levels expressed by wild-type, AIDS38A, and AIDT140A B cells were indistinguishable when measured by Western blotting (Fig. 3 A), and heterozygous AID+/−, AIDS38A/−, and AIDT140A/− expressed half as much AID as their homozygous counterparts (Fig. 3 A). AIDS38A and AIDT140A B cells divided normally in response to LPS and IL-4, but were impaired in switch recombination to IgG1 (Fig. 3, B and C). AIDS38A mutant B cells showed 32% the level of CSR to IgG1 of wild-type controls, whereas AIDT140 produced a milder defect resulting in 87% of wild-type CSR (Fig. 3, B and C). In both cases, the defect was exacerbated in haploinsufficient mice. Although AID+/− B cells displayed IgG1 switching, 80% of wild-type B cells, AIDS38A/−, and AIDT140A/− showed 4 and 45% the level of wild-type B cells, respectively (Fig. 3, B and C). This relative decrease was consistent within each experiment, despite overall switching rates varying from experiment to experiment (Fig. 3, B and C). We also measured mutation within the region 5′ of the Sμ switch repeats. Similar to CSR, AIDS38A and AIDT140A had mutation rates of 25 and 85%, respectively, compared with WT (Fig. 3 D). Because different stimulation conditions may result in differential levels of AID phosphorylation (Fig. 1 C), we measured isotype switching to IgG3. After stimulation with LPS or LPS and anti-dextran in culture, AIDS38A B cells showed 19 or 16% the level of CSR to IgG3 of wild-type controls, whereas AIDT140A produced a milder defect resulting in 71 or 52% wild-type CSR (Fig. 3, E and F). We conclude that both AID-S38 and -T140 phosphorylation are required for physiological levels of CSR, but neither is essential for this reaction.


Regulation of class switch recombination and somatic mutation by AID phosphorylation.

McBride KM, Gazumyan A, Woo EM, Schwickert TA, Chait BT, Nussenzweig MC - J. Exp. Med. (2008)

CSR in AIDS38A and AIDT140A mice. (A) Anti-AID immunoblot of AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, wild-type (WT), AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4. Anti-tubulin immunoblot was used as a loading control. (B) Flow cytometric analysis of IgG1 expression and CFSE dye dilution by WT, AID−/−, AIDT140A, and AIDS38A (top) or AID+/−, AID−/−, AIDT140A/−, or AIDS38A/− B cells (bottom) stimulated with LPS and IL-4. The percentage of IgG1+ cells is indicated on the top right of each graph. (C) Isotype switching to IgG1 by WT, AIDT140A, and AIDS38A (top) or AIDT140A/−, AIDS38A/−, and AID+/− cells (bottom) as in B for the indicated number of independent experiments. Solid lines represent means. Bar graphs represent the mean relative efficiency and SD of IgG1 CSR compared with WT. (D) Number of mutations in the 5′ of Sμ region cloned from AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4 sorted for IgM expression and five cell divisions. Pie charts and statistical analysis, as in Fig. 2 D, represent summary of two experiments. The numbers of point mutations were as follows: 3 mutations/59,185 bp mutations for AID−/−; 18 mutations/77,875 bp for WT; 11 mutations/115,255 bp for AIDS38A; and 24 mutations/117,747 bp for AIDT140A. (E) Flow cytometric analysis of IgG3 expression in AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS or LPS and anti-dextran for 6 d. The percentage of IgG3+ cell is indicated on the top left of each graph. (F) Results of E from three independent experiments. Solid line represents the mean.
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fig3: CSR in AIDS38A and AIDT140A mice. (A) Anti-AID immunoblot of AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, wild-type (WT), AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4. Anti-tubulin immunoblot was used as a loading control. (B) Flow cytometric analysis of IgG1 expression and CFSE dye dilution by WT, AID−/−, AIDT140A, and AIDS38A (top) or AID+/−, AID−/−, AIDT140A/−, or AIDS38A/− B cells (bottom) stimulated with LPS and IL-4. The percentage of IgG1+ cells is indicated on the top right of each graph. (C) Isotype switching to IgG1 by WT, AIDT140A, and AIDS38A (top) or AIDT140A/−, AIDS38A/−, and AID+/− cells (bottom) as in B for the indicated number of independent experiments. Solid lines represent means. Bar graphs represent the mean relative efficiency and SD of IgG1 CSR compared with WT. (D) Number of mutations in the 5′ of Sμ region cloned from AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS and IL-4 sorted for IgM expression and five cell divisions. Pie charts and statistical analysis, as in Fig. 2 D, represent summary of two experiments. The numbers of point mutations were as follows: 3 mutations/59,185 bp mutations for AID−/−; 18 mutations/77,875 bp for WT; 11 mutations/115,255 bp for AIDS38A; and 24 mutations/117,747 bp for AIDT140A. (E) Flow cytometric analysis of IgG3 expression in AID−/−, WT, AIDT140A, and AIDS38A B cells stimulated with LPS or LPS and anti-dextran for 6 d. The percentage of IgG3+ cell is indicated on the top left of each graph. (F) Results of E from three independent experiments. Solid line represents the mean.
Mentions: To examine the physiological function of AID phosphorylation at positions S38 and T140, we produced mice that carry S38A or T140A mutations in AID, AIDS38A, and AIDT140A, respectively (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20081319/DC1). AIDS38A and AIDT140A mutant B cells developed normally (not depicted) and were assayed for CSR to IgG1 after stimulation with LPS and IL-4 in culture. Cell division was monitored by labeling cells with CFSE and tracking dye dilution by flow cytometry. AID protein levels expressed by wild-type, AIDS38A, and AIDT140A B cells were indistinguishable when measured by Western blotting (Fig. 3 A), and heterozygous AID+/−, AIDS38A/−, and AIDT140A/− expressed half as much AID as their homozygous counterparts (Fig. 3 A). AIDS38A and AIDT140A B cells divided normally in response to LPS and IL-4, but were impaired in switch recombination to IgG1 (Fig. 3, B and C). AIDS38A mutant B cells showed 32% the level of CSR to IgG1 of wild-type controls, whereas AIDT140 produced a milder defect resulting in 87% of wild-type CSR (Fig. 3, B and C). In both cases, the defect was exacerbated in haploinsufficient mice. Although AID+/− B cells displayed IgG1 switching, 80% of wild-type B cells, AIDS38A/−, and AIDT140A/− showed 4 and 45% the level of wild-type B cells, respectively (Fig. 3, B and C). This relative decrease was consistent within each experiment, despite overall switching rates varying from experiment to experiment (Fig. 3, B and C). We also measured mutation within the region 5′ of the Sμ switch repeats. Similar to CSR, AIDS38A and AIDT140A had mutation rates of 25 and 85%, respectively, compared with WT (Fig. 3 D). Because different stimulation conditions may result in differential levels of AID phosphorylation (Fig. 1 C), we measured isotype switching to IgG3. After stimulation with LPS or LPS and anti-dextran in culture, AIDS38A B cells showed 19 or 16% the level of CSR to IgG3 of wild-type controls, whereas AIDT140A produced a milder defect resulting in 71 or 52% wild-type CSR (Fig. 3, E and F). We conclude that both AID-S38 and -T140 phosphorylation are required for physiological levels of CSR, but neither is essential for this reaction.

Bottom Line: Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140).Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo.This effect is particularly pronounced in haploinsufficient mice where AID levels are limited.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

Show MeSH
Related in: MedlinePlus