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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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SHM in GC B cells from AIDS38A and AIDT140A mice. (A) GC B cells were purified from the lymph nodes of 5 immunized AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, WT, AIDT140A, or AIDS38A mice. Pie charts indicated the number of mutations in the intronic region 3′ of JH4. Pie charts and statistical analysis as in 2D. The numbers of point mutations were as follows: 2 mutations/33,666 bp mutations for AID−/−; 184 mutations/115,116 bp for WT; 41 mutations/96,654 bp for AIDS38A; and 65 mutations/85,794 bp for AIDT140A (top). 2 mutations/43,440 bp mutations for AID−/−; 51 mutations/46,689 bp for AID+/−; 5 mutations/66,246 bp for AIDS38A/−; and 8 mutations/71,676 bp for AIDT140A/− (bottom). (B) GC B cells purified from Peyer's patch analyzed the same as A. The numbers of point mutations were as follows: 2 mutations/43,983 bp mutations for AID−/−; 227 mutations/45,612 bp for WT; 33 mutations/35,295 bp for AIDS38A; and 95 mutations/46,698 bp for AIDT140A (top). 1 mutation/179,191 bp mutations for AID−/−; 34 mutations/20,091 bp for AID+/−; 4 mutations/31,494 bp for AIDS38A/−; and 12 mutations/17,919 bp for AIDT140A/− (bottom). (C) Summary of efficiency of both SHM and isotype switching to IgG1 relative to wild-type by AIDT140A/−, AIDS38A/−, AIDT140A,and AIDS38A/AID+/− cells. Bars represent the means from and statistics are reported in Figs. 3 C, 3 D, and 4 [A and B].
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fig4: SHM in GC B cells from AIDS38A and AIDT140A mice. (A) GC B cells were purified from the lymph nodes of 5 immunized AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, WT, AIDT140A, or AIDS38A mice. Pie charts indicated the number of mutations in the intronic region 3′ of JH4. Pie charts and statistical analysis as in 2D. The numbers of point mutations were as follows: 2 mutations/33,666 bp mutations for AID−/−; 184 mutations/115,116 bp for WT; 41 mutations/96,654 bp for AIDS38A; and 65 mutations/85,794 bp for AIDT140A (top). 2 mutations/43,440 bp mutations for AID−/−; 51 mutations/46,689 bp for AID+/−; 5 mutations/66,246 bp for AIDS38A/−; and 8 mutations/71,676 bp for AIDT140A/− (bottom). (B) GC B cells purified from Peyer's patch analyzed the same as A. The numbers of point mutations were as follows: 2 mutations/43,983 bp mutations for AID−/−; 227 mutations/45,612 bp for WT; 33 mutations/35,295 bp for AIDS38A; and 95 mutations/46,698 bp for AIDT140A (top). 1 mutation/179,191 bp mutations for AID−/−; 34 mutations/20,091 bp for AID+/−; 4 mutations/31,494 bp for AIDS38A/−; and 12 mutations/17,919 bp for AIDT140A/− (bottom). (C) Summary of efficiency of both SHM and isotype switching to IgG1 relative to wild-type by AIDT140A/−, AIDS38A/−, AIDT140A,and AIDS38A/AID+/− cells. Bars represent the means from and statistics are reported in Figs. 3 C, 3 D, and 4 [A and B].

Mentions: To examine the role of AID phosphorylation in SHM, we cloned and sequenced the DNA region downstream of IgJH4 from purified lymph node GC B cells (46). The effect of AIDS38A on somatic mutation was similar to CSR, resulting in 30% of wild-type activity (Fig. 4 A). In contrast, AIDT140A had more profound effects on somatic mutation than CSR, resulting in 45% of wild-type activity, which was not significantly different from AIDS38A (Fig. 4 A). Similar results were obtained from Peyer's patch B cells, where mutation rates for AIDS38A and AIDT140A were 20 and 40% of controls, respectively (Fig. 4 B). Haploinsufficiency by itself resulted in a mild decrease in hypermutation with 70% hypermutation activity in AID+/− versus wild type (Fig. 4 A). Haploinsufficiency magnified the defect in hypermutation of AIDS38A/− and AIDT140A/−, with neither statistically rising above the background levels of mutation found in AID−/− lymph node GC B cells (Fig. 4 A). Similar, but slightly less pronounced, effects were found in chronically stimulated Peyer's patch GC B cells (Fig. 4 B). We conclude that both AID-S38 and -T140 phosphorylation are required for optimal somatic mutation and that the T140 has a more profound effect on this reaction than on CSR (Fig. 4 C).


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)

SHM in GC B cells from AIDS38A and AIDT140A mice. (A) GC B cells were purified from the lymph nodes of 5 immunized AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, WT, AIDT140A, or AIDS38A mice. Pie charts indicated the number of mutations in the intronic region 3′ of JH4. Pie charts and statistical analysis as in 2D. The numbers of point mutations were as follows: 2 mutations/33,666 bp mutations for AID−/−; 184 mutations/115,116 bp for WT; 41 mutations/96,654 bp for AIDS38A; and 65 mutations/85,794 bp for AIDT140A (top). 2 mutations/43,440 bp mutations for AID−/−; 51 mutations/46,689 bp for AID+/−; 5 mutations/66,246 bp for AIDS38A/−; and 8 mutations/71,676 bp for AIDT140A/− (bottom). (B) GC B cells purified from Peyer's patch analyzed the same as A. The numbers of point mutations were as follows: 2 mutations/43,983 bp mutations for AID−/−; 227 mutations/45,612 bp for WT; 33 mutations/35,295 bp for AIDS38A; and 95 mutations/46,698 bp for AIDT140A (top). 1 mutation/179,191 bp mutations for AID−/−; 34 mutations/20,091 bp for AID+/−; 4 mutations/31,494 bp for AIDS38A/−; and 12 mutations/17,919 bp for AIDT140A/− (bottom). (C) Summary of efficiency of both SHM and isotype switching to IgG1 relative to wild-type by AIDT140A/−, AIDS38A/−, AIDT140A,and AIDS38A/AID+/− cells. Bars represent the means from and statistics are reported in Figs. 3 C, 3 D, and 4 [A and B].
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fig4: SHM in GC B cells from AIDS38A and AIDT140A mice. (A) GC B cells were purified from the lymph nodes of 5 immunized AID−/−, AID+/−, AIDT140A/−, AIDS38A/−, WT, AIDT140A, or AIDS38A mice. Pie charts indicated the number of mutations in the intronic region 3′ of JH4. Pie charts and statistical analysis as in 2D. The numbers of point mutations were as follows: 2 mutations/33,666 bp mutations for AID−/−; 184 mutations/115,116 bp for WT; 41 mutations/96,654 bp for AIDS38A; and 65 mutations/85,794 bp for AIDT140A (top). 2 mutations/43,440 bp mutations for AID−/−; 51 mutations/46,689 bp for AID+/−; 5 mutations/66,246 bp for AIDS38A/−; and 8 mutations/71,676 bp for AIDT140A/− (bottom). (B) GC B cells purified from Peyer's patch analyzed the same as A. The numbers of point mutations were as follows: 2 mutations/43,983 bp mutations for AID−/−; 227 mutations/45,612 bp for WT; 33 mutations/35,295 bp for AIDS38A; and 95 mutations/46,698 bp for AIDT140A (top). 1 mutation/179,191 bp mutations for AID−/−; 34 mutations/20,091 bp for AID+/−; 4 mutations/31,494 bp for AIDS38A/−; and 12 mutations/17,919 bp for AIDT140A/− (bottom). (C) Summary of efficiency of both SHM and isotype switching to IgG1 relative to wild-type by AIDT140A/−, AIDS38A/−, AIDT140A,and AIDS38A/AID+/− cells. Bars represent the means from and statistics are reported in Figs. 3 C, 3 D, and 4 [A and B].
Mentions: To examine the role of AID phosphorylation in SHM, we cloned and sequenced the DNA region downstream of IgJH4 from purified lymph node GC B cells (46). The effect of AIDS38A on somatic mutation was similar to CSR, resulting in 30% of wild-type activity (Fig. 4 A). In contrast, AIDT140A had more profound effects on somatic mutation than CSR, resulting in 45% of wild-type activity, which was not significantly different from AIDS38A (Fig. 4 A). Similar results were obtained from Peyer's patch B cells, where mutation rates for AIDS38A and AIDT140A were 20 and 40% of controls, respectively (Fig. 4 B). Haploinsufficiency by itself resulted in a mild decrease in hypermutation with 70% hypermutation activity in AID+/− versus wild type (Fig. 4 A). Haploinsufficiency magnified the defect in hypermutation of AIDS38A/− and AIDT140A/−, with neither statistically rising above the background levels of mutation found in AID−/− lymph node GC B cells (Fig. 4 A). Similar, but slightly less pronounced, effects were found in chronically stimulated Peyer's patch GC B cells (Fig. 4 B). We conclude that both AID-S38 and -T140 phosphorylation are required for optimal somatic mutation and that the T140 has a more profound effect on this reaction than on CSR (Fig. 4 C).

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