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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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Comparison of AID, AIDT140A, and AIDS38A hypermutation and CSR activity in B and non–B cells. (A) The graph shows a log plot of numbers of kanamycin-resistant (KanR) colonies after induction of AID, AIDS38A, AIDT140A, AIDST-AA, or empty vector expression. (B) Anti-p140, -p38, or -AID immunoblot of FLAG-tagged AID, AIDS38A, or AIDT140A purified from B cells stimulated with LPS and IL-4. (C) Accumulation of GFP-expressing 3T3-NTZ cells after transduction with AID-, AIDS38A-, or AIDT140A-expressing PMX-MK retroviruses. The x axis indicates the number of days after transduction, and the y axis indicates the percentage of GFP-positive cells measured by flow cytometry. (D) Number of mutations in the GFP gene cloned from 3T3-NTZ cells 11 d after transduction with retroviruses encoding AID, AIDS38A, AIDT140A, or control. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The total number of independent sequences analyzed is indicated in the center of each chart. Statistical significance was determined by a two-tailed Student's t test assuming unequal variance and comparing AID expressing with AIDS38A- and AIDT140A-expressing cells. P values are indicated. The numbers of point mutations were as follows: 4 mutations/21,643 bp mutations for vector; 59 mutations/13,174 bp for AID; 15 mutations/19,761 bp for AIDS38A; and 45 mutations/21,643 bp for AIDT140A. (E) Schematic of retroviral constructs used (top); anti-AID immunoblot of AID−/− B cells infected with PMX-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (middle); or wild-type B cells (WT) or AID−/− B cells infected with PMX-AID, MK-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (bottom). Anti-tubulin was used as a loading control. (F) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for five independent experiments, and the mean is indicated by a solid line. (G) Relative efficiency of isotype switching to IgG1. A comparison of PMX-AID versus AIDS38A, AIDT140A, AIDST-AA, or vector control. Bars represent the mean and SD from the five independent experiments in F. (H) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, MK-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for three independent experiments, and the mean is indicated by a solid line. (I) Relative efficiency of isotype switching to IgG1. Bars represent the mean and SD from H. (J) Flow cytometric analysis of IgG1 expression in wild-type B cells alone or transduced with MK-AID, PMX-AID, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of GFP− or GFP+ that were IgG1+ is indicated on the top left or right of each graph, respectively. Graphs are representative of three independent experiments.
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fig2: Comparison of AID, AIDT140A, and AIDS38A hypermutation and CSR activity in B and non–B cells. (A) The graph shows a log plot of numbers of kanamycin-resistant (KanR) colonies after induction of AID, AIDS38A, AIDT140A, AIDST-AA, or empty vector expression. (B) Anti-p140, -p38, or -AID immunoblot of FLAG-tagged AID, AIDS38A, or AIDT140A purified from B cells stimulated with LPS and IL-4. (C) Accumulation of GFP-expressing 3T3-NTZ cells after transduction with AID-, AIDS38A-, or AIDT140A-expressing PMX-MK retroviruses. The x axis indicates the number of days after transduction, and the y axis indicates the percentage of GFP-positive cells measured by flow cytometry. (D) Number of mutations in the GFP gene cloned from 3T3-NTZ cells 11 d after transduction with retroviruses encoding AID, AIDS38A, AIDT140A, or control. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The total number of independent sequences analyzed is indicated in the center of each chart. Statistical significance was determined by a two-tailed Student's t test assuming unequal variance and comparing AID expressing with AIDS38A- and AIDT140A-expressing cells. P values are indicated. The numbers of point mutations were as follows: 4 mutations/21,643 bp mutations for vector; 59 mutations/13,174 bp for AID; 15 mutations/19,761 bp for AIDS38A; and 45 mutations/21,643 bp for AIDT140A. (E) Schematic of retroviral constructs used (top); anti-AID immunoblot of AID−/− B cells infected with PMX-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (middle); or wild-type B cells (WT) or AID−/− B cells infected with PMX-AID, MK-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (bottom). Anti-tubulin was used as a loading control. (F) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for five independent experiments, and the mean is indicated by a solid line. (G) Relative efficiency of isotype switching to IgG1. A comparison of PMX-AID versus AIDS38A, AIDT140A, AIDST-AA, or vector control. Bars represent the mean and SD from the five independent experiments in F. (H) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, MK-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for three independent experiments, and the mean is indicated by a solid line. (I) Relative efficiency of isotype switching to IgG1. Bars represent the mean and SD from H. (J) Flow cytometric analysis of IgG1 expression in wild-type B cells alone or transduced with MK-AID, PMX-AID, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of GFP− or GFP+ that were IgG1+ is indicated on the top left or right of each graph, respectively. Graphs are representative of three independent experiments.

Mentions: To determine whether T140 is essential for catalytic activity, we compared wild-type and AID-T140A for their ability to revert an inactivating point mutation in a kanamycin resistance encoding plasmid in Escherichia coli (19). Reversion of the point mutation (CCAP94 to CTAL94) by cytidine deamination confers kanamycin resistance, which is assayed by colony formation (19). AID-T140A, AID-S38A, and the double mutant (AID-ST/AA) were indistinguishable from wild type in this assay (Fig. 2 A) (29). Thus, these mutations do not alter AID catalytic activity as assayed in E. coli.


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)

Comparison of AID, AIDT140A, and AIDS38A hypermutation and CSR activity in B and non–B cells. (A) The graph shows a log plot of numbers of kanamycin-resistant (KanR) colonies after induction of AID, AIDS38A, AIDT140A, AIDST-AA, or empty vector expression. (B) Anti-p140, -p38, or -AID immunoblot of FLAG-tagged AID, AIDS38A, or AIDT140A purified from B cells stimulated with LPS and IL-4. (C) Accumulation of GFP-expressing 3T3-NTZ cells after transduction with AID-, AIDS38A-, or AIDT140A-expressing PMX-MK retroviruses. The x axis indicates the number of days after transduction, and the y axis indicates the percentage of GFP-positive cells measured by flow cytometry. (D) Number of mutations in the GFP gene cloned from 3T3-NTZ cells 11 d after transduction with retroviruses encoding AID, AIDS38A, AIDT140A, or control. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The total number of independent sequences analyzed is indicated in the center of each chart. Statistical significance was determined by a two-tailed Student's t test assuming unequal variance and comparing AID expressing with AIDS38A- and AIDT140A-expressing cells. P values are indicated. The numbers of point mutations were as follows: 4 mutations/21,643 bp mutations for vector; 59 mutations/13,174 bp for AID; 15 mutations/19,761 bp for AIDS38A; and 45 mutations/21,643 bp for AIDT140A. (E) Schematic of retroviral constructs used (top); anti-AID immunoblot of AID−/− B cells infected with PMX-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (middle); or wild-type B cells (WT) or AID−/− B cells infected with PMX-AID, MK-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (bottom). Anti-tubulin was used as a loading control. (F) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for five independent experiments, and the mean is indicated by a solid line. (G) Relative efficiency of isotype switching to IgG1. A comparison of PMX-AID versus AIDS38A, AIDT140A, AIDST-AA, or vector control. Bars represent the mean and SD from the five independent experiments in F. (H) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, MK-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for three independent experiments, and the mean is indicated by a solid line. (I) Relative efficiency of isotype switching to IgG1. Bars represent the mean and SD from H. (J) Flow cytometric analysis of IgG1 expression in wild-type B cells alone or transduced with MK-AID, PMX-AID, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of GFP− or GFP+ that were IgG1+ is indicated on the top left or right of each graph, respectively. Graphs are representative of three independent experiments.
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fig2: Comparison of AID, AIDT140A, and AIDS38A hypermutation and CSR activity in B and non–B cells. (A) The graph shows a log plot of numbers of kanamycin-resistant (KanR) colonies after induction of AID, AIDS38A, AIDT140A, AIDST-AA, or empty vector expression. (B) Anti-p140, -p38, or -AID immunoblot of FLAG-tagged AID, AIDS38A, or AIDT140A purified from B cells stimulated with LPS and IL-4. (C) Accumulation of GFP-expressing 3T3-NTZ cells after transduction with AID-, AIDS38A-, or AIDT140A-expressing PMX-MK retroviruses. The x axis indicates the number of days after transduction, and the y axis indicates the percentage of GFP-positive cells measured by flow cytometry. (D) Number of mutations in the GFP gene cloned from 3T3-NTZ cells 11 d after transduction with retroviruses encoding AID, AIDS38A, AIDT140A, or control. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The total number of independent sequences analyzed is indicated in the center of each chart. Statistical significance was determined by a two-tailed Student's t test assuming unequal variance and comparing AID expressing with AIDS38A- and AIDT140A-expressing cells. P values are indicated. The numbers of point mutations were as follows: 4 mutations/21,643 bp mutations for vector; 59 mutations/13,174 bp for AID; 15 mutations/19,761 bp for AIDS38A; and 45 mutations/21,643 bp for AIDT140A. (E) Schematic of retroviral constructs used (top); anti-AID immunoblot of AID−/− B cells infected with PMX-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (middle); or wild-type B cells (WT) or AID−/− B cells infected with PMX-AID, MK-AID, or -AIDS38A, or -AIDT140A, or –AIDST-AA (bottom). Anti-tubulin was used as a loading control. (F) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for five independent experiments, and the mean is indicated by a solid line. (G) Relative efficiency of isotype switching to IgG1. A comparison of PMX-AID versus AIDS38A, AIDT140A, AIDST-AA, or vector control. Bars represent the mean and SD from the five independent experiments in F. (H) Graph of flow cytometric analysis of IgG1 expression in AID−/− B cells transduced with PMX-AID, MK-AID, -AIDS38A, -AIDT140A, -AIDST-AA, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of IgG1+ cells is indicated for three independent experiments, and the mean is indicated by a solid line. (I) Relative efficiency of isotype switching to IgG1. Bars represent the mean and SD from H. (J) Flow cytometric analysis of IgG1 expression in wild-type B cells alone or transduced with MK-AID, PMX-AID, or control vector and cultured in LPS plus IL-4 for 3 d after infection. The percentage of GFP− or GFP+ that were IgG1+ is indicated on the top left or right of each graph, respectively. Graphs are representative of three independent experiments.
Mentions: To determine whether T140 is essential for catalytic activity, we compared wild-type and AID-T140A for their ability to revert an inactivating point mutation in a kanamycin resistance encoding plasmid in Escherichia coli (19). Reversion of the point mutation (CCAP94 to CTAL94) by cytidine deamination confers kanamycin resistance, which is assayed by colony formation (19). AID-T140A, AID-S38A, and the double mutant (AID-ST/AA) were indistinguishable from wild type in this assay (Fig. 2 A) (29). Thus, these mutations do not alter AID catalytic activity as assayed in E. coli.

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