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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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AID is phosphorylated at position T140. (A) Amino acid sequence of AID showing the location of S38 and T140 within consensus PKA and PKC sites, respectively (gray boxes). (B) Anti-p140 or -AID immunoblot of recombinant AID (rAID) purified from E. coli or FLAG-tagged AID or AID-T140A immunoprecipitated from retroviral infected AID−/− B cells (* denotes 27-kD FLAG-tagged AID). (C) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells stimulated with LPS, LPS and IL-4, CpG, or anti-CD40 and IL-4. (D) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells or NTZ-3T3 cells. (E) Anti-p140 and -AID immunoblot of rAID untreated (-) or treated with PKC or PKA in vitro. (F) Anti-p140, -p38, and -AID immunoblot of rAID untreated (-) or in vitro phosphorylated with the indicated PKC isoforms.
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fig1: AID is phosphorylated at position T140. (A) Amino acid sequence of AID showing the location of S38 and T140 within consensus PKA and PKC sites, respectively (gray boxes). (B) Anti-p140 or -AID immunoblot of recombinant AID (rAID) purified from E. coli or FLAG-tagged AID or AID-T140A immunoprecipitated from retroviral infected AID−/− B cells (* denotes 27-kD FLAG-tagged AID). (C) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells stimulated with LPS, LPS and IL-4, CpG, or anti-CD40 and IL-4. (D) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells or NTZ-3T3 cells. (E) Anti-p140 and -AID immunoblot of rAID untreated (-) or treated with PKC or PKA in vitro. (F) Anti-p140, -p38, and -AID immunoblot of rAID untreated (-) or in vitro phosphorylated with the indicated PKC isoforms.

Mentions: To examine posttranslational modification of AID, we purified the protein from B cells cultured with LPS and IL-4 and subjected the material to mass spectrometry. Analysis of purified AID confirmed phosphorylation at peptides containing S38 (p38), and tyrosine 184 (p184) (29, 37) and revealed additional phosphorylation at T140 (p140; Fig. 1 A).


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)

AID is phosphorylated at position T140. (A) Amino acid sequence of AID showing the location of S38 and T140 within consensus PKA and PKC sites, respectively (gray boxes). (B) Anti-p140 or -AID immunoblot of recombinant AID (rAID) purified from E. coli or FLAG-tagged AID or AID-T140A immunoprecipitated from retroviral infected AID−/− B cells (* denotes 27-kD FLAG-tagged AID). (C) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells stimulated with LPS, LPS and IL-4, CpG, or anti-CD40 and IL-4. (D) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells or NTZ-3T3 cells. (E) Anti-p140 and -AID immunoblot of rAID untreated (-) or treated with PKC or PKA in vitro. (F) Anti-p140, -p38, and -AID immunoblot of rAID untreated (-) or in vitro phosphorylated with the indicated PKC isoforms.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571933&req=5

fig1: AID is phosphorylated at position T140. (A) Amino acid sequence of AID showing the location of S38 and T140 within consensus PKA and PKC sites, respectively (gray boxes). (B) Anti-p140 or -AID immunoblot of recombinant AID (rAID) purified from E. coli or FLAG-tagged AID or AID-T140A immunoprecipitated from retroviral infected AID−/− B cells (* denotes 27-kD FLAG-tagged AID). (C) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells stimulated with LPS, LPS and IL-4, CpG, or anti-CD40 and IL-4. (D) Anti-p140 and -AID immunoblot of FLAG-tagged AID purified from B cells or NTZ-3T3 cells. (E) Anti-p140 and -AID immunoblot of rAID untreated (-) or treated with PKC or PKA in vitro. (F) Anti-p140, -p38, and -AID immunoblot of rAID untreated (-) or in vitro phosphorylated with the indicated PKC isoforms.
Mentions: To examine posttranslational modification of AID, we purified the protein from B cells cultured with LPS and IL-4 and subjected the material to mass spectrometry. Analysis of purified AID confirmed phosphorylation at peptides containing S38 (p38), and tyrosine 184 (p184) (29, 37) and revealed additional phosphorylation at T140 (p140; Fig. 1 A).

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