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The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation

Nature volume 438, pages 508–511 (2005)Cite this article

Abstract

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR)1,2,3. Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID)4,5,6, which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity7,8,9,10,11. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein12, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID–RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells13,14. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

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Figure 1: In vitro phosphorylation of AID 293and determination of AID phosphorylation sites.
Figure 2: AID is phosphorylated by PKA.
Figure 3: AID phosphorylation mutants fail to interact with RPA.
Figure 4: Mutations of the AID phosphorylation site impair CSR in vivo.

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Acknowledgements

We thank T. Honjo for providing AID-deficient mice and a retroviral vector for expression of AID in B cells, and R. Dalla-Favera and L. Pasqualucci for communicating unpublished findings. U.B. is the Prismedical fellow of the Irvington Institute of Immunology; S.D. is supported by a fellowship from the Swiss National Science Foundation; S.R. is supported by a National Research Service Award. This work was supported by an NIH grant to F.W.A. F.W.A. is an Investigator and G.L. an Associate of the Howard Hughes Medical Institute.

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Author notes

  1. Jayanta Chaudhuri

    Present address: Immunology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021, USA

  2. Uttiya Basu and Jayanta Chaudhuri: *These authors contributed equally to this work

Authors and Affiliations

  1. The Howard Hughes Medical Institute, The Children's Hospital, The CBR Institute for Biomedical Research, and Department of Genetics, Harvard Medical School, Massachusetts, 02115, Boston, USA

    Uttiya Basu, Jayanta Chaudhuri, Craig Alpert, Sheila Ranganath, Gang Li, Jason Patrick Schrum & Frederick W. Alt

  2. Joint Program in Transfusion Medicine, Children's Hospital, Department of Pathology, Harvard Medical School, Massachusetts, 02115, Boston, USA

    Shilpee Dutt & John P. Manis

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Supplementary information

Supplementary Figure 1

PKA sites on AID. (PDF 84 kb)

Supplementary Figure 2

AIDS38A mutant fails to deaminate dsDNA. (PDF 572 kb)

Supplementary Figure 3

PKA activity and AID expression in B cells. (PDF 297 kb)

Supplementary Figure 4

PKA inhibition abolishes CSR in B cells. (PDF 491 kb)

Supplementary Notes

This file contains the Supplementary Figure Legends and the Supplementary Methods (DOC 38 kb)

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Basu, U., Chaudhuri, J., Alpert, C. et al. The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature 438, 508–511 (2005). https://doi.org/10.1038/nature04255

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