Abstract
The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.
Highlights
Considerable effort has been devoted to understanding the multiple mechanisms that combine to regulate activation-induced deaminase (AID) and A3G activity
Numerous proteins have been implicated in regulating AID and A3G function (MDM2 [25], replication protein A [26], heat shock protein 90 (HSP90) [27], germinal centerassociated nuclear protein (GANP) [28], calcium and integrinbinding protein 1 (CIB1) [29], beta-catenin-like protein 1 (CTNNBL1) [30]), with protein kinase A (PKA) [31,32,33,34] being most relevant to this work
AID-Thr-27, A3G-Thr-32, and A3G-Thr-218 Are Homologous and Located within a Region of High Sequence and Structural Conservation—Prior studies demonstrated phosphorylation of AID-Thr-27 in vivo and in vitro by mass spectrometry and radiolabeling[31, 33, 35] and A3G-Thr-32 by immunoblotting [34]. We noted that these two threonines are homologous to A3G-Thr-218, whose high-resolution structures have shown to be located within a solvent-accessible loop (4 –7) (Fig. 1A)
Summary
Considerable effort has been devoted to understanding the multiple mechanisms that combine to regulate AID and A3G activity. Protein samples purified from human cells from the above were used for A3G DNA binding reactions. Phospho-mimetic Mutations Inhibit DNA Cytidine Deaminase Activity—To address whether phosphorylation is capable of attenuating the DNA cytidine deaminase activity of AID and A3G, phospho-null and phospho-mimetic derivatives of these proteins were tested in an E. coli-based activity assay.
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