That AID isolated from activated B cells can interact with RPA, whereas AID from 293 cells (which are non-lymphoid) engineered to express AID (denoted AID293) cannot, has been suggested to be important for the B-cell-specific targeting of AID to sites of CSR and SHM. Because the AID–RPA interaction requires phosphorylation of AID, Basu et al. set out to identify the protein kinase that is responsible for this in activated B cells. Nuclear extracts from activated B cells but not 293 cells could phosphorylate AID in vitro, indicating the presence of an AID protein kinase in nuclear extracts from activated B cells. Detailed analysis of these extracts showed that AID-protein-kinase activity was associated with the catalytic subunit of PKA (denoted PKAcat) and that purified PKAcat could phosphorylate AID293 in vitro. This phosphorylation conferred on AID293 the ability to deaminate double-stranded DNA (dsDNA) efficiently in vitro, a property that is associated with CSR and SHM (but freshly isolated AID293 lacks this ability). Importantly, treatment with a PKA-specific inhibitor markedly impaired CSR in a B-cell clone that was activated to undergo CSR.
Mass-spectrometry analysis of AID from activated B cells showed that Ser38 and Tyr184 were phosphorylated, and Ser38 was found to be in a consensus site for phosphorylation by PKA. Thr27 was identified as an additional potential site for phosphorylation by PKA. AID mutants in which either Ser38 or Thr27 had been mutated to Ala were not phosphorylated by PKA, and they failed to interact with RPA and failed to mediate deamination of dsDNA. By contrast, an AID mutant in which Tyr184 had been substituted with Ala was phosphorylated and could mediate deamination of dsDNA. Consistent with these results, when each of these AID mutants was expressed by AID-deficient B cells induced to undergo CSR, only the AID mutant with the Tyr184Ala substitution could restore CSR.
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