Figure 1

PKACA phosphorylates A3B Thr214 in vivo and in vitro. (a) Schematic figure of AID, A3G, A3B and their mutants. A3B WT CTD and A3B T214A CTD are the purified protein constructs. (b,c) A3B physically binds to PKACA. HEK293T cells were transfected with expression vectors for A3B-HA and FLAG-PKACA. Lysates from these cells were co-immunoprecipitated with anti-HA (b) or anti-FLAG antibodies (c), followed by immunoblot with the indicated antibodies. (d) PKACA phosphorylates A3B in HEK293T cells. HEK293T cells were transfected with expression vectors for A3B, A3G, and A3G DM with or without PKACA, as shown above. 36hrs after transfection, we semi-purified the APOBECs by immunoprecipitation, and PKA-mediated phosphorylation was detected by immunoblot with anti-RXXS/T-p antibodies. (e,f) PKACA phosphorylates Thr214 of A3B. (e) Expression vectors for A3B alanine mutants with or without PKACA are introduced in HEK293T cells as indicated. 36 hrs after transfection, cells were collected, lysed, immunoprecipitated with the anti-HA antibody and subjected to immunoblot with the indicated antibodies. The results show that A3B Thr214 is a crucial residue for PKA-mediated phosphorylation. (f) In vitro phosphorylation assays show that PKACA directly phosphorylates A3B-CTD, but not A3B T214A CTD. AID, activation-induced deaminase; WT, wild type; DM, double mutant; IP, immunoprecipitation.