PKA-Mediated Phosphorylation of APOBEC3B Suppresses Its DNA Mutagenic Potential in Myeloma Cells
Authors: Matsumoto Tadahiko , Shirakawa Kotaro , Matsui Hiroyuki , Yamazaki Hiroyuki , Kazuma Yasuhiro , Sarca Anamaria Daniela , Fukuda Hirofumi , Maruyama Wataru , Takaori-Kondo Akifumi 
Published: October 2019

Abstract
Multiple myeloma (MM) is an incurable plasma cell malignancy and the reason for this is not yet completely understood. It might be partially attributable to heterogeneity and clonal evolution through DNA mutation accumulation. Recent next generation sequencing (NGS) studies have revealed that apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) 3B (A3B) is one of the cause of clonal evolution in MM. APOBEC protein family consists of 11 proteins and most of them, including A3B, have cytidine deaminase (CDA) activity on single-strand DNA, which catalyzes deamination of cytosine to uridine, resulting in C-to-T or C-to-G mutations. We have previously shown in human cell line models that A3B induces genomic mutations (Shinohara et al., Sci Rep, 2012). Therefore, controlling A3B CDA activity is an attractive therapeutic strategy to overcome the unfavorable prognosis of patients with these malignancies.
Enzymatic activity of other APBEC family proteins, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G) is regulated by protein kinase A (PKA)-mediated phosphorylation. A3B also has two consensus PKA phosphorylation motifs conserved upstream of each CDA domain. Thus, we hypothesized that PKA phosphorylates A3B which may alter A3B CDA activity.
Using NetPhosK1.0 and ScanSite programs, two A3B residues (serine at the 46th and threonine at the 214th position) are predicted to be PKA phosphorylation sites. Firstly, we examined the binding between A3B and PKA. We transfected HEK293T cells with expression vectors for C-terminal HA-tagged A3B and N-terminal FLAG-tagged PKA catalytic subunit a (PKACA) and used CoIP assays with anti-HA or anti-FLAG antibodies to demonstrate physical binding between A3B and PKACA. Secondly, to determine whether the putative residues are phosphorylated, we overexpressed PKACA and A3B alanine mutants in HEK293T cells and performed immunoblotting analysis with anti-phospho PKA motif antibodies and found that threonine-214 is phosphorylated by PKA. To confirm that PKA phosphorylates A3B directly, we employed in vitro phosphorylation assays using purified C-terminal A3B and PKACA and found that PKACA phosphorylated A3B, but not the A3B T214A mutant, thus we concluded that PKACA directly phosphorylates threonine-214 in A3B.
Next, to investigate the effect of this phosphorylation on the CDA activity of A3B, we constructed phosphomimetic mutants of these residues, T214D and T214E. We carried out in vitro CDA assays using cell lysates from HEK293T cells overexpressing A3B or mutants (T214A, T214D, T214E), and found that T214D and T214E almost completely lost their CDA activity. We also performed in vitro CDA assays using purified C-terminal A3B and mutant proteins. The purified A3B T214D mutant also lost CDA activity, suggesting that PKA-mediated phosphorylation of A3B might also lead to loss of its CDA activity.
To confirm this result in cell lines, we used differential DNA denaturation PCR (3D-PCR) of DNA from HEK293T cells transfected with vectors for uracil DNA glycosylase inhibitor, EGFP and A3B wild type or mutants. 3D-PCR products of the EGFP sequence were amplified at a lower denaturation temperature in cells expressing the wild type A3B compared to those in cells expressing phosphomimetic A3B or catalytically inactivated A3B. These results indicate that phosphomimetic A3B no longer has exogenous DNA editing activity. We further plan to evaluate the PKA phosphorylation of A3B and the consequences on its mutagenic potential in myeloma cell lines.
In conclusion, PKA induces phosphorylation at threonine-214 in A3B, and this phosphorylation suppresses its CDA activity. PKA-mediated phosphorylation of A3B may be an attractive therapeutic target for inhibiting A3B-induced clonal evolution in lymphoid malignancies, especially in MM.

Disclosures
Takaori-Kondo: Kyowa Kirin: Research Funding; Alexion Pharmaceuticals: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.