Journal: bioRxiv
Article Title: Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression
doi: 10.1101/2025.05.28.656747
Figure Lengend Snippet: (A) HeLa cells were collected under asynchronous (Async), mitotically arrested (M; nocodazole-treated), or S phase-arrested (S; thymidine-treated) conditions. Cell lysates were analyzed by SDS-PAGE with (+) or without (–) Phos-tag acrylamide to detect phosphorylated ADAR1p110. λ-Phosphatase treatment was used to confirm phosphorylation dependency. In Phos-tag gels (top panel), ADAR1p110 exhibited a mobility shift that was strongly enhanced in mitotic samples, appearing as multiple slower-migrating bands. This shift was abolished by phosphatase treatment, indicating that the observed shift is phosphorylation-dependent. Conventional SDS-PAGE (bottom panel) was performed to assess total ADAR1p110 and ADAR1p150 protein levels as loading controls. (B) Mass spectrometry-based phosphopeptide mapping was performed on 3×Flag-tagged ADAR1p110 purified from 293T cells under mitotically synchronized conditions. The amino acid sequence starting from residue 514 is shown. Orange marks indicate phosphorylation sites. Below the sequence, a schematic representation of ADAR1p110 is provided, including the Z-DNA binding domain (green), the dsRBDs (blue), and the deaminase domain (red). (C) HeLa cells were transfected with siRNA targeting the 3′-untranslated region (3′-UTR) of ADAR1, followed by transfection with mCherry-tagged ADAR1p110 constructs. The constructs included WT, phospho-mimetic mutants (3×D and S614D), and phospho-deficient mutants (3×A and S614A). Cells were harvested 48 h after transfection, and total lysates were analyzed by western blotting using antibodies against mCherry (exogenous ADAR1p110), endogenous ADAR1p110, phospho-histone H3 (Ser10), and GAPDH. Phospho-histone H3 (S10) band intensity was used as a readout for mitotic accumulation under each condition. (D) HeLa cells were treated with kinase inhibitors under Async or Msync conditions. Cells were exposed to PLK1 inhibitors BI2536 and GSK461364, and CDK12/13 inhibitors SR-4835 and THZ531, across indicated concentrations. Whole-cell lysates were analyzed by Phos-tag SDS-PAGE followed by immunoblotting to detect phosphorylated ADAR1p110. Phosphorylated forms were visualized as slower-migrating bands. A decrease or disappearance of these bands indicates a loss of phosphorylation upon kinase inhibition. (E) HeLa cells were synchronized in Msync using nocodazole and transfected with either control siRNA (siNC1) or CDK13-targeting siRNA (siCDK13). Asynchronous cells were included for reference. Whole-cell lysates were subjected to Phos-tag SDS-PAGE followed by western blotting to assess the phosphorylation status of ADAR1p110. A reduction in the slower-migrating phosphorylated form of ADAR1p110 was observed upon CDK13 knockdown, confirming its role in mitotic phosphorylation.
Article Snippet: The following primary antibodies were used for western blotting and IP: anti-ADAR1 (Santa Cruz, sc-73408, 1:1000), anti-GAPDH (Cell Signaling Technology [CST], #5174, 1:4000), anti-γH2AX (S139) (CST, #9718, 1:500), anti-phospho-Histone H3 (S10) (CST, #53348, 1:1000), anti-phospho-Aurora A/B/C (Thr288/232/198) (CST, #14475, 1:1000), anti-Aurora A (CST, #2914, 1:1000), anti-Aurora B (Abcam, ab2254, 1:1000), anti-phospho-PLK1 (T210) (CST, #9062, 1:1000), anti-PLK1 (Santa Cruz, sc-17783, 1:500), anti-CDC25C (CST, #4688, 1:1000), anti-α-tubulin (CST, #2125, 1:1000), anti-SMC1 (CST, #6892, 1:1000), anti-SMC2 (CST, #5392, 1:1000), anti-SMC3 (CST, #5696, 1:1000), anti-RAD21 (CST, #4321, 1:1000), and anti-DHX9 (Abcam, ab26271, 1:1000).
Techniques: SDS Page, Phospho-proteomics, Mobility Shift, Mass Spectrometry, Purification, Sequencing, Residue, Binding Assay, Transfection, Construct, Western Blot, Inhibition, Control, Knockdown
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