Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Schematic of AURKA activation mechanisms during mitotic entry (left panel) and spindle assembly (right panel) by its allosteric activators phospho-Bora and Tpx2, respectively. In the G2 phase of the cell cycle, AURKA is maintained dephosphorylated on the T-loop (T288) by counteracting phosphatases. During mitotic entry, Cyclin A-Cdk1 phosphorylates Bora, which then binds and activates AURKA. Phospho-Bora preferentially binds unphosphorylated AURKA. Activated by pBora, AURKA phosphorylates the Plk1 T-loop (T210) to activate the kinase and trigger mitotic entry. Then, during mitosis, Tpx2, through its first 43 amino acids, recruits and activates phosphorylated AURKA (pT288) at the microtubules to promote mitotic spindle assembly. B- Domain architecture of Homo sapiens Bora (orange, left panel) and Tpx2 (blue, right panel) with the minimal fragments required for AURKA activation highlighted in dark orange (Bora 18–120 ) and dark blue (Tpx2 – ). Important sequence elements identified in these regions are indicated, including the aromatic anchors in the M1 and M2 motifs of Bora and Tpx2, as well as the phosphorylatable PSP motif (M3) (green) of Bora, located just downstream of the M2 motif. Based on these sequences, we engineered a phosphorylated Tpx2-Bora fusion (phosphopeptide MK51, left panel) that reconstitutes the regulatory properties of both proteins. We also engineered a minimal Bora construct (phosphopeptide GK51, right panel) by directly fusing the putative M1 motif to the M2 and M3 motifs via a Glycine linker. C- Competitive binding assay where fluorescein-labeled Tpx2 – polypeptide, in complex with AURKA T288V , is displaced by increasing amounts of competitor (cold Tpx2 – , pBora 1–224 , pBora fusion, and pTxp2-Bora chimera) and monitored through fluorescence polarization signals. The displayed data points and the half maximal inhibitory concentration (IC 50 ) value represent the average fluorescence polarization for each reaction condition, with standard deviations of the mean as error bars ( N =3 independent experiments, each performed with n= 3 independent experimental samples). ND: not determined. D- Activation of AURKA T288V ATPase activity by different activators as assessed using the ADP Glo assay with Kemptide substrate. Displayed data points and EC 50 values represent the average luminescence for each reaction condition with standard deviations of the mean as error bars ( N =3 independent experiments, each performed with n =3 independent experiment samples). RLU: relative light unit.

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Activation Assay, Sequencing, Phospho-proteomics, Construct, Competitive Binding Assay, Labeling, Fluorescence, Concentration Assay, Activity Assay, Glo Assay

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Schematic of the two-step in vitro reconstitution of Histone H3 phosphorylation on S10 by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera. In step 1, Bora 1–224 phosphorylated by ERK (noted pBora) or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 minutes. In step 2, the reaction mix from step 1 is incubated in the presence of Mg/ATP with Histone H3 for 5, 10, 20, 30, and 60 minutes. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-S10 Histone H3, or pan Histone H3, and AURKA (from top to bottom). Note that during step 2, pBora 1–224 itself is phosphorylated by activated AURKA T288V during the reaction, which is manifested by a mobility shift in SDS-PAGE, as reported previously . ppBora thus denotes Bora phosphorylated by ERK during step 1 and by activated AURKA during step 2 in this and other Figures. B- Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of Plk1 (pT210) by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera. In step 1, pBora 1–224 or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 minutes. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with Plk1 kinase domain catalytically dead mutant (Plk1 K82R ) for 5, 10, 20, 30, and 60 minutes. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). C- Same experiment as in panel B, except that AURKA T288V activated by pBora 1–224 or the pTpx2-Bora chimera is incubated with the isolated Plk1 T-loop fused to GST as substrate. Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom) D- Side-by-side comparison of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of Plk1 (pT210) by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera when the Plk1 kinase domain or only the isolated T-loop is used as a substrate. In step 1, pBora 1–224 or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 minutes. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with Plk1 kinase domain catalytically dead mutant (Plk1 K82R ) or the isolated Plk1 T-loop for 30 and 60 minutes. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-T210 Plk1, pan Plk1, or GST, and AURKA (from top to bottom). E -Schematics summarizing the main results. The pTpx2-Bora chimera and pBora 1–224 can similarly activate AURKA T288V toward Histone H3 (red arrows). However, in sharp contrast to pBora 1–224 , the pTpx2-Bora chimera is unable to activate AURKA T288V toward the Plk1 T-loop embedded in the kinase domain (crossed arrow). Both the pTpx2-Bora chimera and pBora 1–224 can stimulate AURKA T288V activity towards the isolated T-loop, but with poor efficacy (thin red arrows).

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: In Vitro, Phospho-proteomics, Incubation, Western Blot, Mobility Shift, SDS Page, Mutagenesis, Isolation, Comparison, Activity Assay

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Schematic of MITOKINAC: E. coli BL21 are transformed with three plasmids containing different replication origins, antibiotic resistance, and expressing i) the active ERK kinase (MAPKK constitutively activated S118E, S222D, which phosphorylates and activates the MAPK ERK), ii) Bora 1–224 and AURKA T288V expressed from the same plasmid, and iii) the Plk1 kinase domain (kinase-dead K82R but phosphorylable on the T-loop (T210)) or the isolated T-loop fused to GST as substrates. After co-transformation, culture, and protein induction with IPTG, Plk1 T-loop phosphorylation (T210) is monitored by Western blot directly on total bacterial extracts. B -Schematic of the mitotic kinase cascade reconstituted in E. coli. The activated ERK kinase phosphorylates Bora at multiple sites including S112 (M3 motif), which binds and activates non-phosphorylated AURKA (AURKA T288V ) towards the Plk1 kinase domain. C-D- Western blot analysis of total bacterial extracts reconstituting pBora 1–224 and AURKA T288V -dependent T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) ( C ) or the isolated T-loop fused to GST (GST T-loop ) ( D ). Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1 ( C ) or GST ( D ), and AURKA (from top to bottom). ERK K-dead: kinase dead. Asterisk denotes a nonspecific band. E -Schematic of MITOKINAC using Genetic Code Expansion to produce Bora 1-224 , uniquely phosphorylated at Serine 112, thus bypassing the need for a priming phosphorylation by ERK. The schematic illustrates the translation of Bora 1–224 (orange) by ribosomes (grey) in E. coli, which inserts a phospho-Serine (orange) at the amber codon UAG. When produced and phosphorylated at S112, pBora 1–224 binds to and activates AURKA, which subsequently phosphorylates the Plk1 T-loop (T210). F -Western blot analysis of total bacterial extracts reconstituting pBora and AURKA T288V -dependent T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using Genetic code expansion. Bacteria were transformed with a plasmid expressing wild-type Bora 1–224 (lanes 1-2) or Bora 1–224 harboring the amber codon TAG at position S112 (lanes 3-4). Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). ERK K-dead: kinase dead.

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Transformation Assay, Expressing, Plasmid Preparation, Isolation, Phospho-proteomics, Western Blot, Produced, Bacteria

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Multiple protein sequence alignments of the M1 motif of Bora. Identical residues are in dark blue. The mutated residues and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutation on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles mean no effect, while red rectangles indicate a loss of function. B- Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants of the M1 motif. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. C- View of the extended M1 motif of Bora bound to AURKA predicted by AlphaFold3. The M1 motif of Bora is anchored at one end by F25 binding to the F pocket of and V44, and F45 binding to the Y pocket of AURKA, respectively. D- Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). E- Multiple protein sequence alignments of the M2 and M3 motifs of Bora. Identical residues are shown in dark blue. The mutated residues and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutation on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles indicate no effect, while red rectangles indicate a loss of function. F- Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants of the M2 and M3 motifs. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. Note that Bora F103D and F104D mutants are expressed in E. coli (see panel H) but not detected by our anti-Bora antibody. G- View of the M2 and M3 motifs of Bora bound to AURKA predicted by AF3. AURKA is shown as a surface colored according to electrostatic potential, with blue indicating positive and red indicating negative. Bora is shown as an orange ribbon. Bora residues F103 and F104 bind to the W pocket of AURKA, while pS112 engages an electropositive pocket. H- Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to MBP, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). ERK K-dead: kinase dead. As membranes were sequentially revealed with different antibodies, the orange asterisk indicates the signal revealed by the anti-Bora antibody.

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Sequencing, Mutagenesis, Western Blot, Phospho-proteomics, Binding Assay

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Multiple protein sequence alignments of the Bora region located between the M1 and M2/M3 motifs. Identical residues are shown in dark blue. The residues mutated, and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutations on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles indicate no effect, while red rectangles indicate a loss of function. B- Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. C- Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of the Plk1 kinase domain (pT210) by AURKA T288V and pBora 18–120 . In step 1, pBora 18-120 is incubated 1h at 30°C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and Plk1 KDom for 30 minutes at 30°C. D- Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). As membranes were sequentially revealed with different antibodies, the orange asterisk indicates the signal revealed by the anti-Bora antibody. E- Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of the isolated Plk1 T-loop fused to GST by AURKA T288V and pBora 18–120 . In step 1, pBora 18–120 is incubated 1h at 30°C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and the isolated Plk1 T-loop fused to GST for 30 minutes at 30°C. F- Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of the T-loop of Plk1 (aa190-225) fused to GST (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, GST, and AURKA, as indicated (from top to bottom). G- Schematic of the two-step in vitro reconstitution of Histone 3 phosphorylation on Serine 10 (pS10) by AURKA T288V and pBora 18–120 . In step 1, pBora 18–120 is incubated 1h at 30°C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and Histone H3 for 30 minutes at 30°C. H- Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Histone H3 (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoSer10 Histone H3, or pan Histone H3, and AURKA, as indicated (from top to bottom).

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Sequencing, Western Blot, Phospho-proteomics, In Vitro, Incubation, Mutagenesis, Isolation

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- (i) View of the M1 and M2 motifs of Bora bound to AURKA predicted by AlphaFold 3. The Bora residues F25, V44, and F45 of the M1 motif binding to the F (green) and Y (yellow) pockets, as well as the residues F103 and F104 of the M2 motif binding to the W pocket (magenta), are shown. (ii) View of the Bora-specific motif bound to AURKA. I60 and I71 (blue arrows) anchor the motif to AURKA, whereas other hydrophobic and aromatic residues such as F56, W58, and I66 (red arrows) are solvent-exposed. (iii) View of the Bora segment comprising amino acids 60 to 79 bound to AURKA. B- Western blot analysis of total bacterial extracts reconstituting pBora and AURKA T288V -dependent T-loop (T210) phosphorylation of the isolated Plk1 T-loop fused to GST (GST T-loop ). Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). ERK K-dead: kinase dead.

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Binding Assay, Solvent, Western Blot, Phospho-proteomics, Isolation

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A -AlphaFold 3 model of the trimeric complex composed of pBora 18-120 . (orange), AURKA (grey), and the Plk1 kinase domain (blue). The residue T210 of the Plk1 T-loop, T288 of the AURKA T-loop, and the phosphorylated residue S112 of Bora are highlighted in red. B-C -Zoom in view of the AURKA/Bora/Plk1 interface, highlighting contacting residues. Color scheme as in panel A. D -Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of the Plk1 kinase domain (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phospho-T210 of Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). E -The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 from n=3 independent experiments.

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Residue, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1101/2025.07.25.666770

Figure Lengend Snippet: A- Schematic for the structure-function analysis of Bora during mitotic entry in Xenopus egg extracts. Interphase extracts (red) were depleted with Bora antibodies, and 30 min later supplemented with Bora 18–120 fragments (pre-phosphorylated by the ERK kinase) and with the recombinant human Gwl K72M hyperactive kinase to force mitotic entry. Samples were collected at different time points and analyzed by Western blot (0, 20, 40 min) using mitotic markers to determine whether the extracts entered mitosis (green). The Western blot shows endogenous Bora levels before (Input) and after (Sn: supernatant) immunodepletion (Ip) from Xenopus egg extracts. B- Interphase extracts were immunodepleted using anti-Bora antibodies and 30 min later supplemented with recombinant hyperactive human Gwl kinase (Gwl K72M ) and Bora 18–120 wild-type or variants pre-phosphorylated by ERK. A fraction of the extracts was collected at the indicated time-points (0, 20, 40 min) and analyzed by Western blot using specific antibodies to monitor the levels of Hu and Xe Gwl, Plk1, and Hu Bora as well as phosphorylation of Plx1 on T201 (pPlk1), and Cdk on Tyr15 (pTyr).

Article Snippet: The antibodies used in this study are the following: mouse anti-Bora 1/400 (Santa-Cruz Cat#sc-393741), rabbit anti-AURKA 1/2000 (Cell Signaling Technologies Cat#91590), rabbit anti-Phospho-Plk1 (Thr210) 1/1000 (Cell Signaling, Technologies Cat#5472), rabbit anti-human Plk1 1/2000 (Bethyl Cat#A300-250A), rabbit anti-GST 1/1000 (Cell Signaling Cat#2622), rabbit anti-Phospho-Histone H3 (Ser10) 1/1000 (Cell Signaling Cat#3377S), rabbit anti-Histone H3 1/1000 (Abcam, Cat#AB1791), mouse anti-MBP 1/5000 (NEB biolabs cat#E8032S).

Techniques: Recombinant, Western Blot, Immunodepletion, Phospho-proteomics

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) Flow cytometry analysis showing DNA content (DRAQ5) on the x-axis and phospho-Histone H3 (Ser10) on the y-axis. HeLa cells were transfected with control siRNA (siControl, upper panels) or siRNA targeting ADAR1 (siADAR1, lower panels), and collected at 48, 60, and 72 h post-transfection (left to right). Percentages of cells in each cell cycle phase (G1, S, G2/M) and mitotic population (M, boxed area) are indicated. Green dots represent cells positive for phospho-Histone H3 (Ser10), indicating mitotic cells, while purple dots represent non-mitotic populations. (B) Western blotting analysis of mitotic and DNA damage markers following ADAR1 knockdown. HCT116 (left) and HeLa (right) cells were transfected with control siRNA (siControl) or two independent siRNAs targeting ADAR1 (siADAR1-1 and siADAR1-2). Protein lysates were collected and subjected to immunoblotting for ADAR1, cleaved PARP, phospho-DNA-PKcs, phospho-RPA32, RPA32, γH2AX, Cyclin B1, phospho-histone H3 (Ser10), phospho-PLK1 (T210), PLK1, phospho-Aurora A (T288), Aurora A, phospho-CDC2 (T15), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). Markers were selected to assess cell cycle status during mitosis and the presence of DNA damage.

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: Flow Cytometry, Transfection, Control, Western Blot, Knockdown

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) Identification of mitotic regulators interacting with ADAR1 by co-immunoprecipitation (co-IP). HeLa cells expressing Flag-tagged ADAR1p110 or wild-type (WT) controls were harvested under asynchronous (Async) or mitotically synchronized (Msync) conditions. Cell lysates were subjected to anti-Flag IP, and both input, flow-through (FT), and IP fractions were analyzed by western blotting. Immunoblots were probed for candidate interacting proteins, including DExD/H-box helicase (DHX9), Aurora kinases (Aurora A, B, C), PLK1, CDC25C, SMC3, SMC2, and phospho-specific forms of Aurora kinases and PLK1. GAPDH served as a negative control for nonspecific binding. (B) Lysates from Flag-ADAR1p110-expressing cells were subjected to IP using an anti-Flag antibody, and eluted protein complexes were re-immunoprecipitated using either anti-SMC3 or control IgG antibodies.

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: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot, Negative Control, Binding Assay, Control

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