Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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 in a dephosphorylated state 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 1–43 ). 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, right panel) that reconstitutes the regulatory properties of both proteins. We also engineered a minimal Bora construct (phosphopeptide GK51, left 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 1–43 polypeptide, in complex with AURKA T288V , is displaced by increasing amounts of competitor (cold Tpx2 1–43 , 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: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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 min. 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 min. 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 (Tavernier et al, ). ppBora thus denotes Bora phosphorylated by ERK during step 1 and by activated AURKA during step 2 in this and other Figures. The graph presents the normalized quantification of pS10 Histone H3 signal over Histone H3 from n = 3 independent experiments. Error bars display the standard deviation. ( B ) 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 min. 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 min. 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). The graph presents the normalized quantification of pT210 Plk1 signal over either Plk1 KDom or GST-Plk1 T-loop from n = 3 independent experiments. Error bars display the standard deviation. ( C ) 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: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) 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 min. 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 min. 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). The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. ( B ) Same experiment as in panel A, 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). The graph presents the normalized quantification of pT210 Plk1 signal over GST-Plk1 T-loop from n = 3 independent experiments. ( C ) 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 1 h 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 min at 30 °C. 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 (aa 190-225) 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). The graph presents the normalized quantification of pT210 Plk1 signal over GST-Plk1 T-loop from n = 3 independent experiments. Error bars display the standard deviation.

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Structural alignment of the five pBora•AURKA•Plk1 KDom complex predictions generated by Alphafold 3 in a 1:1:1 binding stoichiometry. AURKA is in gray, Plk1 is in blue, and Bora is in orange. The predicted template modeling (pTM) score and the interface predicted template modeling (ipTM) scores are indicated. ( B ) Predicted Alignment Error (PAE) map calculated by AlphaFold3 showing the confidence of the distances between the residues in contact in the H. sapiens AURKA (126–403) (gray) • Bora (18–116) (orange) • Plk1 (1–332) (blue) complex generated by AlphaFold 3 in a 1:1:1 binding stoichiometry. ( C ) Per-residue confidence (pLDDT) of the pBora•AURKA•Plk1 KDom complex predicted by AlphaFold 3.

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Generated, Binding Assay, Residue

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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 (gray) in E. coli , which inserts a phosphoserine (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 (lane 1) or Bora 1–224 harboring the amber codon TAG at position S112 (lane 2). Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). ERK K-dead: kinase dead. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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). The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. Error bars display the standard deviation. ( 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 ( 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. The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. Error bars display the standard deviation. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

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 1 h 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 min 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. The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. ( E ) 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 1 h 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 min 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 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). The graph presents the normalized quantification of pS10 Histone H3 signal over Histone H3 from n = 3 independent experiments. Error bars display the standard deviation. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

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

Journal: The EMBO Journal

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

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) AlphaFold 3 model of the trimeric complex composed of pBora 18–120 (orange), AURKA (gray), and the Plk1 kinase domain (blue). The residue T210 of the Plk1 T-loop, T288 of the AURKA T-loop are highlighted in red, and the phosphorylated residue S112 of Bora is highlighted in green. ( B , C ) Zoom in view of the AURKA/Bora/Plk1 interface, highlighting contacting residues. Color scheme as in ( 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: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Residue, Western Blot, Mutagenesis