Journal: bioRxiv

Article Title: PP6-regulated Aurora A activity towards NDC80 limits mitotic spindle size to prevent micronucleation

doi: 10.1101/2022.05.23.492953

Figure Lengend Snippet: ( A ) DNA sequence of the PPP6C genomic locus showing the sequence of 3 candidate PPP6C KO alleles. ( B ) Western blot of parental eHAP and candidate PPP6C KO alleles showing loss of PPP6C protein and elevation of active Aurora A pT288. ( C ) Parental eHAP and PPP6C KO cells stained for Aurora A pT288, tubulin, and DNA. Arrowheads mark spread of active Aurora A on the mitotic spindle, and arrows indicate chromosome missegregation. ( D ) Growth rate of parental eHAP and PPP6C KO cell lines. ( E ) Parental eHAP and PPP6C KO cells stained for nuclear pore proteins (NUPs) and DNA. Arrowheads indicate micronuclei. ( F ) Frequency of micronucleation in parental and PPP6C KO eHAP clones.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs) were used for pT288 Aurora A (rabbit mAb; Cell Signalling Technology, 3079S), Aurora A (rabbit mAb; Cell Signalling Technology, 4718S), pan Aurora (Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198); rabbit mAb, Cell Signalling Technology, 2914S), Aurora B (mouse mAb, BD Transduction labs, 611083), pT232 Aurora B (rabbit pAb, Rockland, 600-401-677), PPP6C (rabbit, mAb, AbCam, ab131335), NDC80 pS55 (rabbit pAb, GeneTex, GTX70017), NDC80 (for immunoprecipitation and immunoblotting: mouse mAb, Santa Cruz, sc-515550; for immunoprecipitation only: rabbit pAb, ProteinTech, 18932-1-AP and mouse mAb, AbCam, ab3613, for immunofluorescence: mouse mAb, AbCam, ab3613), MPS1 (rabbit pAb, ProteinTech, 10381-1-AP), Cyclin B1 (mouse mAb; Millipore, 05-373), pT320 PPP1CA (rabbit mAb, AbCam, ab62334), PPP1CA (rabbit pAb, Bethyl Laboratories, A300-904A), pT481 PRC1 (rabbit mAb, AbCam, ab62366), tubulin (mouse mAb; Merck, T6199), BUB1 (rabbit pAb, Bethyl Laboratories, A300-373A), BUBR1 (mouse mAb, Merck, MAB3612), CENP-A (mouse mAb, AbCam, ab13939; mouse mAb, GeneTex, GTX13939), CENP-C (guinea pig pAb, MBL life science, PD030), CREST (human pAb, Antibodies Inc., 15-234), Mab414 (mouse mAb, AbCam, ab24609), NUP153 (rabbit pAb, Bethyl Laboratories, A301-788A), TPX2 (mouse mAb, AbCam, ab32795), Mad1 (rabbit pAb, GeneTex, GTX105079).

Techniques: Sequencing, Western Blot, Staining, Clone Assay

Journal: bioRxiv

Article Title: PP6-regulated Aurora A activity towards NDC80 limits mitotic spindle size to prevent micronucleation

doi: 10.1101/2022.05.23.492953

Figure Lengend Snippet: ( A ) Genome-wide CRISPR screens were performed in parental and PPP6C KO eHAP cells for equal time of growth (Screen or equal cell divisions (Screen 2). Significance (-logP) for negatively selected genes in PPP6C KO compared to the parental background is plotted for screen 1 against screen 2. Dotted red lines mark p = 0.01 significance level. NDC80 is highlighted. ( B ) Parental and PPP6C KO cells depleted of NDC80 were stained for DNA, tubulin and the nuclear pore marker NUP153. Arrows mark micronuclei in NDC80 depleted parental cells. ( C ) Mitotic lysates of parental and PPP6C KO HeLa cells were blotted for the proteins listed in the figure. Overall NDC80 phosphorylation was monitored using a Phos-tag gel. Relative levels of NDC80 S55 phosphorylation, active Aurora A (pT288) and active Aurora B (pT232) were measured (mean ± SEM). ( D ) Mitotic HeLa cell lysates mock (-) or lambda-phosphatase treated (+λ-PPase) were blotted for NDC80, NDC80 pS55 and pS62. ( E ) Mitotic lysates of parental and PPP6C KO HeLa cells treated with Aurora A (AurA-i) and B (AurB-i) kinase inhibitors in the combinations shown were blotted for the proteins listed in the figure. ( F ) HeLa cells in different stage of mitosis stained for NDC80 pS55 or pS62, DNA and CENP-A. ( G ) HeLa cells in mitosis were treated with CDK-inhibitor (CDK-i) to promote entry into anaphase and mitotic exit. NDC80 phosphorylation was followed using NDC80 pS55 and Phos-tag gels. Mitotic exit was confirmed by blotting for cyclin B, the inhibitory pT320 modification of PP1, and active Aurora A pT288.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs) were used for pT288 Aurora A (rabbit mAb; Cell Signalling Technology, 3079S), Aurora A (rabbit mAb; Cell Signalling Technology, 4718S), pan Aurora (Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198); rabbit mAb, Cell Signalling Technology, 2914S), Aurora B (mouse mAb, BD Transduction labs, 611083), pT232 Aurora B (rabbit pAb, Rockland, 600-401-677), PPP6C (rabbit, mAb, AbCam, ab131335), NDC80 pS55 (rabbit pAb, GeneTex, GTX70017), NDC80 (for immunoprecipitation and immunoblotting: mouse mAb, Santa Cruz, sc-515550; for immunoprecipitation only: rabbit pAb, ProteinTech, 18932-1-AP and mouse mAb, AbCam, ab3613, for immunofluorescence: mouse mAb, AbCam, ab3613), MPS1 (rabbit pAb, ProteinTech, 10381-1-AP), Cyclin B1 (mouse mAb; Millipore, 05-373), pT320 PPP1CA (rabbit mAb, AbCam, ab62334), PPP1CA (rabbit pAb, Bethyl Laboratories, A300-904A), pT481 PRC1 (rabbit mAb, AbCam, ab62366), tubulin (mouse mAb; Merck, T6199), BUB1 (rabbit pAb, Bethyl Laboratories, A300-373A), BUBR1 (mouse mAb, Merck, MAB3612), CENP-A (mouse mAb, AbCam, ab13939; mouse mAb, GeneTex, GTX13939), CENP-C (guinea pig pAb, MBL life science, PD030), CREST (human pAb, Antibodies Inc., 15-234), Mab414 (mouse mAb, AbCam, ab24609), NUP153 (rabbit pAb, Bethyl Laboratories, A301-788A), TPX2 (mouse mAb, AbCam, ab32795), Mad1 (rabbit pAb, GeneTex, GTX105079).

Techniques: Genome Wide, CRISPR, Staining, Marker, Phospho-proteomics, Modification

Journal: bioRxiv

Article Title: PP6-regulated Aurora A activity towards NDC80 limits mitotic spindle size to prevent micronucleation

doi: 10.1101/2022.05.23.492953

Figure Lengend Snippet: ( A ). Parental and candidate PPP6C KO cell lines were stained for DNA and NUP153, arrowheads indicate micronuclei or nuclear morphology defects, or ( B ) western blotted for tubulin and PPP6C. ( C ) Parental (P) and PPP6C KO clone # 34 in asynchronous culture or arrested in mitosis for 15h or 18h with nocodazole were blotted for cyclin B and the activating pT288 phosphorylation on Aurora A. ( D ) Metaphase and ( E ) anaphase parental and PPP6C KO HeLa cells stained for Aurora A, the Aurora A activating protein TPX2, active Aurora A pT288, and DNA. Arrowheads indicate the spread of active Aurora A on metaphase and anaphase spindles in PPP6C KO cells. Note the enlarged metaphase plate in PPP6C KO cells.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs) were used for pT288 Aurora A (rabbit mAb; Cell Signalling Technology, 3079S), Aurora A (rabbit mAb; Cell Signalling Technology, 4718S), pan Aurora (Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198); rabbit mAb, Cell Signalling Technology, 2914S), Aurora B (mouse mAb, BD Transduction labs, 611083), pT232 Aurora B (rabbit pAb, Rockland, 600-401-677), PPP6C (rabbit, mAb, AbCam, ab131335), NDC80 pS55 (rabbit pAb, GeneTex, GTX70017), NDC80 (for immunoprecipitation and immunoblotting: mouse mAb, Santa Cruz, sc-515550; for immunoprecipitation only: rabbit pAb, ProteinTech, 18932-1-AP and mouse mAb, AbCam, ab3613, for immunofluorescence: mouse mAb, AbCam, ab3613), MPS1 (rabbit pAb, ProteinTech, 10381-1-AP), Cyclin B1 (mouse mAb; Millipore, 05-373), pT320 PPP1CA (rabbit mAb, AbCam, ab62334), PPP1CA (rabbit pAb, Bethyl Laboratories, A300-904A), pT481 PRC1 (rabbit mAb, AbCam, ab62366), tubulin (mouse mAb; Merck, T6199), BUB1 (rabbit pAb, Bethyl Laboratories, A300-373A), BUBR1 (mouse mAb, Merck, MAB3612), CENP-A (mouse mAb, AbCam, ab13939; mouse mAb, GeneTex, GTX13939), CENP-C (guinea pig pAb, MBL life science, PD030), CREST (human pAb, Antibodies Inc., 15-234), Mab414 (mouse mAb, AbCam, ab24609), NUP153 (rabbit pAb, Bethyl Laboratories, A301-788A), TPX2 (mouse mAb, AbCam, ab32795), Mad1 (rabbit pAb, GeneTex, GTX105079).

Techniques: Staining, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: PP6-regulated Aurora A activity towards NDC80 limits mitotic spindle size to prevent micronucleation

doi: 10.1101/2022.05.23.492953

Figure Lengend Snippet: ( A ) HeLa cells were arrested in mitosis with nocodazole and then released to allow mitotic spindle formation in the presence of Aurora A and Aurora B kinase inhibitors as indicated. Cell lysates were prepared and NDC80 isolated by immune precipitation (NDC80 IP). Lysates and IP samples were western blotted with antibodies to Aurora A and B, activated Aurora A pT288, a pan-phospho-Aurora antibody detecting pT288 and pT232, NDC80, NDC80 pS55 and pT61-pS62 (rabbit, R; sheep, Sh). ( B ) Parental and PPP6C KO cells were arrested in mitosis with nocodazole and then released to allow mitotic spindle formation in the presence of Aurora A inhibitor (AurA-i) for the times indicated. Cell lysates were prepared and NDC80 isolated by immune precipitation (NDC80 IP). Lysates and IP samples were western blotted with antibodies to Aurora A and B, activated Aurora A pT288, a pan-Aurora antibody detecting pT288 and pT232, NDC80 pS55 and pS62. ( C ) Aurora A pT288 and NDC80 pS55 dephosphorylation in parental and PPP6C KO cells. Aurora A pT288 is stabilized following Aurora A inhibition in PPP6C KO cells (mean ± SEM). ( D ) HeLa cells were arrested in mitosis with nocodazole and then released to allow mitotic spindle formation in the presence of Aurora B inhibitors (AurB-i) for the times indicated. Cell lysates were prepared and western blotted with antibodies to Aurora A and B, activated Aurora A pT288, a pan-phospho-Aurora antibody detecting pT288 and pT232, NDC80 pS55. Both Aurora A pT288 and NDC80 pS55 were stable under these conditions.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs) were used for pT288 Aurora A (rabbit mAb; Cell Signalling Technology, 3079S), Aurora A (rabbit mAb; Cell Signalling Technology, 4718S), pan Aurora (Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198); rabbit mAb, Cell Signalling Technology, 2914S), Aurora B (mouse mAb, BD Transduction labs, 611083), pT232 Aurora B (rabbit pAb, Rockland, 600-401-677), PPP6C (rabbit, mAb, AbCam, ab131335), NDC80 pS55 (rabbit pAb, GeneTex, GTX70017), NDC80 (for immunoprecipitation and immunoblotting: mouse mAb, Santa Cruz, sc-515550; for immunoprecipitation only: rabbit pAb, ProteinTech, 18932-1-AP and mouse mAb, AbCam, ab3613, for immunofluorescence: mouse mAb, AbCam, ab3613), MPS1 (rabbit pAb, ProteinTech, 10381-1-AP), Cyclin B1 (mouse mAb; Millipore, 05-373), pT320 PPP1CA (rabbit mAb, AbCam, ab62334), PPP1CA (rabbit pAb, Bethyl Laboratories, A300-904A), pT481 PRC1 (rabbit mAb, AbCam, ab62366), tubulin (mouse mAb; Merck, T6199), BUB1 (rabbit pAb, Bethyl Laboratories, A300-373A), BUBR1 (mouse mAb, Merck, MAB3612), CENP-A (mouse mAb, AbCam, ab13939; mouse mAb, GeneTex, GTX13939), CENP-C (guinea pig pAb, MBL life science, PD030), CREST (human pAb, Antibodies Inc., 15-234), Mab414 (mouse mAb, AbCam, ab24609), NUP153 (rabbit pAb, Bethyl Laboratories, A301-788A), TPX2 (mouse mAb, AbCam, ab32795), Mad1 (rabbit pAb, GeneTex, GTX105079).

Techniques: Isolation, Western Blot, De-Phosphorylation Assay, Inhibition

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Left, the crystal structure of the Aurora A kinase domain in an active conformation, shown in grey ribbons representation, adopts the canonical kinase domain fold with N-terminal and C-terminal lobes. The ATP binding pocket containing bound AMP-PNP, shown in stick representation (with carbons colored dark grey, oxygens red, and phosphorus orange) is located within the active site cleft in between the two lobes. Side chains of Cys247, Cys290, and phosphorylated Thr288 are also shown in stick representation. Right, the crystal structure of the Aurora A kinase domain obtained with cacodylate buffer, shown in blue ribbons representation, depicting covalent modification of Cys247 and Cys290 (shown in stick representation with cyan carbons, overlayed with transparent space filled rendering) and a large displacement of the activation segment (shown in orange). TPX2 residues 7–20 are shown in yellow. (B) The crystal structure of the activation segment-swapped dimer of the cacodylate-modified Aurora kinase domain. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon indicating the relative orientation of each monomer in the upper right of the structure. The activation segment is orange. Symmetry-related kinase domain monomers within the crystal structure can be seen in a dimeric arrangement with their activation segments exchanged between the monomers at the dimer interface. The monomers are oriented with their N-terminal lobes pointing in near-orthogonal directions, as indicated by the dotted axis lines in the cartoon representation.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Binding Assay, Modification, Activation Assay

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Superposition of the active sites in the cacodylate-modified Aurora A kinase domain (blue and cyan) with the corresponding unmodified structure (grey and dark grey) shows spatial overlap between Phe275 in the active DFG-in conformation (dark grey, in stick representation) and the dimethyl arsenic adduct of the cacodylate-modified Cys247 (cyan and purple in stick representation, overlayed with transparent space filled rendering). AMP-PNP in the active structure (stick representation with dark grey carbons) spatially overlaps with Phe275 in the inactive cacodylate-modified DFG-out structure (cyan), and the side chain of Glu181 is also displaced in the cacodylate-modified structure (cyan) relative to the unmodified structure (dark grey). (B) The displacement of the Glu181 side chain expands the ATP-binding pocket, allowing an oxidized DTT molecule to occupy this space in the cacodylate-modified Aurora A structure. Superposition of the previous structure of the Aurora A kinase domain in complex with a 5-aminopyrimidinyl quinazoline inhibitor (beige and yellow; PDB code 2C6E) shows how this expanded ATP-binding pocket accommodates existing Aurora A-selective inhibitors. (C) Schematic of the experiment in which an Aurora A kinase domain construct containing a single cysteine (Cys247) was used for a mass spectrometry-based high throughput tethering screen of 880 disulfide-containing compounds (listed in data file S1) to identify additional covalent modifiers of Cys247. Each compound was incubated separately with Aurora A kinase domain, to allow thiol-disulfide exchange with the Cys247 side chain thiol in the presence of ß-mercaptoethanol, a non-specific disulfide reducing agent. Stable covalent labeling of Cys247 under these mildly reducing conditions requires additional stabilizing contacts between the particular compound and residues in Aurora A in close proximity to Cys247. These stable disulfide adducts were then detected by an increased total mass of the protein using mass spectrometry (data file S1).

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Modification, Binding Assay, Construct, Mass Spectrometry, High Throughput Screening Assay, Incubation, Labeling

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A and B) Structures of the Aurora A kinase domain modified with compounds 7–80 (A) and 8–34 (B) at Cys290 show an activation segment-swapped dimer, in which the monomers are oriented with their N-terminal lobes pointing in the same direction, as indicated by the dotted axis lines in the cartoon representation in the center bottom. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. This dimer configuration is distinct from the near orthogonal arrangement seen in the cacodylate-modified structure (Fig. 1B). (C) Superposition of the active site regions of the 7–80 modified Aurora A structure with the Akt kinase domain (dark green and chartreuse) in complex with AMP-PNP and a GSK3ß substrate peptide (beige and yellow) from PDB code 1O6L. The active site of one monomer of the 7–80 modified Aurora A is shown in blue and cyan, with the Thr288/Cys290 activation loop from the other monomer shown in purple and magenta. Sulfur atoms of Cys290 are colored bright green. (D and E) Structure of the 7–80-modified structure, superimposed and contrasted with each of the monomers from a structure of a fully reduced and unphosphorylated Aurora A kinase domain in a similar dimer configuration (PDB code 4C3P). Large differences in the positioning of the phopsho acceptor residue (Thr288) and of the catalytic base (Asp256) in the 4C3P structure (emphasized with black outlines) can be seen between the 7–80-modified and the unmodified structures.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Modification, Activation Assay

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) The structure shows an activation segment-swapped dimer with the disulfide bond formed between Cys290 in each monomer (with sulfur atoms colored green) at the center of the dimer interface. Molecules are shown in cartoon representation with the monomers colored blue and magenta, and with a cartoon representation shown above the structure indicating the relative orientations of the monomers. (B) The cacodylate-modified Aurora A kinase domain dimer in an identical orientation as in (A), shows a similar overall dimer configuration but with conformational differences in the activation segments [green in (B) vs orange in (A)]. (C) Monomers of Aurora A from (A) and (B) are shown superimposed. The DFG-in active conformation of a monomer of the Aurora A kinase domain disulfide homodimer (Phe shown in stick representation, colored cyan) contrasts with the inactive DFG-out conformation of the cacodylate-modified kinase domain (colored yellow), and also with the inactive DFG-out conformation of the CoAlated disulfide-linked dimer structure (Fig. 6E).

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Activation Assay, Modification

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Sequence alignment of human Aurora A (residues 246–293) with X. laevis Aurora A (residues 253–300). The highlighted amino acid residues denote residues mutated in the constructs used in the assays described in the remainder of this figure (B to D). (B to D) Xenopus egg extracts were used for xAurora A activation assays to examine the requirement for the activation loop cysteine (Cys297) of xAurora A in autophosphorylation. Endogenous xAurora A was depleted from Xenopus egg extracts using an immobilized xAurora A-binding fragment of xCEP192. Wild-type and mutant xAurora A constructs were then added to this depleted extract and assayed for activation by addition of either sperm nuclei as a source of centrosomes (B) or by addition of an antibody to xAurora A (C). In (D), xAurora A activation was also assayed using undepleted Xenopus egg extract and selective activation of exogenous wild-type and mutant FLAG-tagged xAurora A constructs using an antibody to FLAG. To the right of each experimental schematic, total and autophosphorylated (pThr295) Aurora A was detected by Western blotting. Blots in (B and D) are representative of 5 independent experiments, and the blots in (C) are representative of 3 independent experiments.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Sequencing, Construct, Activation Assay, Binding Assay, Mutagenesis, Western Blot

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Experimental schematic of the creation of stable HeLa cell lines by incorporating a doxycycline-inducible shRNA against endogenous Aurora A and transfected with or without shRNA-resistant, FLAG-tagged, wild-type and mutant Aurora A constructs driven by a native Aurora A promoter fragment. Following induction of shAurora A, the cells were nocodazole arrested. (B) Western blotting to assess Aurora A autophosphorylation at pThr288 in the nocodazole-arrested lysates described in (A). Total Aurora A and β-tubulin were blotted for reference. Blots are representative of 2 independent experiments.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: shRNA, Transfection, Mutagenesis, Construct, Western Blot

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Western blotting for Aurora A autophosphorylation at pThr288 to assess xAurora A activation in Xenopus egg extracts supplemented with demembranated sperm nuclei and exposed to DTT (or buffer, control) for the indicated times. Blots are representative of 4 independent experiments. (B) Schematic for Aurora A kinase domain constructs CoAlated on Cys290 and crystallized in the presence of AMP-PNP. (C) Structure of a wild type Aurora A kinase domain construct CoAlated on Cys290 shows an activation segment-swapped dimer. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon shown above the structure. The activation segments (orange and red) are swapped between the monomers. The CoA adduct of one monomer is bound in the ATP-binding of pocket of the opposing monomer. No electron density was observed for the TPX2 fragment fused to the N-terminus of the Aurora A kinase domain construct used to determine this structure. (D) Structure of a single-cysteine human Aurora A kinase domain construct CoAlated on Cys290 and crystallized in complex with AMP-PNP shows an activation segment-swapped dimer with monomers oriented with their N-terminal lobes pointing in near orthogonal directions (dotted axis lines in the cartoon representation shown in the upper right of the panel), in contrast to the monomer orientation shown in panel C. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. (E) The CoAlated Aurora A kinase domain dimer from (D), with the activation segments colored orange and the DFG phenylalanine in cyan, is shown superimposed on the cacodylate-modified dimer, colored yellow/gold with activation segments colored green. Both structures show a catalytically inactive DFG-out activation segment conformation. (F) A 2mFo-DFc map contoured at 0.5σ shows electron density consistent with a subpopulation of the Aurora A kinase domain molecules in the crystal containing a Cys290-Cys290 symmetric disulfide.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Western Blot, Activation Assay, Construct, Binding Assay, Modification

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Catalytically active Aurora A kinase domain constructs treated with the disulfide-promoting Ellman’s reagent and incubated with ATP show robust Thr288 autophosphorylation as assayed by Western blotting. Phosphorylation of Thr288 is detected in the bands corresponding to dimers for both the wild type and the C247V + C319V mutant construct. Inclusion of DTT in the kinase assay abrogates Thr288 phosphorylation. Blots are representative of 2 independent experiments. (B) Upper panel, purification scheme for disulfide-linked heterodimers containing an MBP-tagged kinase dead and untagged catalytically active Aurora A kinase domain. Lower panel, following incubation with ATP, the samples were analyzed by SDFS-PAGE under non-reducing or reducing conditions, and probed for autophosphorylation by Western blotting. Blots are representative of 8 independent experiments. (C) Proposed model of redox and dimerization-dependent activation of Aurora A. We posit that increased protein cysteine oxidation during mitosis results in increased levels of disulfide modifications of proteins, such as CoAlation of Aurora A. Clustering of Aurora A molecules upon centrosomal recruitment promotes dimerization and thiol-disulfide exchange between kinase domains to form a disulfide homodimer that facilitates autophosphorylation. Resolution of the disulfide homodimer releases activated (pThr288) Aurora A monomers.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Construct, Incubation, Western Blot, Mutagenesis, Kinase Assay, Purification, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 promotes Golgi unlinking along with Aurora A recruitment to the centrosomes . A , HeLa S3 cells were fixed with formaldehyde and stained for LAT1 ( green ) and TGN46 ( red ) or calnexin ( red ). Representative images are shown. Scale bar, 10 μm. B–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 19 h and washed with PBS(−). B and C , the cells were cultured for a further 9 h or 10 h in siControl or siLAT1 cells, respectively, to analyze the Golgi structure in prophase. The cells were fixed with MeOH and stained for TGN46 ( gray or green ) and DNA ( red ). B , representative z-stack images are shown, and Golgi objects based on TGN46 staining are numbered (see “ ”). Scale bar, 10 μm. C , the number of Golgi object within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 40). Statistical analysis was performed using Welch’s ANOVA ( F = 319, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , The cells were cultured for a further 9.5 h or 10.5 h in siControl or siLAT1 cells, respectively, to analyze Aurora A recruitment in prometaphase cells. Then, the cells were fixed with MeOH and stained for Aurora A ( green ), phospho-Aurora A (pT288) ( red ), and DNA ( cyan ). D , representative z-stack images are shown. Scale bar, 10 μm. E , the fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured (see “ ”), and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Statistical analysis was performed using Welch’s ANOVA ( F = 99.8, p = 0.000 in the left panel ; F = 88.6, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). F and G , HeLa S3 cells were treated with DMSO or 30 μM SP600125 for 2 h. F , the cells were fixed with MeOH and stained for TGN46 and DNA. The number of Golgi objects within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). G , the cells were fixed with MeOH and stained for Aurora A, phospho-Aurora A (pT288), and DNA. The fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured, and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Asterisks indicate significant differences [Student’s t test in panel ( F and G ), ∗∗ p < 0.01; ∗∗∗ p < 0.001].

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Staining, Transfection, Control, Cell Culture, Fluorescence