Fig 2: AURKA has extensive proximity interactions with centrosomes and centriolar satellites A, BComparison of the AURKA proximity interactome with the proximity interactome of the centrosome (Gupta et al, 2015), the proximity interactome of centriolar satellites (Gheiratmand et al, 2019), and the physical interactome of centriolar satellites (Quarantotti et al, 2019).CAURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3, and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18-h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4, and SSX2IP.DAURKA interacts with PCM1 in centriole-less cells. GFP-PCM1 or GFP-PCM1 was co-expressed with FLAG-AURKA in HEK293T cells treated with DMSO (control) or 500 nM centrinone B for 7 days. Complexes were precipitated with GFP-Trap beads. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against GFP and FLAG.EEndogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.FAURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1–269 a.a.)-FRB and treated with 500 nM rapamycin for 1 h followed by fixation at 6 h. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA, and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 µm.GQuantification of pericentrosomal levels of AURKA and gamma-tubulin for F. n > 25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****P < 0.0001; **P < 0.01; unpaired Student's t-test.).

Fig 3: Identification, validation, and networking analysis of high‐confidence AURKA proximity interactors AURKA proximity interactome consists of both overlapping and distinct interactors with the published AURKA interactors. AURKA proximity interactome was compared with published physical interactors/substrates and proteins curated from the BioGRID repository. Venn diagram shows the identity of the overlapping 52 proteins and the number of proteins specific to the AURKA proximity interactome and BioGRID dataset.GO‐enrichment analysis of the AURKA proximity interactors based on their biological process and cellular compartment. The x‐axis represents the log‐transformed p‐value (Fisher’s exact test) of GO terms.Sub‐interaction networks of AURKA based on biological process and cellular compartment. The AURKA proximity interactors were grouped using DAVID functional annotation tool and literature mining. The interaction networks visualized for the biological processes include cell cycle, centriole duplication, primary cilium biogenesis, microtubule organization, and cell adhesion. The interaction networks visualized for cellular compartments include the centrosome and the centriolar satellites. The interconnectedness among the proteins of each network was determined by the STRING database. Edges in green color indicate the published physical AURKA interactors/substrates, and edges in red color indicate the proximate interactors validated in this study.

Fig 4: Validation of PCM1 depletion and regulation of AURKA abundance by satellites Immunoblotting analysis of PCM1 depletion by RNAi. RPE1 cells were transfected with control or PCM1 siRNA #1 and #2 for 72 h. Cells were lysed and immunoblotted with PCM1 and Actin (loading control). Band intensities for immunoblots were quantified from two experimental replicates. Scale bar, 5 µm.Immunofluorescence analysis of PCM1 depletion and loss of satellites by RNAi. RPE1 cells were transfected with control or PCM1 siRNA #1 and #2 for 72 h. Cells were fixed and stained with PCM1, gamma-tubulin, and DNA. DNA was stained with DAPI. Scale bar, 10 µm.Confirmation of CCDC66 depletion by RNAi and quantification of AURKA and p-AURKA levels upon CCDC66 depletion. RPE1 cells were transfected with control or CCDC66 siRNA for 72 h. Cells were lysed and immunoblotted for AURKA, p-AURKA, CCDC66, vinculin (loading control), and alpha-tubulin (loading control).Depletion of PCM1 in RPE1 GFPCCDC66 line. RPE1 cells were transfected with control or PCM1 siRNA 72 h. Cells were fixed and immunostained with GFP, PCM1, and centrin3. DNA was stained with DAPI. Scale bar, 10 µm.Total AURKA in control and RPE1 PCM1 KO cells. RPE1 WT and RPE1 PCM1 KO cells were serum-starved for 24 h, cell lysates were prepared and run on an SDS–PAGE gel. Proteins were detected by immunoblotting with antibodies against AURKA and vinculin (loading control). The band intensities were measured from two replicates (unpaired Student's t-test).Representative images and quantification of basal body levels of AURKA and phospho-AURKA (p-AURKA) in control and RPE1 PCM1 KO cells. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies and DAPI. Scale bar, 10 µm.Quantification of AURKA and p-AURKA levels in RPE1 WT and PCM1 KO cells for G. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n > 100 cells per experiment. Data represent mean value from two experiments per condition ± SEM (**P < 0.01, ns: non-significant, unpaired Student's t-test). Data information: (B, D, F) Sizes of the scale bars are indicated in the figure legends.

Fig 5: Validation and phenotypic characterization of HEK293T cells stably expressing V5-BirA*-AURKA ARepresentative immunofluorescence images of HEK293T::V5-BirA*-AURKA fixed and stained for gamma-tubulin, alpha-tubulin, and DAPI. Centrosome number higher than 2 was quantified as “centrosome amplification”. Cells with more than one nucleus were quantified as “multinucleated”). Scale bar, 5 µmB, CQuantification for (B) centrosome amplification and (C) multinucleation for A. Data represent mean value from two experiments per condition ± SEM (ns: non-significant, unpaired Student's t-test). n > 100 cells per experiment.DRepresentative immunofluorescence images of mitotic HEK293T::V5-BirA*-AURKA fixed and stained for centrin 2 (centrioles), alpha-tubulin, and DAPI. Scale bar, 5 µm.E, FQuantification for (E) mitotic index (F) percentage of multipolar spindles for D. Data represent mean value from two experiments per condition ± SEM (ns: non-significant, unpaired Student's t-test). n > 100 cells per experiment.GCell cycle analysis of asynchronous HEK293T::V5-BirA*-AURKA cells. Cells were fixed and stained with propidium iodide (PI) and in flow cytometer. Data represent mean value from two experiments per condition ± SEM (ns: non-significant, unpaired Student's t-test).HRelative expression of Caspase3 in control cells andHEK293T::V5-BirA*-AURKA cells. Cells were lysed and immunoblotted with antibodies against Caspase3 and vinculin (loading control).IRepresentative images of HEK293T::V5-BirA*AURKA cells stained for V5, AURKA, and gamma-tubulin. DNA was stained with DAPI. Scale bar is 10 µm.JExpression and biotinylation of BirA* (control) and BirA*-AURKA in the absence and the presence of biotin (50 µM biotin, 18 h). Cells were lysed, run on an SDS–PAGE gel, and immunoblotted with streptavidin. Arrows indicate the bait proteins V5-BirA* and V5-BirA*-AURKA.