Quantitative Phosphoproteomics Identifies Substrates and Functional Modules of Aurora and Polo-Like Kinase Activities in Mitotic Cells

Arminja N. Kettenbach, Devin K. Schweppe, Brendan K. Faherty, Dov Pechenick, Alexandre A. Pletnev, Scott A. Gerber*

*To whom correspondence should be addressed. E-mail: scott.a.gerber{at}dartmouth.edu

This PDF file includes:

• Materials and Methods
• Fig. S1. Spindle and chromosome morphology of inhibitor-treated HeLa cells.
• Fig. S2. Plk1 and Plk4 inhibition by BI2536.
• Fig. S3. Experimental setup and schematic of time line of HeLa cell synchronization.
• Fig. S4. Flow cytometry analysis of inhibitor-treated HeLa cells.
• Fig. S5. Ratio distributions.
• Fig. S6. Comparison of the heavy-to-light ratios in AZD1152- and ZM447439-treated cells.
• Fig. S7. Candidate Aurora kinase A versus B targets.
• Fig. S8. Cluster and motif analysis of ambiguous Aurora substrates.
• Fig. S9. Log2 ratio distribution of known Aurora kinase A, Aurora kinase B, and Plk1 substrates identified in this analysis.
• Fig. S10. Candidate Plk targets.
• Fig. S11. In vitro peptide kinase motif assay.
• Fig. S12. Regulation of Aurora A by Plk.
• Fig. S13. Prediction of phosphorylation sites in structured and ordered regions of proteins.
• Fig. S14. Evolutionary motif conservation.
• Fig. S15. T loop sequence alignments.
• Fig. S16. STRING and MCODE analysis of proteins predicted to be phosphorylated by Aurora A, Aurora B, or Plk.
• Fig. S17. Western blot and flow cytometry analysis of nocodazole-arrested HeLa cells and HeLa cells collected by mitotic shake-off after thymidine release.
• Fig. S18. Reciprocal plots of mass spectrometry results from in vitro kinase reactions and cellular proteomics analysis.
• Fig. S19. The effect of NuMA phosphorylation on NuMA localization.
• Details Regarding Data Availability
• Description for Tables S1 to S6
• References
  

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Other Supplementary Material for this manuscript includes the following:

• Table S1 (Microsoft Excel format). All ModSites and representative peptides.
• Table S2 (Microsoft Excel format). ModSites assigned to the Aurora kinase substrate cluster.
• Table S3 (Microsoft Excel format). ModSites assigned to the Plk substrate cluster.
• Table S4 (Microsoft Excel format). Analysis of site and motif conservation for candidate Aurora A, Aurora B, and Plk substrates across evolution.
• Table S5 (Microsoft Excel format). ModSite assignments to Aurora A, Aurora B, Aurora ambiguous, and Plk clusters.
• Table S6 (Microsoft Excel format). Plk1-interacting proteins.

[Download Tables S1 to S6 (Compressed)]

Technical Details

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© 2011 American Association for the Advancement of Science