Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Funabiki Laboratory

Keywords

mitosis, Aurora B, Vespera, nuclear formation, SUMOylation

Abstract

Upon completion of mitosis, the disassembly of spindle components and reassembly of nuclear structures occur simultaneously around chromatin. Previous studies have suggested that an important step in this process is the inactivation of the Aurora B kinase by the Triple A‐ATPase Cdc48/p97, which physically extracts the protein from chromatin at anaphase. Aurora B is the catalytic subunit of the Chromosome Passenger Complex (CPC), which promotes microtubule polymerization and spindle formation from mitotic chromosomes. Removal of the CPC from chromosomes at anaphase is required for proper nuclear reassembly, but the molecular basis for this requirement remains unclear. On the whole, the orchestration of structural events at the end of mitosis is poorly understood. Using Xenopus egg extracts, we endeavored to identify uncharacterized proteins that bind to purified mitotic chromosomes. In doing so we discovered Vespera, a protein which is functionally antagonistic to the CPC. We show that Vespera promotes microtubule depolymerization from chromosomes and is required on chromatin for proper nuclear formation. We initially identified Vespera as a protein that is specifically SUMOylated upon interaction with chromatin, a modification that is dependent on the SUMO E3 ligase, PIASy. Immunodepletion of Vespera from Xenopus egg extracts interferes with proper nuclear formation and nuclear transport, and rescue of this defect requires Vespera SUMOylation. Addition of excess amounts of Vespera to metaphase extracts disassembles spindle microtubules, and this activity is also dependent on SUMOylation of Vespera and PIASy. Importantly, the Vespera depletion defect in nuclear formation can be rescued by nocodazole or by co‐depletion of the CPC. These manipulations depolymerize microtubules, suggesting that the microtubule‐depolymerizing activity of SUMO‐Vespera is central to its role in ensuring proper nuclear formation. Our results suggest that microtubules must be actively depolymerized around chromosomes to permit proper nuclear reassembly, a process that is promoted by SUMOylated Vespera. Ultimately, we would like to understand how various post‐translational modifications on chromosomal proteins signal events through the cell cycle. Towards this end, we have developed a chemical method for the identification of phosphorylation using mass spectrometry, and have applied it to identify novel phosphorylation sites on Vespera as well as on members of the CPC. Furthermore, although phosphorylation has been shown to play a central role during mitosis, we hypothesized that the interplay between phosphorylation and other covalent modifications may also prove to be important in this context. Here, we observed that, in the absence of SUMOylated Vespera, protein phosphatase 1 does not fully accumulate on chromosomes and dephosphorylation of Aurora B substrates is delayed. The interaction between protein phosphatases and SUMO may represent an interesting general principal in crosstalk between covalent modifications, and provides intriguing opportunities for future studies.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/367

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