EDD, a Ubiquitin-protein Ligase of the N-end Rule Pathway, Associates with Spindle Assembly Checkpoint Components and Regulates the Mitotic Response to Nocodazole

Flavia Scialpi,David Mellis,Mark Ditzel

doi:10.1074/jbc.m114.625673

Flavia Scialpi, David Mellis + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m114.625673

Copy DOI

Abstract

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G2/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.

Highlights

Mitotic progression is regulated by the spindle assembly checkpoint (SAC) to prevent aneuploidy and chromosome damage
E3 identified by differential display (EDD) and BUBR1 siRNA Increase ␥ H2AX Levels—To investigate what might be responsible for the increased cell death, we looked for signs of DNA damage
We reveal the ability of EDD to govern the cellular response to a mitotic spindle poison (Noc), document the dynamic relationship of EDD with mitotic chromatids/chromosomes during mitosis (Fig. 2), and support the mass spectrometry-based observations that identified the association of EDD with mitotic chromosomes [33]

Summary

Background

Mitotic progression is regulated by the spindle assembly checkpoint (SAC) to prevent aneuploidy and chromosome damage. Copurification studies identified physical interactions with CDC20, BUBR1, and other components of the SAC Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis. The SAC is a multiprotein complex that comprises mitotic arrest deficient 2 (MAD2), Bub1-related protein kinase (BUBR1), and budding uninhibited by benzimidazoles 3 (BUB3). Acting together, they provide an essential mitotic checkpoint that maintains chromosomal integrity, ensures correct chromosome separation, and prevents aneuploidy [11]. We identify physical interactions between EDD, CDC20, and components of the SAC and reveal the potential role of EDD promoting mitotic arrest in response to Noc

EXPERIMENTAL PROCEDURES

RESULTS

C EDD siRNA

DISCUSSION