The mitotic checkpoint is specified by an evolutionarily conserved group of six proteins whose functions are to prevent cells with unaligned chromosomes from prematurely exiting mitosis. The mechanisms by which these proteins detect misaligned chromosomes, and then generate and transduce an inhibitory signal throughout the cell to block the Anaphase Promoting Complex/cyclosome (APC/C) from promoting mitotic exit are major questions that remain to be solved. A major focus of this proposal is to examine the biochemical mechanism(s) by which the mitotic checkpoint inhibits the APC/C. To accomplish this, we have developed a somatic cell-free system that allows us to identify and characterize factors responsible for inhibiting the APC/C. Amongst the factors to be studied is the Mitotic Checkpoint Complex (MCC), a complex consisting of checkpoint proteins hBUBR1, hBUB3, Mad2 and Cdc20, that is the most potent inhibitor of the APC/C reported to date. We discovered that formation of the MCC is linked to mitotic entry and exit and is critically dependent on another checkpoint protein, hMps1 kinase. In addition, we identified a new subunit in the MCC. Ro52 is an autoantigen that is present in patients with Sjogren's syndrome. Ro52 is itself an E3 ubiquitin ligase that may regulate the function of the MCC by modifying some of its subunits. We propose to study the regulation of MCC by focusing on hMps1 and Ro52. We will characterize how hMps1 is regulated in mitosis and how hMps1 contributes to MCC assembly in vivo and in vitro. Preliminary data show that MCC purified from mitotic cells exhibits ubiquitin ligase activity and that BubR1 is one of the substrates within the MCC. We therefore plan to test if Ro52 is responsible for this ubiquitin ligase activity in MCC and whether this modification affects MCC composition and thus function in vitro and in vivo. The possibility that the mitotic checkpoint is regulated by a ubiquitin ligase is supported by recent studies that showed the antagonistic actions of ubiquitin ligase and deconjugase regulates the ability of checkpoint proteins to inhibit the APC/C.

Public Health Relevance

The broad objective of our lab is to understand the key mechanical and regulatory events that specify accurate chromosome segregation in human cells. This is directly relevant to human health as defects in this process results in chromosome imbalance that can lead to tumor formation or developmental defects. This topic is also relevant to understanding how cancer cells survive treatment with drugs such as paclitaxel that are commonly used in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086877-06
Application #
7846792
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Hamlet, Michelle R
Project Start
2003-07-07
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
6
Fiscal Year
2010
Total Cost
$380,061
Indirect Cost
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Martz, Matthew K; Grabocka, Elda; Beeharry, Neil et al. (2013) Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission. Mol Biol Cell 24:2785-94
Beeharry, Neil; Rattner, Jerome B; Caviston, Juliane P et al. (2013) Centromere fragmentation is a common mitotic defect of S and G2 checkpoint override. Cell Cycle 12:1588-97
Michal, Allison M; So, Christopher H; Beeharry, Neil et al. (2012) G Protein-coupled receptor kinase 5 is localized to centrosomes and regulates cell cycle progression. J Biol Chem 287:6928-40
Beeharry, Neil; Rattner, Jerome B; Bellacosa, Alfonso et al. (2012) Dose dependent effects on cell cycle checkpoints and DNA repair by bendamustine. PLoS One 7:e40342
Ratushny, V; Pathak, H B; Beeharry, N et al. (2012) Dual inhibition of SRC and Aurora kinases induces postmitotic attachment defects and cell death. Oncogene 31:1217-27
Tipton, Aaron R; Wang, Kexi; Link, Laura et al. (2011) BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional mitotic checkpoint complex. J Biol Chem 286:21173-9
Tipton, Aaron R; Tipton, Michael; Yen, Tim et al. (2011) Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC). Cell Cycle 10:3740-50
Wan, Xiaohu; O'Quinn, Ryan P; Pierce, Heather L et al. (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137:672-84
Beeharry, Neil; Yen, Tim J (2009) p53-dependent apoptosis in response to spindle damage is linked to loss of Bub1. Cancer Biol Ther 8:645-7
Huang, Haomin; Yen, Timothy J (2009) BubR1 is an effector of multiple mitotic kinases that specifies kinetochore: microtubule attachments and checkpoint. Cell Cycle 8:1164-7

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