Aberrant regulation of cell cycle progression results in genomic instability that ultimately leads to cancer development. Proper cell cycle transitions are driven by coordinated waves of ubiquitin-dependent degradation of key cell cycle regulators by APC and SCF, the two major E3 ubiquitin ligase complexes. However, the critical mechanisms mediating ordered SCF and APC activities have not yet been identified. Previously we demonstrated that the APC/Cdh1 complex ubiquitinates and thus targets the SCF component Skp2 for degradation, hence gaining important insight as to why SCF and APC activities are mutually exclusive. More recently, we accumulated evidence suggesting that the F-box protein ?-TRCP can target Cdh1 for degradation, thereby creating a negative feedback loop to repress APC activity. This finding extends our understanding of the underlying mechanisms that tightly orchestrate the activity of the SCF and APC complexes.
In Specific Aim #1, we will utilize both genetic and biochemical approaches to explore the underlying molecular mechanisms through which ?-TRCP controls Cdh1 abundance and activity. We will further define how sequential phosphorylation of Cdh1 by Cyclin A/Cdk2 and Plk1 triggers the interaction with, and subsequent ubiquitination, by ?-TRCP. The proposed studies are expected to reveal the important function of ?-TRCP in governing S phase entry via timely destruction of Cdh1. Recent studies indicate that beside its cell cycle regulatory function, ?-TRCP has also emerged as a critical player in S and G2 DNA damage response checkpoints, mainly through destruction of its downstream targets Cdc25A and Claspin. However, further investigation will be necessary to fully understand the function of ?-TRCP in DNA damage response, especially in the regulation of the G1 damage response checkpoint, primarily regulated by the p53 pathway. Mdm2 is the major negative regulator of p53 and frequently overexpressed in tumors, yet the underlying mechanisms are unclear. We recently reported that SCF?-TRCP is a novel E3 ubiquitin ligase targeting Mdm2 for ubiquitination and destruction in a CK1?-dependent manner. But it remains largely unknown how CK1? is activated following DNA damage to govern the Mdm2/p53 pathway.
In Specific Aim #2, we intend to continue this innovative research by using multi-disciplinary approaches to investigate how ATM modulates Mdm2 stability by regulating CK1? activity and cellular localization. We will also examine whether Mdm2 is the primary physiological signaling pathway by which ?-TRCP regulates the p53 pathway to govern the DNA damage response, and whether non-degradable Mdm2 (?p1-23A) displays elevated oncogenic activities. Moreover, we will explore whether clinically, disruption of the components of Mdm2 destruction pathways (?-TRCP/ATM/CK1?) facilitates tumorigenesis, which may be responsible for Mdm2 accumulation often seen in tumors. Our proposed studies will provide new insight into the signaling pathways controlling Mdm2 destruction. It also provides the rationale for developing CKI and ATM agonists as anti-cancer agents.

Public Health Relevance

SCF?-TRCP has been implicated in DNA damage repair and cell cycle progression while the underlying molecular mechanisms are unknown. We plan to elucidate whether ?-TRCP plays a critical role in governing S phase entry by regulating the stability of Cdh1. Furthermore, we will also investigate a potential role for ?-TRCP in DNA damage response through its modulation of the activity of the Mdm2/p53 pathway, which plays a pivotal role in the establishment of DNA damage checkpoints.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM094777-01A1
Application #
8295160
Study Section
Molecular Oncogenesis Study Section (MONC)
Program Officer
Hamlet, Michelle R
Project Start
2012-05-01
Project End
2016-01-31
Budget Start
2012-05-01
Budget End
2013-01-31
Support Year
1
Fiscal Year
2012
Total Cost
$328,425
Indirect Cost
$139,675
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
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