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Department of Molecular Medicine
 

YewR P. Renee  Yew,  Ph.D.

Associate Professor

Profile and Contact Information | Research | Laboratory

RESEARCH

 

Research Program

Cell division is one of the most basic processes of living organisms, yet the mechanisms that regulate many steps of this complicated process remain unclear. Understanding the molecular mechanisms that regulate different stages of cell division is critical for determining how and why cells lose normal controls and checkpoints, resulting in transformation and oncogenesis. This understanding can lead the way toward identifying new drugs, new drug targets, or novel therapies to fight cancer. The research in our laboratory is focused on understanding the processes which regulate the onset and progression of DNA replication or S phase in vertebrates. This point in the cell cycle is a major checkpoint of cell division where cells must correctly initiate the replication of the genome following mitosis while ensuring that replication occurs completely and correctly, only once per cell cycle. Premature entry into S phase, incomplete replication, or re-initiation of replication can result in genomic instability and the development of cancer. For our studies, we use several model systems including (1) the eggs of the frog, Xenopus laevis, to study the biochemical, molecular, and cellular events that regulate DNA replication during the simple embryonic cell cycle; and (2) mammalian cells, to study the cellular, molecular, and environmental events which regulate DNA replication during the somatic cell cycle.

Our laboratory currently works on four different projects. The first is focused on understanding how cyclin-dependent kinase (CDK) inhibitors negatively regulate cell division in the vertebrate. In cancer cells, CDK inhibitors are not expressed or are expressed at abnormally low levels. CDK inhibitors of the Cip/Kip-type are known to be regulated by protein turnover, but how their destruction is coordinated at the molecular level with the events of DNA replication initiation or environmental stress responses is still unclear. To understand the underlying mechanisms regulating CDK inhibitor function, we study the function and regulation of the Xenopus CDK inhibitor called p27Xic1 (Xic1). Our previous studies demonstrate that Xic1 is targeted for proteolysis only in the nucleus and in a manner dependent upon cell cycle phase, PCNA binding, ubiquitination, and the proteasome. Our recent findings suggest that Xic1 proteolysis is also regulated during the cell cycle by phosphorylation and during a DNA replication checkpoint. The main goals of this project are to elucidate the molecular mechanism of Xic1 ubiquitination and degradation during the normal cell cycle and during a cell cycle checkpoint and to characterize how Xic1 phosphorylation and dephosphorylation regulate Xic1 function during the cell cycle.

The second project focuses on understanding the role of a protein complex containing the ubiquitin conjugating enzyme, Cdc34. Our previous studies have shown that this Cdc34 complex is required for the onset of DNA replication, but how Cdc34 and its as yet unidentified associated components function to regulate DNA replication in a vertebrate is not known. We hypothesize that Cdc34 and its associated proteins function to ubiquitinate substrate proteins to trigger the start of S phase. The specific goals of this project are to identify and functionally characterize the Cdc34-associated protein complex that regulates the onset of DNA replication and to identify and characterize the Cdc34 substrates that must be ubiquitinated to allow the start of DNA replication. Additional studies are also being conducted in order to understand the biochemistry of human Cdc34 function during ubiquitin transfer to a substrate. Our recent findings suggest that the acidic C-terminal tail domain of Cdc34 is critical for processive substrate ubiquitination. The role of Cdc34 and its tail domain in ubiquitin transfer and ubiquitin chain synthesis is the focus of future studies.

The third project is focused on understanding the role of the Retinoblastoma (Rb) protein in regulating the onset of DNA replication. Mutational inactivation of the Rb tumor suppressor gene is the most common cause of primary malignant intraocular tumors in children and contributes to cancer formation in many other tissues. Past studies have demonstrated that Rb negatively regulates cell proliferation at the restriction point through the binding and sequestration of the transcriptional activator, E2F. Recent studies by several groups indicate that Rb possesses an alternative activity which directly regulates DNA replication at a site of initiation. We hypothesize that Rb negatively regulates the pre-replication complex at an origin as part of a G1 to S phase checkpoint to prevent the initiation of DNA replication until an optimal point. We use the Xenopus system to study the mechanism of Rb function and its effect on the temporal regulation of DNA replication onset. Our preliminary findings indicate that full-length Xenopus Rb inhibits DNA replication in the Xenopus egg extract in the presence of cycloheximide and that Xenopus Rb associates with MCM pre-replication proteins. Our future studies will be focused on understanding the molecular mechanism and regulation of Rb function at an origin of DNA replication.

The fourth project is focused on understanding the function of the tumor suppressor gene, BRCA1, which is frequently mutated in a large percentage of hereditary breast cancer. BRCA1 is proposed to play an important role in DNA repair. During DNA replication, errors occur which must be corrected in order to prevent the accumulation of mutations. Additionally, environmental assaults can also cause damage to the DNA, which must then be repaired. However, although BRCA1 has been implicated to function in DNA repair as well as a host of other cellular processes, it is still unclear how BRCA1 biochemically mediates its cellular functions. Recently, BRCA1 in association with another tumor suppressor protein, BARD1, has been shown to function as an ubiquitin protein ligase or E3 enzyme in the ubiquitination of proteins, but how this biochemical activity of BRCA1 may mediate the functions of BRCA1 in breast cancer prevention has yet to be addressed. Our hypothesis is that BRCA1 mediates its biological function by targeting specific proteins for ubiquitination. Our goal is to identify the physiologically relevant BRCA1 substrates and to determine how the E3 activity of BRCA1 contributes to the prevention of breast cancer. To this end, we have purified a BRCA1-BARD1 complex from mammalian cells and have identified a number of BRCA1-BARD1-associated proteins by Mass Spectrometry. These BRCA1-BARD1-associated proteins include proteins involved in mismatch repair, non-homologous end joining, DNA unwinding, and are putative substrates of the BRCA1-BARD1 ubiquitin ligase. Our work is now focused on determining the biological significance of these interactions and on identifying potential targets of the BRCA1-BARD1 ubiquitin ligase.

 

Selected Publications

  1. Yew, P.R. and Kirschner, M.W. (1997) Proteolysis and DNA replication: The CDC34 requirement in the Xenopus egg cell cycle. Science 277: 1672-1676.

  2. Chuang, L.-C. and Yew, P.R. (2001) Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1. J. Biol. Chem. 276: 1610-1617.

  3. Yew, P.R. (2001) Ubiquitin-mediated proteolysis of vertebrate G1 and S phase regulators. J. Cell. Phys. 187: 1-10.

  4. Block, K., Boyer, T.G. and Yew, P.R. (2001) Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by Casein Kinase 2. J. Biol. Chem. 276: 41049-41058.

  5. Philpott, A. and Yew, P.R. (2003) The Xenopus Cell Cycle. Protocols in Cell Cycle Control: Methods and Techniques. Methods in Molecular Biology. 296:95-112. Humana Press. Editors: Gavin Brooks and Tim Humphrey.

  6. Chauhan, D., Li, G., Hideshima, T., Podar, K., Shringarpure, R., Mitsiades, C., Munshi, N., Yew, P.R. and Anderson, K.C. (2004) Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341. Oncogene 23: 3597-3602.

  7. Chuang, L.-C, Zhu, X.-N., Herrera, C.R., Tseng, H.-M., Pfleger, C.M., Block, K. and Yew, P.R. (2005) The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis. J. Biol. Chem. 280: 35290-35298.

  8. Chuang, L.-C. and Yew, P.R., (2005) Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching. J. Biol. Chem. 280: 35299-35309. (Selected as a JBC Paper of the Week)

  9. Block, K., Appikonda, S., Lin, H.-R., Bloom, J., Pagano, M. and Yew, P.R. (2005) The acidic tail domain of human Cdc34 is required for p27Kip1 ubiquitination and complementation of a cdc34 temperature sensitive yeast strain. Cell Cycle 4(10):1421-1427.

  10. Lin, H.R., Chuang, L.C., Boix-Perales, H., Philpott, A. and Yew, P.R. (2006) Ubiquitination of cyclin-dependent kinase inhibitor, Xic1, is mediated by the Xenopus F-box protein xSkp2. Cell CycleFeb 5(3):304-314.

  11. Boix-Perales, H., Lin, H.-R., Chuang, L.-C., Yew, P.R., and Philpott, A. (2007) The E3 ubiquitin ligase Skp2 regulates neural differentiation independent from the cell cycle.Neural Development.Dec. 14. 2(1): 27.

  12. Philpott, A. and Yew, P.R. (2008) The Xenopus Cell Cycle: An Overview. Molecular Biotechnology.February 12.

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