This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A.
SPECIFIC AIMS Protein ubiquitination regulates numerous cellular functions through proteasome dependent proteolysis and other pathways. The specificity of the ubiquitination is conferred by an E3 ubiquitin ligase that interacts with both a ubiquitin conjugatinig enzyme and a specific substrate to ligate a ubiquitin to the substrate. In this aspect, E3 ubiquitin ligases are the key components for regulating numerous cellular functions through ubiquitin-signaling pathways. The cullin family ubiquitin ligases are evolutionarily conserved multi-subunit E3 ubiquitin ligases that consist of three modules, a cullin family protein as a scaffold, a small ring finger protein as a catalytic subunit, and receptor molecules for targeting a specific substrate. This modular feature of the cullin ligases allows them to target virtually hundreds of substrates and regulate their cellular functions through the ubiquitin signaling-pathway. Although genetic analysis in different organisms has linked the function of individual cullins to diverse physiological processes such as cell cycle control, tumor suppression, and development, substrates and regulations of individual cullin ligases are largely unknown. Our long-term research goal is to understand functions and regulations of cullin family ligases in homeostasis and human diseases including cancers. We previously demonstrated that CUL3 ligases target their specific substrates through one of a large number of BTB domain containing proteins for ubiquitination (named BTB for Drosophila broad-complex C, Tramtrack, and Bric-a-brac). This study leads us to hypothesize that CUL3 ligases target a large number of specific substrates through BTB containing proteins. Although the cellular functions of CUL3-BTB ligases are expected to be pleiotropic, very little is currently known. In our preliminary study, knocking down CUL3 results in cells accumulating in G2/M phase with multipolar spindles. This study let us further hypothesize that human CUL3 ligase functions in organizing the spindle pole in G2/M transition. The multipolar spindle is commonly observed in human cancers and is linked to chromosome instability, one of the most important characteristics in human cancer cells. However, the precise mechanism in spindle pole organization during cell cycle is largely unknown. Our rationale for this proposed study is that elucidating a function of CUL3 in mitosis, especially organizing the spindle pole, will fill the gap between cell cycle control in mitosis and the ubiquitin signaling-pathway and bring us better understanding of the regulation of spindle pole organization in normal and tumor cells. We are especially well-prepared to undertake this project since, in addition to our strong preliminary data, we have substantial experience and success in studying the CUL3 based E3 ubiquitin ligases using the biological, molecular biological and, proteomic approaches. We propose the following specific Aims:
Aim 1. Determine the cellular function of human CUL3 in mitosis Based on our preliminary study on human CUL3, we hypothesize that CUL3 ligase functions in cell cycle control especially in organizing the spindle pole during mitosos.
Aim 2. Identify the substrate of CUL3 ligase that regulates mitosisOur working hypothesis, based on our preliminary study and genetic study on C.elegans CUL3 ortholog, is that CUL3 targets microtubule-severing proteins, katanins, for regulating mitosis.
Aim 3. Determine the function of human TOGp in CUL3 ligase in mitosisOur working hypothesis, based on our preliminary results, is that a conserved microtubule-associated protein, Tumor Over-expressed Gene product (TOGp), physically interacts with CUL3 and is involved in mitotic function of the CUL3 ligase.Many important cell cycle regulatory proteins and pathways are regulated post-transcriptionally by ubiquitin-mediated proteolysis to restrict the window of accumulation of those proteins to the specific time when the function of that protein is required. Although some of those cell cycle regulatory proteins have been linked to a specific E3 ligase, such as the CUL1 ligases, the regulation of cell cycle by the ubiquitin signaling pathway is still largely unknown. Our proposed study takes a very unique approach to reveal those cell cycle regulatory mechanisms since it is based on our observation on a unique and specific cell cycle phenotype caused by the inactivation of the CUL3 ligase. In addition, our preliminary data links the CUL3 ligase to a microtubule-associated and tumor over-expressed gene product TOGp. The proposed study will shed light on the cell cycle regulatory pathway controlled by an E3 ligase, especially microtubule organization and tumorigenesis. We also expect that greater understanding of the relationship between ubiquitin-mediated proteolysis and cell-cycle control is certain to provide new opportunities for therapeutic intervention.Summary: Research in this proposal combines tools from molecular biology, cell biology, RNAi technology and proteomic analysis to address the following questions about CUL3 ligase in mitosis and spindle pole organization: (1) What is the function of the mitotic CUL3 ligase? (2) What are the substrate and the substrate receptor BTB protein for the mitotic CUL3 ligase? (3) What is the function of TOGp in the mitotic CUL3 ligase? Since multipolar spindles as well as TOGp over expression are frequently observed in human cancers and the ubiquitin proteasome pathway is the major regulatory pathway in controlling cell cycle, the information obtained from this research should provide critical knowledge about the cell cycle regulatory mechanism and tumorigenesis controlled by the ubiquitin signaling-pathway. This knowledge may also provide insights for the better design of cancer therapeutic agents.
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