Considerable progress has been made in elucidating the critical regulatory role of cullin-based E3 ubiquitin ligases over a wide array of biological processes in eukaryotic cells. However, we are far from understanding their intricacies and we have only begun to appreciate their complex regulatory networks. While defective regulation of the cullin-dependent proteolysis pathways is manifest in human diseases including cancers, we are still in our infancy with respect to developing effective pharmacologic agents due to insufficient mechanistic understanding of these processes. Thus, if we are to understand aberrant growth, we must have fundamental knowledge of how cullins mediate proteolysis and how these processes are regulated. Recently, we have identified a novel member of the cullin protein family called CUL7, containing both the cullin and DOC signature domains. Remarkably, CUL7 assembles an SCF-like E3 Ub ligase complex composed of Skp1, CUL7, ROC1, and a previously uncharacterized F-box protein, Fbx29. We propose to elucidate the biological function of the CUL7/Fbx29-based E3-1ike complex in mediating protein degradation by identifying substrate(s) of Fbx29. Nedd8 is a specific cullin modifier that up-regulates the activity of cullin-based E3 Ub ligases. Recently, we have cloned a potential regulator of the Nedd8 pathway called hDEN-1, a novel Nedd8 isopeptidase. We will investigate the biological function of hDEN-1 in Ub-dependent protein degradation pathways by assessing the function of hDEN-1 in regulating Nedd8-dependent SCF activities in mammalian cells, as well as by examining the biological role of this novel protease using S. pombe and Drosophila model systems. The pVHL tumor suppressor functions by assembling a cullin 2-containing E3 ligase that targets HIF-1alpha for ubiquitination and degradation. Recent studies have revealed that a novel post-translational modification, prolyl hydroxylation, plays a central role in regulating the pVHL/HIF-1 pathway. Hydroxylation of HIF-1alpha at proline residue 564, by the PHO1/SM20 family prolyl hydroxylase, is required for its interaction with pVHL to initiate the ubiquitination. More recently, we have identified a cellular activity that stimulates the function of the PHD1/SM20 prolyl hydroxylase. We propose to determine the molecular identity of this novel activity, which may prove to be yet another critical regulator of the pVHL/HIF-1 pathway.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095634-03
Application #
6944288
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Perry, Mary Ellen
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$301,710
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
Wu, Kenneth; Chong, Robert A; Yu, Qing et al. (2016) Suramin inhibits cullin-RING E3 ubiquitin ligases. Proc Natl Acad Sci U S A 113:E2011-8
Chong, Robert A; Wu, Kenneth; Kovacev, Jordan et al. (2015) Generation of a proteolytic signal: E3/E2-mediated polyubiquitination of I?B?. Methods Mol Biol 1280:339-54
Scheufele, Florian; Wolf, Benjamin; Kruse, Michael et al. (2014) Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signaling. Cell Signal 26:233-239
Chong, Robert A; Wu, Kenneth; Spratt, Donald E et al. (2014) Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination. Proc Natl Acad Sci U S A 111:8434-9
Kovacev, Jordan; Wu, Kenneth; Spratt, Donald E et al. (2014) A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination. J Biol Chem 289:7068-81
Hartmann, Thomas; Xu, Xinsong; Kronast, Mira et al. (2014) Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen. Proc Natl Acad Sci U S A 111:3371-6
Xu, Xinsong; Keshwani, Malik; Meyer, Kathleen et al. (2012) Identification of the degradation determinants of insulin receptor substrate 1 for signaling cullin-RING E3 ubiquitin ligase 7-mediated ubiquitination. J Biol Chem 287:40758-66
Spratt, Donald E; Wu, Kenneth; Kovacev, Jordan et al. (2012) Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase. J Biol Chem 287:17374-85
Sarikas, Antonio; Hartmann, Thomas; Pan, Zhen-Qiang (2011) The cullin protein family. Genome Biol 12:220
Wu, Kenneth; Yan, Hua; Fang, Lei et al. (2011) Mono-ubiquitination drives nuclear export of the human DCN1-like protein hDCNL1. J Biol Chem 286:34060-70

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