Most, if not all, cellular processes are regulated by timely degradation of regulatory proteins through the ubiquitin system. Specific substrate proteins are targeted for degradation through the activity of E3 ubiquitin ligases. We previously discovered that two small RING finger proteins, ROC1 and ROC2, function as the essential catalytic components of the largest family of E3 ligase complexes, the cullin-RING E3 ligases (CRLs). We also discovered that cullin 3 and cullin 4 could assemble as many as 200 and 100 distinct CRL3 and CRL4 E3 complexes, respectively. In this application, we propose two lines of research aimed at understanding the function and mechanism of two specific CRL4 E3 ubiquitin ligase complexes, CRL4G?2, and CRL4WDR5, and how alterations of these CRL4 complexes contribute to two major human diseases, heart failure and X-linked mental retardation. In the first Aim, we will examine the molecular function of CRL4 G?2 and its role in cardiovascular disease. We present data demonstrating that a WD40 protein, G-protein ? subunit 2 (G?2), associates with DDB1- CUL4A to target G protein-coupled receptor kinases (GRK2), for ubiquitylation. Elevated GRK2 has previously been associated with myocardial infarction, heart failure, portal hypertension, insulin resistance, and Alzheimer's disease. We propose first to explore how broadly CRL4 is involved in the regulation of G- protein coupled receptor (GPCR) signaling by determining the interaction of CRL4 with other G? and GRK proteins. We will then focus on one well-characterized GPCR signaling pathway, ?-adrenergic receptors (? - AR) signaling, to determine the mechanism of CRL4G?2 mediated GRK2 ubiquitylation. We will also determine whether loss of Cul4a impairs heart function in mouse. In the second Aim, we present data demonstrating that CUL4B, one of the most frequently mutated genes in X-linked mental retardation (XLMR) patients, but not CUL4A, is a nuclear E3 ligase of WDR5, a WD40 protein and an essential component for histone H3K4 trimethylation. We propose to determine the function of CUL4B-mediated WDR5 ubiquitylation in neuronal cell proliferation, survival and differentiation. We also propose to develop and characterize a novel mouse model, conditional Cul4b mutant mouse strain, to determine the function of Cul4b in brain. Finally, we propose to determine broadly the function of CUL4B in the regulation of chromatin modification and gene expression.

Public Health Relevance

Based on the discoveries made during the last funding cycle, we propose to determine the mechanism and function of two distinct cullin-based ubiquitin ligase complexes, each linked with a specific human disease: CRL4GNB2 in cardiovascular disease and CRL4WDR5 in X-linked mental retardation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067113-12
Application #
8642184
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Krasnewich, Donna M
Project Start
2003-02-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
12
Fiscal Year
2014
Total Cost
$358,269
Indirect Cost
$113,481
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Seifert, Bryce A; Xiong, Yue (2014) Out of the F-box: reawakening the pancreas. Cell Stem Cell 15:111-2
Pei, Xin-Hai; Bai, Feng; Li, Zhijun et al. (2011) Cytoplasmic CUL9/PARC ubiquitin ligase is a tumor suppressor and promotes p53-dependent apoptosis. Cancer Res 71:2969-77
Liu, Chen-Ying; Lv, Xianbo; Li, Tingting et al. (2011) PP1 cooperates with ASPP2 to dephosphorylate and activate TAZ. J Biol Chem 286:5558-66
Nakagawa, Tadashi; Xiong, Yue (2011) X-linked mental retardation gene CUL4B targets ubiquitylation of H3K4 methyltransferase component WDR5 and regulates neuronal gene expression. Mol Cell 43:381-91
Lee, Hyun O; Zacharek, Sima J; Xiong, Yue et al. (2010) Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase. Mol Biol Cell 21:3639-53
Jackson, Sarah; Xiong, Yue (2009) CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem Sci 34:562-70
Kotake, Yojiro; Zeng, Yaxue; Xiong, Yue (2009) DDB1-CUL4 and MLL1 mediate oncogene-induced p16INK4a activation. Cancer Res 69:1809-14
Hu, Jian; Zacharek, Sima; He, Yizhou Joseph et al. (2008) WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev 22:866-71
McCall, Chad M; Miliani de Marval, Paula L; Chastain 2nd, Paul D et al. (2008) Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development. Mol Cell Biol 28:5621-33
van Buuren, Nick; Couturier, Brianne; Xiong, Yue et al. (2008) Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex. J Virol 82:9917-27

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