Global Identification of Modular Cullin-RING Ligase Substrates

Abstract

Summary Cullin Ring Ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. One substrate, NUSAP1, we demonstrate is an SCFCyclin F substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology, and provides a set of proteins likely to be key indicators of cellular physiology.

Regulation of cancer-related pathways by protein NEDDylation and strategies for the use of NEDD8 inhibitors in the clinic

“…CRLs control the stability of a vast variety of targets with established roles in cell cycle progression (p21, p27 and cyclin D/E), DNA replication (CDT1), the oxidative response (NFR2) and the response to hypoxia (HIF1a) (Freed et al 1999, Karin & Ben-Neriah 2000, Kondo & Kaelin 2001, Ohh et al 2002, Bloom et al 2003, Hu et al 2004, Li & Kong 2009. A study has identified hundreds of potential CRL targets, where functional inactivation of cullins was combined with genetic and proteomic approaches, displaying the diversity of CRLs to control protein stability (Emanuele et al 2011). Different models exist for the role of NEDD8 in the regulation of CRL function, including cullin dimerisation, dissociation of cullins from its negative regulator CAND1, conformational changes that bring the E3-RING ligases RBX1/2 in close proximity to the substrate protein, stabilisation of the active CRL state, or control the CRL binding with other E3-ligases and components of the p97 pathway (Duda et al 2008, Saha & Deshaies 2008, Merlet et al 2009, Deshaies et al 2010, Duda et al 2011, Bandau et al 2012, den Besten et al 2012, Kelsall et al 2013, Pierce et al 2013, Wu et al 2013, Zemla et al 2013.…”

Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

“…Gel shift analysis on a proteomic scale has been proposed and applied (16), but a relatively high false positive rate is observed, and analysis may be confounded by other post-translational modifications of the observed proteins. Recently, the use of an antibody directed against the diglycine moiety left on a ubiquitinated lysine residue after trypsin digestion has been reported to enrich ubiquitinated peptides (17)(18)(19)(20). This strategy is implicitly specific for modification with ubiquitin or a ubiquitin-like protein but has not yet been reported for the association of substrates with a particular E3 ligase.…”

VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1

“…We recovered FoxM1 in a global screen for substrates of the modular CRLs, which represent the largest E3 ligase family in humans (33). CRL assembly is based on a common molecular scaffold and relies on a cullin backbone that simultaneously engages substrates and E2 ubiquitin-conjugating enzymes.…”

“…for substrates of the Cul4-based cullin ring ligase (CRL4) (33). The GPS expression system relies on a viral vector that expresses a bicistronic mRNA encoding both DsRed and an enhanced green fluorescent protein-open reading frame (EGFP-ORF) fusion protein.…”