Elsevier

Methods in Enzymology

Volume 399, 2005, Pages 287-309
Methods in Enzymology

Identification of Substrates for F‐Box Proteins

https://doi.org/10.1016/S0076-6879(05)99020-4Get rights and content

Abstract

F‐box proteins serve as specificity factors for a family of ubiquitin protein ligases composed of Skp1, Cu11, and Rbx1. In SCF complexes, Cu11 serves as a scaffold for assembly of the catalytic components composed of Rbx1 and a ubiquitin‐conjugating enzyme and the specificity module composed of Skp1 and an F‐box protein. F‐box proteins interact with Skp1 through the F‐box motif and with ubiquitination substrates through C‐terminal protein interaction domains such as WD40 repeats. The human genome contains ∼68 F‐box proteins, which fall into three major classes: Fbws containing WD40 repeats, Fbls containing leucine‐rich repeats, and Fbxs containing other types of domains. Most often, F‐box proteins interact with their targets in a phosphorylation‐dependent manner. The interaction of F‐box proteins with substrates typically involves a phosphodegron, a small peptide motif containing specific phosphorylation events whose sequence is complementary to the F‐box protein. The identification of substrates of F‐box proteins is frequently a challenge because of the relatively weak affinity of substrates for the requisite F‐box protein. Here we describe approaches for the identification of substrates of F‐box proteins. Approaches include stabilization of ubiquitination targets by Cu11‐dominant negatives, the use of shRNA hairpins to disrupt F‐box protein expression, and the use of collections of F‐box proteins as biochemical reagents to identify interacting proteins that may be substrates. In addition, we describe approaches for the use of immobilized phosphopeptides to identify F‐box proteins that recognize particular phosphodegrons.

Introduction

F‐box proteins function as specificity factors for a family of ubiquitin ligases that use Cul1 as a scaffold (reviewed in Deshaies 1999, Koepp 1999, Patton 1998a). These complexes, referred to as SCF ubiquitin ligases, contain four major components (Feldman 1997, Patton 1998b, Skowyra 1997). The C‐terminus of Cul1 binds the Ring‐finger protein Rbx1 (also called Roc1 and Hrt1) to form the core E3 ubiquitin ligase, which binds and activates the E2 ubiquitin conjugating enzyme (Seol 1999, Skowyra 1999, Zheng 2002) (Fig. 1). Cul1 is also modified by the ubiquitin‐like protein Nedd8 (Deshaies, 1999). This modification greatly stimulates ubiquitin ligase activity. The specificity component is composed of Skp1 and a member of the F‐box family of proteins (Bai et al., 1999). Skp1 binds to the N‐terminus of Cul1 and also interacts with the F‐box motif. F‐box proteins also contain additional protein interaction domains that bind substrates. In many cases, the interaction of an ubiquitylation substrate with the F‐box protein is phosphorylation dependent.

Currently, 68 F‐box–containing genes have been identified in the human genome (Jin et al., 2004). However, only a small fraction of these have been examined biochemically or genetically. Most frequently, individual F‐box proteins interact with multiple ubiquitylation substrates. For example, β‐TRCP (Fbw1) functions in the ubiquitylation of β‐catenin (Latres 1999, Winston 1999), IκBα (Spencer 1999, Winston 1999, Yaron 1998), Emi1 (Margottin‐Goguet et al., 2003), and Cdc25A (Busino 2003, Jin 2003). In contrast, Fbw7/Sel‐10 functions in the ubiquitylation of cyclin E, c‐myc, notch‐1, and c‐jun (Hubbard 1997, Koepp 2001, Strohmaier 2001, Welcker 2004, Yada 2004). Given that there is a large number of F‐box proteins in eukaryotic genomes, a major emphasis of current research in the field concerns the identification of targets of ubiquitin ligases in general and SCF complexes in particular. In this chapter, we describe several approaches that can be used to identify F‐box proteins that control the turnover of proteins whose degradation is known to be controlled in a phosphorylation‐dependent manner.

Section snippets

Overview of Approaches

Several approaches have been used to identify F‐box protein involved in the degradation of particular substrates. Typically, the identification of relevant F‐box proteins has relied on prior knowledge concerning turnover of a particular ubiquitylated protein. In most cases, the ubiquitylation protein is known to be rapidly turned over in cells, either constitutively or in response to particular signals. Frequently, protein kinases important for turnover are known, because they are the identity

Use of Dominant‐Negative Cul1 to Identify SCF Targets

A truncated cDNA version of Cul1—containing the first 452 amino acid residues followed by a TGA stop codon—is cloned into a mammalian expression vector with a strong promoter (e.g., pcDNA3 with a CMV promoter; Jin et al., 2003). This truncated protein acts as a dominant‐negative (DN) version of Cul1, because even though it has lost its ability to interact with Rbx1 and the E2 Ub–conjugating enzyme, it retains its interaction with Skp1 and, indirectly, the F‐box proteins (Fig. 1). Accordingly,

Screening F‐Box Proteins for Interaction with Substrates In Vivo

Having validated the involvement of the SCF pathway in degradation of the protein of interest using Cul1DN, it is then possible to begin a search for relevant specificity components (i.e., F‐box proteins). Because F‐box proteins bind tightly and specifically to their targets, protein interaction approaches can be used to identify candidate F‐box proteins. Several F‐box proteins have been shown to bind tightly enough to their targets to be immunoprecipitated as a complex from extracts of

Use of RNAi to Validate the In Vivo Requirement of Particular F‐Box Proteins in Targeted Degradation

Once candidate F‐box proteins are identified, it is critical that further genetic and biochemical evidence of functional interactions be obtained. Of particular interest is the reconstitution of ubiquitin ligase activity toward the substrate of interest, as described later. In addition, loss‐of‐function experiments are required to demonstrate a requirement for a particular F‐box protein in degrading the protein of interest. An alternative to RNAi is the use of dominant‐negative F‐box proteins,

Use of Synthetic Phosphodegrons to Identify F‐Box Proteins for Particular Substrates

The term “degron” is used to refer to a minimal peptide sequence required to recruit an ubiquitylation substrate to its cognate E3 and was first used in the context of the N‐end Rule Pathway (Dohmen et al., 1994). SCF ubiquitin ligases often interact with short phosphopeptide motifs referred to as “phosphodegrons.” The crystal structure of the β‐TRCP1‐Skp1 and β‐catenin phosphodegron complex has demonstrated that the phosphoserine, aspartic acid, and hydrophobic residues in the β‐catenin

Reconstitution of Ubiquitin Ligase Activity

Several approaches have been used to demonstrate specific ubiquitylation of proteins by candidate SCF complexes. One approach involves reconstitution of ubiquitin ligase activity in reticulocyte lysate. We and others have found that F‐box proteins synthesized by in vitro translation will assemble with Skp1/Cul1/Rbx1 present in the reticulocyte extract to form active E3s (Jin 2003, Welcker 2004, Wu 2003). Interestingly, although neddylated Cul1 represents a small fraction of Cul1 in the

Conclusions

Available data indicate the existence of a large number of F‐box proteins, which presumably ubiquitylate an even larger number of cellular proteins. To date, substrates for only a small fraction of F‐box proteins have been identified. A further understanding of the role of the SCF pathway in protein homeostasis will require a detailed understanding of the proteins whose stability is regulated by F‐box proteins. In this chapter, we have described several biochemical approaches that should

Acknowledgments

This work is supported by NIH grant AG11085 and by the Department of Defense (DAMD17‐01‐1‐0135) to J. W. H., and by the Department of Defense (DAMD17‐02‐1‐0284) to J. J.

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