Identification of substrates for F-box proteins

Jianping Jin, Xiaolu Cambronne, Takahiro Shirogane, J. Wade Harper

Research output: Contribution to journalReview article

70 Citations (Scopus)

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.

Original languageEnglish (US)
Article number20
Pages (from-to)287-309
Number of pages23
JournalMethods in Enzymology
Volume399
DOIs
StatePublished - Dec 20 2005
Externally publishedYes

Fingerprint

F-Box Proteins
Substrates
Phosphorylation
Ubiquitination
F-Box Motifs
Ubiquitin-Conjugating Enzymes
Protein Interaction Domains and Motifs
Phosphopeptides
Ubiquitin-Protein Ligases
Human Genome
Scaffolds
Leucine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Identification of substrates for F-box proteins. / Jin, Jianping; Cambronne, Xiaolu; Shirogane, Takahiro; Wade Harper, J.

In: Methods in Enzymology, Vol. 399, 20, 20.12.2005, p. 287-309.

Research output: Contribution to journalReview article

Jin, Jianping ; Cambronne, Xiaolu ; Shirogane, Takahiro ; Wade Harper, J. / Identification of substrates for F-box proteins. In: Methods in Enzymology. 2005 ; Vol. 399. pp. 287-309.
@article{3272bc2bc4dd4cf6b153b4340c9802ed,
title = "Identification of substrates for F-box proteins",
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.",
author = "Jianping Jin and Xiaolu Cambronne and Takahiro Shirogane and {Wade Harper}, J.",
year = "2005",
month = "12",
day = "20",
doi = "10.1016/S0076-6879(05)99020-4",
language = "English (US)",
volume = "399",
pages = "287--309",
journal = "ImmunoMethods",
issn = "1046-2023",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Identification of substrates for F-box proteins

AU - Jin, Jianping

AU - Cambronne, Xiaolu

AU - Shirogane, Takahiro

AU - Wade Harper, J.

PY - 2005/12/20

Y1 - 2005/12/20

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=28944448413&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=28944448413&partnerID=8YFLogxK

U2 - 10.1016/S0076-6879(05)99020-4

DO - 10.1016/S0076-6879(05)99020-4

M3 - Review article

C2 - 16338364

AN - SCOPUS:28944448413

VL - 399

SP - 287

EP - 309

JO - ImmunoMethods

JF - ImmunoMethods

SN - 1046-2023

M1 - 20

ER -