Identification of substrates for F-box proteins

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.