SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.     

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development

F-box proteins, components of the Skp1-Cullin1-F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.     

A novel route for F-box protein-mediated ubiquitination links CHIP to glycoprotein quality control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.     

The abundance of cell cycle regulatory protein Cdc4p is controlled by interactions between its F box and Skp1p

Posttranslational modification of a protein by ubiquitin usually results in rapid degradation of the ubiquitinated protein by the proteasome. The transfer of ubiquitin to substrate is a multistep process. Cdc4p is a component of a ubiquitin ligase that tethers the ubiquitin-conjugating enzyme Cdc34p to its substrates. Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, we have sought evidence of mechanisms that modulate F-box protein regulation. Here we demonstrate that the abundance of Cdc4p is subject to control by a peptide segment that we term the R-motif (for "reduced abundance"). Furthermore, we show that binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities.     

SCFFBXO28-mediated self-ubiquitination of FBXO28 promotes its degradation

The F-box protein is the substrate recognition subunit of SCF (SKP1/CUL1/F-box) E3 ubiquitin ligase complex, a multicomponent RING-type E3 ligase involved in the regulation of numerous cellular processes by targeting critical regulatory proteins for ubiquitination. However, whether and how F-box proteins are regulated is largely unknown. Here we report that FBXO28, a poorly characterized F-box protein, is a novel substrate of SCF E3 ligase. Pharmaceutical or genetic inhibition of neddylation pathway that is required for the activation of SCF stabilizes FBXO28 and prolongs its half-life. Meanwhile, FBXO28 is subjected to ubiquitination and cullin1-based SCF complex promotes FBXO28 degradation. Moreover, deletion of F-box domain stabilizes FBXO28 and knockdown of endogenous FBXO28 strongly upregulates exogenous FBXO28 expression. Taken together, these data reveal that SCF^FBXO28 is the E3 ligase responsible for the self-ubiquitination and proteasomal degradation of FBXO28, providing a new clue for the upstream signaling regulation for F-box proteins.     

Two distinct proteolytic systems responsible for glucose-induced degradation of fructose-1,6-bisphosphatase and the Gal2p transporter in the yeast Saccharomyces cerevisiae share the same protein components of the glucose signaling pathway.

Addition of glucose to Saccharomyces cerevisiae inactivates the galactose transporter Gal2p and fructose-1,6-bisphosphatase (FBPase) by a mechanism called glucose- or catabolite-induced inactivation, which ultimately results in a degradation of both proteins. It is well established, however, that glucose induces internalization of Gal2p into the endocytotic pathway and its subsequent proteolysis in the vacuole, whereas FBPase is targeted to the 26 S proteasome for proteolysis under similar inactivation conditions. Here we report that two distinct proteolytic systems responsible for specific degradation of two conditionally short-lived protein targets, Gal2p and FBPase, utilize most (if not all) protein components of the same glucose sensing (signaling) pathway. Indeed, initiation of Gal2p and FBPase proteolysis appears to require rapid transport of those substrates of the Hxt transporters that are at least partially metabolized by hexokinase Hxk2p. Also, maltose transported via the maltose-specific transporter(s) generates an appropriate signal that culminates in the degradation of both proteins. In addition, Grr1p and Reg1p were found to play a role in transduction of the glucose signal for glucose-induced proteolysis of Gal2p and FBPase. Thus, one signaling pathway initiates two different proteolytic mechanisms of catabolite degradation, proteasomal proteolysis and endocytosis followed by lysosomal proteolysis.     

FBXL5 interacts with p150Glued and regulates its ubiquitination

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150(Glued) is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao, Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394-402]. Here we show that F-box protein FBXL5 interacts with p150(Glued) and orchestrates its turnover via ubiquitination. FBXL5 binds to p150(Glued)in vitro and in vivo. FBXL5 and p150(Glued) co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150(Glued) and protein turnover of p150(Glued). Our findings provide a potential mechanism by which p150(Glued) protein function is regulated by SCFs.     

F-box蛋白质在植物生长发育中的功能

在真核生物中, 泛素介导的蛋白降解途径参与了许多生物学过程。SCF复合体是一种非常重要的E3泛素连接酶, 在植物中研究的最为深入。F-box蛋白包含一个F-box 基序, 是SCF复合体的一个亚基, 它决定了底物识别的特异性。目前, 从各种植物中已鉴定出大量的F-box蛋白质, 它们参与了植物激素(乙烯, 生长素, GA, JA)的信号传导以及自交不亲和、花器官发育等生物学过程, F-box蛋白还参与了植物的胁迫反应。最新研究结果显示, 一个F-box蛋白TIR1是生长素的受体。因此, F-box蛋白质介导的泛素化蛋白质降解途径可能是植物基因表达调控的重要机制。     

The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination

Human cIAP1 protein is a member of the inhibitor of apoptosis proteins (IAPs) that are involved in apoptosis regulation and an increasing number of other functions, including cell cycle and intracellular signal transduction. In order to identify novel proteins involved in cIAP1 regulation, we performed a yeast two-hybrid screen and identified an F-box protein Fbxo7 as a cIAP1 interacting protein. Co-immunoprecipitation assay showed that cIAP1 can interact with Fbxo7 in human cells. When co-expressed in cells, cIAP1 and Fbxo7 co-localized remarkably both in the cytoplasm and nucleus, and considerable amounts of these often co-localized at one or few distinct Golgi-like structures close to the nucleus. Furthermore, we showed that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin protein ligases, our results suggest that Fbxo7 can mediate the ubiquitination of cIAP1 by SCF ubiquitin protein ligase and thus have important implication in the regulation of cIAP1 function.     

Glucose-induced monoubiquitination of the Saccharomyces cerevisiae galactose transporter is sufficient to signal its internalization

In Saccharomyces cerevisiae, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole. Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase. Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin. An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway.     

The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction

The ubiquitin system of intracellular protein degradation controls the abundance of many critical regulatory proteins. Specificity in the ubiquitin system is determined largely at the level of substrate recognition, a step that is mediated by E3 ubiquitin ligases. Analysis of the mechanisms of phosphorylation directed proteolysis in cell cycle regulation has uncovered a new class of E3 ubiquitin ligases called SCF complexes, which are composed of the subunits Skp1, Rbx1, Cdc53 and any one of a large number of different F-box proteins. The substrate specificity of SCF complexes is determined by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34. F-box proteins have a bipartite structure--the shared F-box motif links F-box proteins to Skp1 and the core complex, whereas divergent protein-protein interaction motifs selectively bind their cognate substrates. To date all known SCF substrates are recognised in a strictly phosphorylation dependent manner, thus linking intracellular signalling networks to the ubiquitin system. The plethora of different F-box proteins in databases suggests that many pathways will be governed by SCF-dependent proteolysis. Indeed, genetic analysis has uncovered roles for F-box proteins in a variety of signalling pathways, ranging from nutrient sensing in yeast to conserved developmental pathways in plants and animals. Moreover, structural analysis has revealed ancestral relationships between SCF complexes and two other E3 ubiquitin ligases, suggesting that the combinatorial use of substrate specific adaptor proteins has evolved to allow the regulation of many cellular processes. Here, we review the known signalling pathways that are regulated by SCF complexes and highlight current issues in phosphorylation dependent protein degradation.     

Isolation and characterization of HRT1 using a genetic screen for mutants unable to degrade Gic2p in saccharomyces cerevisiae

Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required for degradation of many regulatory proteins involved in cell cycle progression, morphogenesis, and signal transduction. Using a genetic screen, we have isolated a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y). hrt1-C81Y mutant cells exhibited an aberrant morphology but were viable at all temperatures. The cells displayed multiple genetic interactions with mutations in known SCF components and were defective for the degradation of several SCF targets including Gic2p, Far1p, Sic1p, and Cln2p. In addition, they also failed to degrade the F-box proteins Grr1p, Cdc4p, and Met30p. Wild-type Hrt1p but not Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent manner. Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is conserved in subunits of distinct E3 ligases. Finally, Hrt1p was localized in both nucleus and cytoplasm and despite a short half-life was expressed constitutively throughout the cell cycle. Taken together, these results suggest that Hrt1p is a core subunit of multiple SCF complexes.     

Stability of homologue of Slimb F-box protein is regulated by availability of its substrate

The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCF(HOS)) E3 ubiquitin ligase, which mediates ubiquitination and degradation of beta-catenin and the inhibitor of NFkappaB, IkappaB. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCF(HOS) complex in vitro, the addition of phosphorylated IkappaBalpha inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated IkappaBalpha) by activating IkappaB kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate.     

The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability

Ubiquitin-mediated degradation plays a crucial role in many fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin ligase complexes, called SCF complexes, found throughout eukaryotes, is involved in a variety of biological pathways. In Saccharomyces cerevisiae, an SCF complex contains a common set of components, namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a variable component called an F-box protein. The F- box is a approximately 40-amino-acid motif that allows the F-box protein to bind Skp1p. Each SCF complex recognizes different substrates according to which F-box protein is associated with the complex. In yeasts, three SCF complexes have been demonstrated to associate with the ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity. F-box proteins are not abundant and are unstable. As part of the SCF(Met30p) complex, the F-box protein Met30p represses methionine biosynthetic gene expression when availability of L-methionine is high. Here we demonstrate that in vivo SCF(Met30p) complex activity can be regulated by the abundance of Met30p. Furthermore, we provide evidence that Met30p abundance is regulated by the availability of L-methionine. We propose that the cellular responses mediated by an SCF complex are directly regulated by environmental conditions through the control of F-box protein stability.