Oncogene (2005) 24, 2860–2870 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc SCF-mediated protein degradation and cell cycle control Xiaolu L Ang1 and J Wade Harper*,1 1Program in Biological and Biomedical Sciences, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA The regulatory step in ubiquitin (Ub)-mediated protein established that targeted protein degradation is a key degradation involves recognition and selection of the conduit for regulating protein levels, what is now most target substrate by an E3 Ub-ligase.E3 Ub-ligases evoke important is being able to understand the specific sophisticated mechanisms to regulate their activity mechanisms as to how this proteolysis contributes to temporally and spatially, including multiple post-transla- the regulation of cell cycle states and phase transitions. tional modifications, combinatorial E3 Ub-ligase path- This review aims to examine how various E3Ub- ways, and subcellular localization.The phosphodegrons of ligases – which are responsible for substrate recognition many substrates incorporate the activities of multiple in the regulatory step of the Ub-mediated proteolytic kinases, and ubiquitination only occurs when all necessary pathway – recognize their targets, and we focus on three phosphorylation signals have been incorporated.In this particular emerging themes in the regulation of Ub- manner, the precise timing of degradation can be mediated proteolysis. Using exemplary findings, we will controlled.Another way that the Ub pathway tightly discuss: (1) how the interaction between the E3Ub- controls the timing of proteolysis is with multiple E3 Ub- ligase and its substrate is often dependent upon ligases acting upon a single target.Lastly, subcellular phosphorylation; (2) how multiple E3Ub-ligases are localization can either promote or prevent degradation by often involved in the targeting of cell cycle proteins; and regulating the accessibility of kinases and E3 Ub-ligases. (3) how the subcellular localization of substrates and E3 This review highlights recent findings that exemplify these Ub-ligases is used to regulate proteolysis. emerging themes in the regulation of E3 Ub-ligase substrate recognition. Oncogene (2005) 24, 2860–2870. doi:10.1038/sj.onc.1208614 Ub-mediated degradation and the SCF Keywords: proteolysis; ubiquitin; SCF; APC; E3ubiquitin- (Skp1/Cullin/F-box) complex in cell cycle control ligase; phosphodegron Regulated proteolysis occurs through the Ub-mediated proteolytic pathway (reviewed in Hershko and Ciechan- over, 1998). This pathway governs the formation of a covalent multi-Ub chain, which serves as a cellular degradation signal, onto a target substrate. The Introduction ubiquitination pathway sequentially involves the E1 Ub-activating enzyme, E2 Ub-conjugating enzymes, and Cellular duplication and differentiation requires me- E3Ub-ligases. Of particular interest is the last step of chanisms that integrate cellular growth, genomic repli- the pathway where an E3Ub-ligase facilitates transfer of cation, and equal nuclear division. Misregulation of any Ub – a small molecule comprised of 76-amino-acid aspect of the cell cycle may understandably lead to residues – onto a lysine residue on the target substrate or developmental defects and diseases such as cancer. onto Lys-48 of a growing Ub chain extending from the Given that the regulation of the cell cycle is so critical substrate. Given that this multi-Ub chain is sufficient to to an organism’s well being, coordinated regulatory promote the protein’s unfolding and degradation by the mechanisms have evolved to establish a specific config- 26S proteasome, substrate ubiquitination must occur in uration of interacting proteins that define a particular a controlled and specific manner. Insight into how E3 cell cycle phase or that mediate transitions between these Ub-ligases recognize specific substrates and what con- phases. In addition to transcriptional upregulation and trols this interaction is vital for understanding the protein activation, targeted ubiquitin (Ub)-mediated regulation of the Ub pathway. proteolysis provides a swift and precise means by which The E3Ub-ligases that perform the rate-limiting to regulate the abundance of specific proteins and selectivity step of direct substrate recognition are the therefore the level of their activities (reviewed in Tyers most numerous and varied collection in the Ub path- and Jorgensen, 2000; Reed, 2003). While it has been well way. They can be classified into HECT domain, Ring- H2-finger domain, and U-box domain superfamilies (Kamura et al., 1999; Ohta et al., 1999; Seol et al., 1999; *Correspondence: JW Harper; Skowyra et al., 1999; Joazeiro and Weissman, 2000; E-mail: [email protected] Hatakeyama et al., 2001; Fang et al., 2003; Ingham SCF-mediated protein degradation and cell cycle control XL Ang and J Wade Harper 2861 et al., 2004). The two principal E3Ub-ligases involved in of phosphorylated residues on the substrate that directly cell cycle control are the SCF complex and the APC/C interact with a protein–protein interaction domain in an (anaphase-promoting complex/cyclosome). They are E3Ub-ligase (e.g. an F-box protein), thereby linking the both members of the Ring-H2-finger-containing E3 substrate to the conjugation machinery (Winston et al., Ub-ligase family. SCF complexes are used throughout 1999c; Nash et al., 2001). Additionally, phosphorylation the cell cycle, whereas APC functions primarily during allows temporal regulation of substrate degradation, mitosis and G1 phase. even if SCF complexes are constitutively active through- The SCF complex has three primary subunits: Skp1, out the cell cycle. Cullin, and Rbx1/Roc1 (reviewed in Deshaies, 1999; The best characterized example of phosphodegron Koepp et al., 1999). This complex can interact with an utilization involves the G1/S transition in S. cerevisiae, assortment of modular F-box proteins, which all share where the WD40 propeller structure of the F-box an F-box motif that was first identified in yeast Cdc4 protein Cdc4p recognizes sequences in Sic1p after their and cyclin F and that was shown to interact directly with phosphorylation by Cln1/2-Cdc28 kinases (Nash et al., Skp1 (Bai et al., 1996). The Cul1/Rbx1 components 2001; Orlicky et al., 2003). Sic1p is a Cdk inhibitor, and form the E3Ub-ligase core that associates with E2 Ub- its degradation frees Cln5/6-Cdc28 to initiate DNA conjugating enzymes. F-box proteins directly recruit replication. Sic1p contains as many as nine Cdk ubiquitination substrates and bridge the interaction phosphorylation sites, which individually bind weakly between the E2 and the substrate (Feldman et al., to Cdc4p. However, upon phosphorylation of a mini- 1997; Skowyra et al., 1997). Thus, the identity of the mum of six Cdk sites, Sic1p is efficiently recognized by F-box protein determines the target of the SCF. There Cdc4p, ubiquitinated, and degraded (Nash et al., 2001). are a variety of F-box proteins including subfamilies In this manner, Sic1p acts as a gauge for Cln-Cdc28 with WD40 domains (Fbw) or with leucine-rich repeats activity by accumulating phosphates until a threshold (Fbl) (Cenciarelli et al., 1999; Winston et al., 1999b; Jin number of phosphorylation events promotes its turn- et al., 2004). Moreover, F-box proteins frequently over (Deshaies and Ferrell, 2001; Harper, 2002). More- recognize their substrates in a manner dependent upon over, suboptimal phosphorylation sites confer particular modifications, for example, phosphorylation ultrasensitivity to the system, as individual Sic1 proteins or glycosylation (Feldman et al., 1997; Skowyra et al., ‘resist’ ubiquitination when Cln-Cdc28 activity is 1997; Nash et al., 2001; Yoshida et al., 2002; Orlicky increasing, but still too low to signal for Sic1p to release et al., 2003; Wu et al., 2003). The SCF complex itself is Clb-Cdc28 from inhibition. Thus, the system avoids also subject to post-translational modifications in the ‘noise’ or premature Clb-Cdc28 activation, and instead form of neddylation on the Cullin subunit (Lammer creates a switch-like transition where Clb-Cdc28 will be et al., 1998). Neddylation describes the linkage of Nedd8 activated in a uniform manner. – a small 76-residue protein with sequence similarity to In the case of Sic1p, phosphorylation by kinase Cln- Ub – to a lysine residue on the substrate. Neddylation Cdc28 appears to be sufficient for recognition by Cdc4p. occurs on the winged-helix B domain of Cullins, which is In contrast, many targets of mammalian SCF complexes part of the domain that forms a globular tail binding require a complex interplay between multiple kinases or Rbx1 and the E2-conjugating enzyme (Zheng et al., kinase pathways in order to generate the requisite 2000; Kawakami et al., 2001). Although Nedd8 con- phosphodegron on the SCF target. For example, jugation is known to dramatically increase Ub transfer SCFb-TRCP substrates IkBa, b-catenin, Emi1 (early by E2’s bound to the Rbx1/Cullin complex, the precise mitotic inhibitor 1), Cdc25A, and Wee1 require the mechanism for this stimulation is unknown. combined activities of multiple kinases to create their phosphodegron motifs (Figure 1) (Winston et al., 1999c; Liu et al., 2002; Jin et al., 2003; Margottin-Goguet et al., 2003; Watanabe et al., 2004). Early studies identified a Substrate recognition by the SCF complex is dependent small peptide motif (i.e. phosphodegron) on both IkBa upon phosphorylation and b-catenin that was able to bind to b-TRCP in a phosphorylation-dependent manner (Winston et al., Most substrates require phosphorylation to interact 1999c). The motif or a closely related sequence with the F-box protein in an SCF complex. The SCF (DpSGFXpS, where F is a hydrophobic amino acid pathway, in fact, was elucidated through efforts to and pS is phosphoserine) has been found to be involved understand how phosphorylation controls the abun- in the majority of SCFb-TRCP substrates. In these cases, dance of cyclin and Cdk inhibitors in the Saccharomyces dual phosphorylation within the motif is important for cerevisiae cell cycle (Bai et al., 1996; Feldman et al., high-affinity binding.