Figure 1 (below) depicts a very simplified model emphasizing the contribution of ubiquitin ligases to the regulation of CDKs. There are, of course, crucial contributions from transcription, translation, translocation mechanisms, etc., which were omitted here to focus on how SCF and APC/C complexes work in concert to control and modulate CDK activity. In brief, SCFSkp2 is an activator of both Cdk1 and Cdk2; SCFFbw7 is an inhibitor of Cdk2; and SCFßTrcp contributes to turning Cdk1 activity off during S and G2 phase, and turning it on at G2/M. APC/C functions as an antagonist of CDK activity. Notably, SCF ligases and APC/C reciprocally control each other: in G1, APC/CCdh1 promotes the degradation of Skp2, thereby maintaining the G1 state, whereas in early mitosis SCFßTrcp activates APC/CCdc20 by inducing the degradation of its inhibitor, Emi1. Thus, a picture emerges highlighting waves of different ubiquitin ligases that modify components of the CDK regulatory network by maintaining, activating or attenuating distinct cell cycle regulatory proteins during defined time windows. At the same time, the ubiquitin ligases themselves are subject to direct or indirect control by CDKs.

SCFSkp2, SCFß-Trcp and SCFFbw7 also regulate important transcription factors: FoxO1 (SCFSkp2); Myc, Jun and Notch 1 and 4 (SCFFbw7); and Tcf1/ß-catenin, Atf4 and NFKB (SCFß-Trcp) (Ang and Harper, 2005; Guardavaccaro and Pagano, 2004; Petroski and Deshaies, 2005). Similarly, APC/CCdh1 controls the degradation of SnoN, a corepressor of SMAD. In turn, these transcription factors, among other functions, control CDK activities by inducing or suppressing the transcription of genes that encode certain CDK subunits and their regulators.

In summary, despite the large number of F-box proteins, only three human SCF ubiquitin ligases have well-established substrates, many of which are involved in cell cycle control.

Black color signifies activated forms of the respective proteins, turquoise indicates inactive forms or degraded proteins. Asterisks denote the contribution to this model derived from work in our laboratory. 

Fig. 1A. G1 phase: APC/CCdh1 maintains the attenuation of both Cdk1 and Cdk2 during G1 by targeting Skp2 for degradation (Bashir et al., 2004; Wei et al., 2004) and completing the degradation of cyclins A and B, which was initiated by APC/CCdc20 during mitosis. Thus, the first action of APC/CCdh1 upholds the inhibition of Cdk1 and Cdk2 by p27 and p21 --key inhibitors of both CDKs (Aleem et al., 2005; Bashir and Pagano, 2005; Nakayama et al., 2004; Sherr and Roberts, 1999)--, while the second keeps cyclin A and B levels low. During G1 APC/CCdh1 furthermore ensures that the key CDK-activating phosphatase Cdc25A (Donzelli et al., 2002) as well as a number of other substrates such as Plk1, Aurora A, Nek2A, Cdc6, Geminin, and Cdc20 do not accumulate prematurely (Peters, 2002).

Fig. 1B. G1/S, S and G2 phases: During the G1/S transition a crucial switch in the regulation of Cdk1 and Cdk2 involves the inactivation of APC/CCdh1 simultaneously releasing Skp2 and cyclins A and B from APC/CCdh1–mediated destabilization. Seen through the prism of CDK-activity profiles during the cell cycle, the finding that SCFSkp2 is responsible for the degradation of p27 (Carrano et al., 1999; Nakayama et al., 2000; Sutterluty et al., 1999; Tsvetkov et al., 1999) and p21 (Bornstein et al., 2003; Wang et al., 2005; Yu et al., 1998) reveals an important role of this SCF ligase in executing the transition to S-phase. However, this begs the following question: How is Cdk1 kept in an attenuated state given that its inhibitors are degraded and its activating cyclin subunits are stabilized? This is where another F-box protein comes into play. For full activation, Cdk1 requires the action of Cdc25A that counteracts an inactivating phosphorylation by the Wee1 kinase. SCFßTrcp has been shown to target phosphorylated Cdc25A in S and early G2 phase for degradation (Busino et al., 2003; Jin et al., 2003). Hence, a balanced picture with maximal Cdk2 activity and low Cdk1 activity emerges: cyclin E-Cdk2 and cyclin A-Cdk2 activities are maintained by the action of Skp2 on p21 and p27; and Cdk1 activity is kept low by the action of SCFßTrcp on Cdc25A (apparently low levels of Cdc25A in S and G2 are sufficient to sustain full Cdk2 activity but only low Cdk1 activity). Thus, G1/S is characterized by inactivation of APC/CCdh1 and activation of CDKs. But how is APC/CCdh1 inactivated? The F-box protein Emi1 accumulates at G1/S and inhibits APC/CCdh1 by a non-proteolytic mechanism (Reimann et al., 2001). Cdk2 contributes to the inactivation of APC/CCdh1 by phosphorylating Cdh1 and consequently inhibiting its binding to APC/C (Peters, 2002). This places Emi1 and Cdk2 both upstream and downstream of APC/CCdh1, suggesting that some unknown factor (X? in Fig. 1B) initiates the inactivation of APC/CCdh1. It has been proposed that this unknown factor is APC/CCdh1 itself, which, by mechanisms not yet completely understood, induces the degradation of both Cdh1 (Listovsky et al., 2004) and its major UBC, namely Ubc10 (Rape and Kirschner, 2004).

Fig. 1C. Late G2 and G2/M transition:  Interpreting the CDK activity profile at the G2/M transition through the action of ubiquitin ligases is similarly revealing. During G2, Cdk2 attenuation is now maintained through SCFFbw7–mediated degradation of cyclin E (Koepp et al., 2001; Moberg et al., 2001; Strohmaier et al., 2001), although some Cdk2 activity remains due to the presence of cyclin A. Cdk1 activity instead increases through the continued inhibition of APC/C, and the renewed availability of Cdc25A. In fact, Cdc25A is not recognized any longer by SCFßTrcp as the kinase that generates the phospho-degron for this recognition (Chk1) is inactivated in late G2. Remarkably, SCFßTrcp is also involved in the inactivation of Chk1 as it targets Claspin (a mediator of Chk1 activation) for degradation (Peschiaroli et al., 2006). Just a bit later, at G2/M, SCFßTrcp continues to have a role, namely to induce the degradation of the Cdk1 inhibitory kinase Wee1 (Watanabe et al., 2004).

Fig. 1D. Mitosis: In prometaphase, the block on APC/C is removed by yet another activity of SCFßTrcp: the degradation of Emi1 (Guardavaccaro et al., 2003; Margottin-Goguet et al., 2003). As a consequence, APC/CCdc20 is released. Later, APC/CCdc20, in addition to mediating the anaphase-promoting degradation of securin, actively initiates the degradation of cyclins A and B, thereby quenching Cdk1 activity and resetting the cell cycle machinery to low CDK activity. Interestingly, Cdk1 promotes the activity of APC/CCdc20 by phosphorylating certain APC/C subunits and therefore contributes to its own attenuation (Peters, 2002).

Deregulation of the ubiquitin system in cancer and other proliferative diseases.             Several ubiquitin ligases have been shown to play a critical role in regulating cell proliferation, differentiation or apoptosis. For this reason, the ubiquitin system is often the target of cancer-related deregulation and involved in processes such as oncogenic transformation and tumor progression. Indeed, increased stability of positive regulators of proliferation can be achieved by lowering the activity or the levels of the specific enzymes necessary for their degradation. For example, proto-oncoproteins such as cyclin E, c-Myc, c-Jun and Notch are stabilized by inactivating mutations in Fbw7, which are found in certain epithelial cancers. Similarly, Hif-1α and Hif-2α are stabilized by germline mutations in the VHL gene, inducing the cancer-predisposing Von Hippel-Lindau syndrome. Thus, the ubiquitinylating enzymes specific for proto-oncoproteins can act as tumor suppressors. In contrast, some ubiquitinylating enzymes (e.g., Mdm2) display oncogenic properties based on their specific function to target tumor suppressors (e.g., p53). Recent reviews (Ang and Harper, 2005; Bashir and Pagano, 2003; Pagano and Benmaamar, 2003; Reed, 2003; Yamasaki and Pagano, 2004) summarize many established examples of overactivation or inactivation of the ubiquitin pathway (with particular emphasis on ubiquitin ligases) in human tumors.

F-box proteins also play a role in human diseases. Skp2 is the product of a proto-oncogene, Fbw7 functions as a tumor suppressor [reviewed in (Pagano and Benmaamar, 2003; Yamasaki and Pagano, 2004)], and overexpression of ßTrcp can contribute to transformation, at least in some epithelial tissues (He et al., 2005; Koch et al., 2005; Kudo et al., 2004; Ougolkov et al., 2004; Zhou and Hung, 2005). Emi1 has been found overexpressed in tumor cell lines and certain breast tumors [(Hsu et al., 2002; van 't Veer et al., 2002), our unpublished data and P. Jackson, pers. comm.]. Fbw4 is encoded by SHFM3, the split hand-foot malformation syndrome gene 3 (Basel et al., 2003). The expression of Fbx3 is increased in the proliferating synovium of patients with rheumatoid arthritis (Masuda et al., 2002). Finally, Fbx32/Atrogin-1 has been reported to be upregulated during muscle atrophy (Bodine et al., 2001; Gomes et al., 2001). Thus, F-box proteins are attractive candidates for drug discovery because they play crucial roles in many important signaling pathways.

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