Ubiquitin-mediated proteolysis of many cellular regulators is controlled by SCF ubiquitin ligases, formed by four subunits: Skp1, Cul1, Rbx1, and a variable F-box protein that provides substrate specificity.  Using a yeast two-hybrid screen of Skp1 binding proteins in conjunction with state-of-the-art bioinformatics tools, we identified and annotated a family of 69 human F-box proteins (see list and Current Biol 9: 1177, 1999).

In initial studies of the SCF complexes, our group focused on the roles of the F-box proteins Skp2 and beta-Trcp, producing numerous studies establishing these proteins as key regulators of cyclin-dependent kinases (CDKs, reviewed in 6).  We demonstrated that Skp2 functions as an activator of Cdk1 and Cdk2 by directing the degradation of the CDK inhibitors p21 and p27 (Genes & Dev, 22:2496, 2008, Mol Cell, 27:462, 2007; Cell, 115:71, 2003; JBC,278: 25752, 2003; Nature Cell Biol, 3:321, 2001; J Cell Biol,153:1381, 2001; Genes & Dev, 13:1181, 1999; and Nature Cell Biol, 1:193, 1999) (Figure 1).  Additionally, we found a dual role for beta-Trcp in controlling Cdk1 activity, attenuating its activity by inducing Cdc25A degradation in S phase and contributing to its activation by inducing Claspin degradation at G2/M (Mol Cell, 23: 319, 2006 and Nature, 426:87, 2003) (Figure 2).

Beyond simply examining SCF function, we have investigated the regulation of SCF complexes, discovering that Skp2 is degraded via the Anaphase-Promoting Complex/Cyclosome (APC/C) (Nature, 428:190, 2004), an SCF-like ubiquitin ligase.  Additionally, our studies of the APC/C revealed that p21, Claspin, and Cdc25A are substrates for both SCF and APC/C, albeit during different cell cycle stages (Mol Cell, 27:462, 2007 and Cell, 134:256, 2008).  This convergence of interests is exemplified in our studies of the F-box protein Emi1.  Using a mouse model, we established that Emi1, which plays an SCF-independent role in APC/C regulation, is targeted for degradation by beta-Trcp (Dev Cell 4: 799, 2003).  Our findings illustrate that a degradation cycle, in which the APC/C and SCF ligases reciprocally regulate each other, controls the tempo of cell cycle progression, attenuating or activating distinct components of the CDK regulatory network during defined time windows.

We have also studied the contribution of SCF and SCF-like ligases to the DNA damage checkpoints.  While it is established that the major function of APC/CCdh1 is the maintenance of the G0/G1 state, we have demonstrated that in response to DNA damage, APC/CCdh1 is reactivated to allow the degradation of the pro-mitotic kinase Plk1, and this event is essential for the establishment and maintenance of an efficient G2 checkpoint (Cell, 134:256, 2008).  The reactivation of APC/CCdh1 depends on Cdh1 dephosphorylation by Cdc14B, which is released from the nucleolus upon genotoxic stress.  Our findings indicate that the Cdc14B-Cdh1-Plk axis is a hub in the G2 DNA damage response that is crucial for preventing entry into mitosis.  In different studies, we have implicated beta-Trcp in the establishment of the S/G2 checkpoints in response to genotoxic stresses (in cooperation with Chk1 and Chk2) (Nature, 426:87, 2003) as well as in the recovery from the G2 checkpoint (in cooperation with Plk1) (Mol Cell, 23: 319, 2006).

We have also expanded our studies of Skp2 and beta-Trcp to the clinic (Figure 3 and Figure 4), finding that Skp2 levels directly correlate with poor prognosis and correlate inversely with p27 expression in human epithelial cancers and lymphomas (Nature Med, 3:231, 1997 and J of Clinical Invest, 110:633-641, 2002).  Accordingly, Skp2 cooperates with activated N-Ras in a mouse model of lymphomagenesis (PNAS, 98:2515, 2001). beta-Trcp also behaves as an oncoprotein, at least in some tissues, as shown by the development of mammary carcinomas when constitutively expressed in breast epithelium (MCB 24:8184, 2004).  

By analogy to Skp2 and beta-Trcp, we believe that other F-box proteins are important regulators of the three key dimensions of cellular life:  growth/proliferation, survival, and differentiation.  Notably, only six out of 69 human F-box proteins have well-established substrates  (reviewed in 7-12) (Figure 5).  Therefore, one of the goals of our lab is to study the cellular functions of orphan F-box proteins and systematically identify their biologically significant substrates.       

As most SCF substrates require phosphorylation for recognition by their specific F-box, we are also interested in identifying the kinases that facilitate substrate recognition. The requirement for substrate phosphorylation indicates that SCF ligases contribute to cellular processes in part by “sensing” the activity of kinases, and, in many cases, this “sensing” can be combinatorial, requiring the cooperative phosphorylation of a substrate by kinases from two distinct pathways.  Therefore, elucidation of both the kinase(s) and F-box protein for each substrate is required to fully understand substrate targeting and degradation. 

In deciding which F-box proteins to investigate, we prioritized our effort using five criteria (based on biochemical data, siRNA screens, and gene expression profiles) that could indicate a role in the key dimensions of cellular life.  By applying these five criteria, we found that 16 F-box proteins meet at least three criteria.  We are actively pursuing these proteins using an interdisciplinary approach similar to our previous characterizations of beta-Trcp and Skp2.  One of our primary goals is to identify substrates in order to understand the molecular mechanism by which these 16 F-box proteins control cellular functions. 

Our laboratory has successfully utilized two techniques for unbiased substrate identification.  Although we have utilized traditional tandem affinity purifications to identify novel SCF substrates, we have also developed a novel immunoaffinity/enzymatic assay that enriches for ubiquitylated substrates based on the ability of SCF complexes to ubiquitylate co-purified substrates in vitro.  These unbiased screens have broadened our research interests from a CDK-centric view of cell proliferation to a more far-reaching vision that links together many (at first glance) disparate cellular pathways controlling cell proliferation, DNA-damage checkpoints, the circadian clock, protein synthesis, ribosomal biogenesis, apoptosis, cytoskeleton organization, and neurogenesis.  A few examples are detailed below.

βTrcp and cell survival
The BimEL tumor suppressor is a potent pro-apoptotic BH3-only protein. We found that in response to survival signals BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F-box protein βTrCP (Mol Cell, 33:109-116, 2009). Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. We had previously shown that when cells are stimulated with mitogens, βTrCP allows efficient protein synthesis and cell growth in cooperation with the PI3K-S6K pathway (see bwlow). Our findings reveal that βTrCP promotes cell survival in cooperation with the ERK-RSK pathway, by targeting BimEL for degradation. 

βTrcp, the spindle checkpoint and chromosome stability
We identified the transcriptional repressor Rest (RE1-silencing transcription factor) as a novel beta-Trcp interactor, and found that Rest is degraded via beta-Trcp in G2 to allow transcription of Mad2, an essential component of the spindle checkpoint.  Expression of a stable Rest mutant that is unable to bind beta-Trcp inhibited Mad2 expression and resulted in defective activation of the spindle checkpoint in cultured cells.  Furthermore, expression of Rest-FS, a mutant lacking the beta-Trcp degron that was identified in human cancers, leads to chromosome instability through inhibition of Mad2 expression. Thus, mutations causing increased Rest stability generate chromosomal instability, a mechanism that may contribute to tumor development.  (Nature, 452, 365-369, 2008). 

Fbxl3 and the circadian clock
We discovered that the F-box protein Fbxl3 induces the ubiquitylation and consequent degradation of Cry1 and Cry2, two repressors of the heterodimeric transcription factor Clock:Bmal1, a central component of the circadian clock (Science, 316:900-904, 2007).  This regulation by Fbxl3 is a prerequisite for the efficient and timely reactivation of Clock:Bmal1 and the consequent expression of Per1 and Per2, two tumor suppressors that control fundamental processes, such as the timing of cell cycle progression and checkpoint activation. 

Fbxl10 epigenetically controls the expression of ribosomal genes
We found that Fbxl10 is a nucleolar protein and demonstrated that Fbxl10 preferentially binds ribosomal DNA to repress transcription of rRNA genes (Nature, 450, 309-313, 2007).  Furthermore, we showed that repression of rRNA genes by Fbxl10 is dependent on its JmjC-domain, which is necessary to specifically demethylate H3K4me3 in the nucleolus. In agreement with the coupling of rRNA synthesis and cell proliferation, we showed a negative effect of Fbxl10 on cell growth and cell cycle progression. 

βTrcp, protein translation, and control of cell size
We found that beta-Trcp plays a role at the G0/G1 transition.  In response to mitogens, the tumor suppressor PDCD4, which inhibits the translation initiation factor eIF4A, is rapidly degraded in a beta-Trcp- and S6K1-dependent manner, allowing efficient protein translation and cell growth (Science, 314:467-471, 2006).

In summary, our research program involves the study of the human F-box protein family to gain an understanding of the molecular mechanisms through which SCF and SCF-like ubiquitin ligases control basic cellular processes, such as cell growth/ division, cell survival, and differentiation.  To this end, we are using a comprehensive and interdisciplinary approach, including biochemical methods, as well as somatic cell and mouse genetics.  These tools, together with our expertise in the ubiquitin system, will ensure that our laboratory will continue to contribute to the understanding of cell functions.  

Relevant review articles:

1.  Bloom, J. and Pagano, M. Deregulated degradation of the cdk inhibitor p27 and malignant transformation. Seminars in Cancer Biology 13:41-47 (2003).

2.  Pagano, M. and Benmaamar, R. When protein destruction runs amok, malignancy is on the loose. Cancer Cell 4:251-256 (2003).

3.  Guardavaccaro, D. and Pagano, M. Oncogenic Aberrations of Cullin-Dependent Ubiquitin Ligases. Oncogene 23:2037-49 (2004).

4.  Yamasaki L. and Pagano M. Cell cycle, proteolysis and cancer. Curr Op in Cell Biol, 16:623-628 (2004).

5.  Nakayama K.I., Nakayama, K. Ubiquitin ligases: cell-cycle control and cancer. Nature Rev Cancer 6:369-381 (2006).

6.  Pagano, M. and Jackson, P. Wagging the Dogma: Tissue specific Cell Cycle Control in the Mouse Embryo. Cell 118:535-538 (2004).

7.  Reed, S. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Rev Mol Cell Biol 4:855-64 (2003).

8.  Cardozo T. and Pagano M. The SCF ubiquitin ligase: insights into a molecular mechanism. Nature Rev Mol Cell Biol 5:739-753 (2004).

9.  Petroski M.D., Deshaies R.J. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Bio 6:9-20 (2005).

10. Ang X.L. and Harper J.W. SCF-mediated protein degradation and cell cycle control. Oncogene 24:2860-2870 (2005).

11. Guardavaccaro, D. and Pagano, M. Stabilizers and destabilizers controlling cell cycle oscillators. Mol Cell 22:1-4 (2006).

12. Frescas, D. and Pagano, M. Deregulated proteolysis by the F-box proteins Skp2 and ß-TrCP: Tipping the scales of cancer. Nature Rev Cancer 8:438-449 (2008).

“No matter how counter-intuitive it may seem, basic research has proven over and over to be the lifeline of practical advances in medicine.”