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Regulation of Mammalian Cell Division by the Ubiquitin System
Summary: Michele Pagano explores the roles that the ubiquitin system plays in cell growth, proliferation, and survival and how the deregulation of this system contributes to causing cancers.
Cells depend on the proper functioning of an ensemble of networked, molecular machines to control diverse processes—from cell proliferation to differentiation to cell death. The ubiquitin system can rapidly degrade modular regulatory components of these machines, contributing to the precision and synchronization of complex cellular processes. Given this critical role, the ubiquitin system is often deregulated in disease.
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.
In initial studies of the SCF complexes, our group focused on two F-box proteins, Skp2 and βTrcp, producing numerous studies establishing these proteins as the prototypical F-box proteins. We demonstrated that Skp2 functions as an activator of the cyclin-dependent kinases Cdk1 and Cdk2 by directing the degradation of p21 and p27 (Figure 1). Additionally, we found a dual role for βTrcp in controlling Cdk1 activity, turning it off in S phase and turning it on at G2/M.
Beyond simply examining SCF function, we investigated the regulation of SCF complexes, discovering that Skp2 is degraded via the anaphase-promoting complex/cyclosome (APC/C), an SCF-like ubiquitin ligase. The interplay between these different ligases 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 βTrcp. Our findings illustrate that a degradation cycle, in which 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 ubiquitin ligases to 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 promitotic kinase Plk1, and this event is essential for the establishment and maintenance of an efficient G2 checkpoint. 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-Plk1 axis is a hub in the G2 DNA damage response that is crucial for preventing entry into mitosis. In other studies, we have implicated βTrcp in the establishment of the S/G2 checkpoints in response to genotoxic stresses (in cooperation with Chk1 and Chk2), as well as in the recovery from the G2 checkpoint (in cooperation with Plk1).
We have extended our studies of Skp2 and βTrcp to the clinic (Figure 2 and Figure 3), finding that Skp2 levels directly correlate with poor prognosis and correlate inversely with p27 expression in human epithelial cancers and lymphomas. Accordingly, Skp2 cooperates with activated N-Ras in a mouse model of lymphomagenesis. βTrcp also behaves as a tissue-specific oncoprotein, as shown by the development of mammary carcinomas from breast epithelium constitutively expressing βTrcp.
By analogy to Skp2 and βTrcp, we believe that other F-box proteins represent key regulators of the three critical dimensions of cellular life: growth/proliferation, survival, and differentiation. Notably, only 6 of 69 human F-box proteins have well-established substrates (Figure 4). Therefore, we are studying the cellular functions of orphan F-box proteins.
In deciding which F-box proteins to investigate, we used five criteria (based on biochemical data, siRNA screens, and gene expression profiles) that could indicate a key role in cellular life. We found that 16 F-box proteins meet at least three criteria. We are using an interdisciplinary approach similar to our previous characterizations of βTrcp and Skp2 to pursue these proteins. One of our goals is to identify biologically significant substrates to understand the molecular mechanism by which these 16 F-box proteins control cellular functions. Another goal is the identification of the kinases that facilitate substrate recognition by SCF.
We have successfully utilized two techniques for unbiased substrate identification. Although we have used 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 copurified substrates in vitro. These unbiased screens have broadened our research interests from a CDK-centric view of cell proliferation to a vision that links 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.
βTrcp, the Spindle Checkpoint, and Chromosome Stability
We identified the transcriptional repressor REST (RE1-silencing transcription factor) as a novel βTrcp interactor and found that REST is degraded via βTrcp 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 βTrcp inhibited Mad2 expression and resulted in defective activation of the spindle checkpoint in cultured cells. Expression of REST-FS, a mutant lacking the βTrcp degradation motif that was identified in human cancers, leads to chromosome instability (Figure 5) through inhibition of Mad2 expression. Thus, mutations causing increased REST stability generate chromosomal instability, a mechanism that may contribute to tumor development.
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 (Figure 6). 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.
βTrcp, Protein Translation, and Control of Cell Size
We found that βTrcp 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 βTrcp- and S6K1-dependent manner, allowing efficient protein translation and cell growth (Figure 7).
Fbxl10 Epigenetically Controls the Expression of Ribosomal Genes
We found that Fbxl10 is a nucleolar protein (Figure 8) that preferentially binds ribosomal DNA to repress transcription of rRNA genes. 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.
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. Our comprehensive and interdisciplinary approach, which includes biochemical methods and somatic cell and mouse genetics, should continue to contribute to the understanding of cell functions.
This research is supported in part by grants from the National Institutes of Health and the Multiple Myeloma Research Foundation.
Last updated July 01, 2008
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