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Regulation of Mammalian Cell Division by the Ubiquitin System

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Michele Pagano explores the roles that the ubiquitin system plays in fundamental cellular processes, such as cell growth, proliferation, and the DNA damage response. His group is also committed to understanding how the deregulation of this regulatory network contributes to malignant transformation.

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 rapidly degrades 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 key cellular regulators is controlled by SCF ubiquitin ligases, formed by four subunits: Skp1, Cul1, Rbx1, and a variable F-box protein (Figure 1). The F-box protein dictates the substrates for each complex, and we initially identified, defined, and annotated the human F-box protein family, which contains 69 members. This large protein family allows the SCF complexes to control an even larger number of substrates and direct the degradation of these substrates in both a spatial and temporal manner.

Following our initial identification of the F-box proteins, we performed in-depth characterizations of two F-box proteins, Skp2 and βTrCP, defining these proteins as the prototypical F-box proteins. Beyond providing a mechanistic understanding of SCF complex function, our studies of Skp2 and βTrCP yielded important insights into the control of cell proliferation. βTrCP serves as a molecular rheostat, responding to multiple stimuli to target both positive and negative regulators of the cell cycle, while Skp2 activates cyclin-dependent kinases by targeting negative regulators of the cell cycle for degradation. Consistent with these results, we found that Skp2 is the product of an oncogene that is overexpressed in multiple cancers, and this overexpression is an indicator of poor prognosis.

Very few members of the F-box protein family have been characterized (Figure 2), so in addition to continuing our studies of Skp2 and βTrCP, we turned our attention to the broader F-box protein family. We have begun to characterize "orphan" F-box proteins, focusing on those that we implicated in vital cellular processes by an siRNA screen.

We are using an interdisciplinary approach to identify and characterize substrates and biological pathways for these orphan F-box proteins, with an emphasis on biochemical purifications, mass spectrometry, somatic cell genetics, and cell biological techniques. Our investigations have linked orphan F-box proteins and their SCF complexes to multiple diverse biological pathways, many of which reveal unexpected connections to the core cell proliferation machinery. Thus, we have evolved from a view of cell proliferation centered on the actions of cyclin-dependent kinases to a vision that links many (at first glance) disparate pathways controlling cell signaling and proliferation, DNA damage checkpoints, gene transcription, protein synthesis, ribosomal biogenesis, apoptosis, and circadian oscillations.

Control of Basic Biological Processes by SCF Ubiquitin Ligases
We have elucidated the role of SCF ubiquitin ligases in controlling multiple basic pathways and processes that are often deregulated in tumors, including the following:

Control of genome stability. Our laboratory discovered important roles for ubiquitin-mediated degradation in controlling genome stability. We demonstrated that cyclin F–mediated degradation of CP110 is required to limit centrosome duplication to once per cell cycle (Figure 3). Moreover, cyclin F mediates the degradation of ribonucleotide reductase member 2 (RRM2) to maintain balanced deoxyribonucleotide triphosphate (dNTP) pools (Figure 4). Failure to degrade CP110 causes centrosomal and mitotic defects, and inhibition of RRM2 degradation leads to increased mutation frequency and genome instability.

DNA damage response. We demonstrated that, in response to DNA damage, APC/CCdh1, an SCF-like ligase, is activated to promote the degradation of the promitotic kinase Plk1, inhibiting the βTrCP- and Plk1-dependent degradation of Claspin (a Chk1 activator) and Wee1 (a Cdk1 inhibitor). This event is essential for the establishment and maintenance of the G2 checkpoint (Figure 5). Moreover, we showed that, following genotoxic stress, cyclin F is degraded in an ATR-dependent manner to allow RRM2 accumulation, dNTP availability, and efficient DNA repair synthesis (Figure 4).

Response to proliferation signals. Our continued study of βTrCP has revealed a key role for this protein at the G0/G1 transition. Upon mitogenic stimulation, 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. Moreover, we found that mTOR cooperates with CK1α and βTrCP to induce the degradation of DEPTOR (an mTOR inhibitor), thereby generating an auto-amplification loop that promotes the full activation of mTOR. Our studies also revealed that βTrCP regulates cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation (Figure 6).

Control of circadian rhythms. We discovered that the F-box protein Fbxl3 induces the ubiquitylation and degradation of Cryptochrome 1 and Cryptochrome 2, two repressors of the Clock:Bmal1 transcription factor, a central component of the circadian clock. Fbxl3-dependent degradation of the Cryptochromes is necessary for the timely and efficient reactivation of Clock:Bmal1 and the consequent expression of Per1 and Per2, two tumor suppressors that, in addition to the circadian clock, control cell cycle progression and checkpoint activation.

From Bench to Bedside: Connections Between SCF Ligases and Cancer
Over the past two years, we have demonstrated a direct functional role for two F-box proteins in the development of specific cancers.

First, we found that FBXO11 targets the BCL6 oncoprotein for degradation. BCL6 overexpression is the primary driver of oncogenesis in diffuse large B cell lymphoma (DLBCL), and FBXO11 is mutated in 20 percent of DLBCLs, resulting in BCL6 overexpression in these tumors. We are continuing to investigate the biological roles of FBXO11 in other tumor types that also contain FBXO11 mutations.

Second, while Fbxw7 is best known as a tumor suppressor, we found a tissue-specific role for Fbxw7 in cell survival. Fbxw7 targets p100/NF-κB2, an inhibitor of NF-κB signaling, for degradation. In multiple myeloma cells, which are addicted to constitutive NF-κB signaling, the disruption of Fbxw7-dependent p100 degradation results in cell death. This dependence on Fbxw7 for survival is likely unique to the B cell lineage, and it is notable that no Fbxw7 mutations have been found in B cell tumors. We are continuing to investigate the potential utility of this context-dependent function of Fbxw7 in targeted therapies.

This research has been supported in part by the National Institute of General Medical Sciences, the National Cancer Institute, the Department of Defense, the American Association for Cancer Research, the American Cancer Society, the International Agency for Research on Cancer, Susan G. Komen for the Cure, the Leukemia and Lymphoma Society, the Lymphoma Research Foundation, the Multiple Myeloma Research Foundation, the Human Frontier Science Program, the New York State Department of Health, the Emerald Foundation, and the Irma T. Hirschl Scholar Program.

As of January 08, 2013

Scientist Profile

Investigator

Michele Pagano, MD

New York University

Biochemistry, Cell Biology

HHMI Investigator Robert Lefkowitz

HHMI Bulletin, Spring 2013

HHMI Professors Competition

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