Faculty Bibliography

Cell cycle transitions are driven by the periodic oscillations of cyclins, which bind and activate cyclin-dependent kinases (CDKs) to phosphorylate target substrates. Cyclin F uses a substrate recruitment strategy similar to that of the other cyclins, but its associated catalytic activity is substantially different. Indeed, cyclin F is the founding member of the F-box family of proteins, which are the substrate recognition subunits of Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complexes. Here, we discuss cyclin F function and recently identified substrates of SCF(cyclin)(F) involved in deoxyribonucleotide triphosphate (dNTP) production, centrosome duplication, and spindle formation. We highlight the relevance of cyclin F in controlling genome stability through ubiquitin-mediated proteolysis and the implications for cancer development.

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.

The Tel2 (also known as Telo2) and Tti1 proteins control the cellular abundance of mammalian PIKKs and are integral components of mTORC1 and mTORC2. Here we report that Tel2 and Tti1 are targeted for degradation within mTORC1 by the SCFFbxo9 ubiquitin ligase to adjust mTOR signalling to growth factor availability. This process is primed by CK2, which translocates to the cytoplasm to mediate mTORC1-specific phosphorylation of Tel2/Tti1, subsequent to growth factor deprivation. As a consequence, mTORC1 is inactivated to restrain cell growth and protein translation whereas relief of feedback inhibition activates the PI(3)K/TORC2/Akt pathway to sustain survival. Significantly, primary human multiple myelomas exhibit high levels of Fbxo9. In this setting, PI(3)K/TORC2/Akt signalling and survival of multiple myeloma cells is dependent on Fbxo9 expression. Thus, mTORC1-specific degradation of the Tel2 and Tti1 proteins represents a central mTOR regulatory mechanism with implications in multiple myeloma, both in promoting survival and in providing targets for the specific treatment of multiple myeloma with high levels of Fbxo9 expression.

Geminin, an essential factor for DNA replication, directly binds to the licensing factor Cdt1 and inhibits pre-replicative complex formation to prevent re-replication. In G1, geminin levels are controlled by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex, which targets geminin for proteasomal degradation to allow pre-replicative complex formation. Conversely, from S to G2, geminin is stabilized due to APC/C ubiquitin ligase complex inhibition, ensuring the inhibition of pre-replicative complex formation. However, mitotic regulation of geminin has hitherto not been described. Here we show that Aurora-A phosphorylates geminin on Thr25 during M phase, and this event induces geminin stabilization by preventing its APC/C ubiquitin ligase complex-mediated degradation during mitosis. In turn, stabilized geminin inhibits SCF(Skp2)-mediated degradation of Cdt1 to ensure pre-replicative complex formation in the ensuing S phase. The Aurora-A-geminin-Cdt1 axis therefore represents a critical regulator of proper DNA replication.

In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein, Cks1, promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type-specific blocks in the G1 or G2/M phases. Designing SCF-Skp2-specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.

Centrosome duplication is a pivotal process required for cell division. In order to avoid genome instability, the duplication of centrosomes must be restricted to once per cell cycle. Different mechanisms that control centrosome duplication impinge on the regulation of CP110, an essential component of the centriole duplication process. Excessive CP110 drives centrosome over-duplication while loss of CP110 inhibits centrosome amplification. CP110 levels are controlled through ubiquitin mediated proteolysis by the SCF(cyclin F) during G2 and M phase of the cell cycle. From published mass spectrometry data, we have identified a de-ubiquitylating enzyme (DUB) as a CP110-interacting protein. We report a new mechanism to regulate centrosome duplication that entails DUB-dependent regulation of CP110 levels. Ubiquitylation and deubiquitylation cycles control CP110 stability and centrosome duplication. We further observe that the levels of this DUB and CP110 are markedly elevated in pancreatic ductal adenocarcinoma (PDAC), suggesting a rationale for inhibiting tumors associated with centrosome amplification. These studies have identified one of the first centriolar deubiquitinating enzymes whose expression regulates centrosome homeostasis by countering cyclin F-mediated destruction of a key centrosomal substrate

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11. The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene

On the basis of differential analysis of affinity purifications by mass spectrometry, we identified the nuclear factor kappaB (NF-kappaB) protein p100 (NF-kappaB2) as an interactor of the F-box protein FBXW7alpha. The NF-kappaB pathway is important for cell growth, differentiation, and survival. p100, which shuttles between the cytoplasm and nucleus, functions as the primary inhibitor of the noncanonical NF-kappaB pathway by sequestering NF-kappaB heterodimers in the cytoplasm. In the absence of NF-kappaB stimulation, the nuclear pool of p100 is constitutively targeted for degradation by FBXW7alpha, which recognizes a conserved motif that is phosphorylated by glycogen synthase kinase 3 (GSK3). Efficient activation of noncanonical NF-kappaB signaling depends on the clearance of nuclear p100, either through FBXW7alpha-mediated degradation or nuclear export mediated by a signal in the C terminus of p100. Upon prolonged stimulation of the NF-kappaB pathway, p100 is stabilized and retained in the nucleus, contributing to the cessation of noncanonical NF-kappaB signaling. The molecular mechanism of p100 degradation has implications in multiple myeloma, a disease with constitutive activation of the noncanonical NF-kappaB pathway. Accordingly, expression of a stable p100 mutant, FBXW7alpha depletion, or chemical inhibition of GSK3 in multiple myeloma cells results in cell death in vitro and in a xenotransplant model. Thus, the FBXW7alpha-dependent degradation of p100 functions as a prosurvival mechanism through control of NF-kappaB activity.

RANTES (CCL5) is a chemokine implicated in many human diseases. We previously showed that the transcription factor Kruppel-like factor 13 (KLF13) controls the late (3-5 days after activation) expression of RANTES in T lymphocytes and that KLF13 itself is translationally regulated through the 5'-untranslated region of its mRNA. Here, we show that KLF13 levels are further regulated by ubiquitination and degradation. KLF13 protein is undetectable in resting human T lymphocytes, but treatment with either proteosomal or lysosomal inhibitors increases KLF13 protein levels. Glycogen synthase kinase 3beta (GSK3beta)-mediated phosphorylation of KLF13 triggers the ubiquitination of KLF13 by the E3 ligase Fbw7gamma, resulting in KLF13 protein degradation. Knockdown of either Fbw7gamma or GSK3beta by small interfering RNA increases KLF13 expression in resting human T lymphocytes. In contrast, in murine T lymphocytes, KLF13 protein is abundant because of the absence of Fbw7gamma. Treatment of unactivated human lymphocytes with lysosomal inhibitors stabilizes KLF13 protein, resulting in an increase of RANTES mRNA and protein. Taken together, these studies found that tightly regulated control of both synthesis and degradation allows rapid changes in the level of KLF13 in human T lymphocytes.

The kinase eEF2K [eukaryotic elongation factor 2 (eEF2) kinase] controls the rate of peptide chain elongation by phosphorylating eEF2, the protein that mediates the movement of the ribosome along the mRNA by promoting translocation of the transfer RNA from the A to the P site in the ribosome. eEF2K-mediated phosphorylation of eEF2 on threonine 56 (Thr(5)(6)) decreases its affinity for the ribosome, thereby inhibiting elongation. Here, we show that in response to genotoxic stress, eEF2K was activated by AMPK (adenosine monophosphate-activated protein kinase)-mediated phosphorylation on serine 398. Activated eEF2K phosphorylated eEF2 and induced a temporary ribosomal slowdown at the stage of elongation. Subsequently, during DNA damage checkpoint silencing, a process required to allow cell cycle reentry, eEF2K was degraded by the ubiquitin-proteasome system through the ubiquitin ligase SCF(betaTrCP) (Skp1-Cul1-F-box protein, beta-transducin repeat-containing protein) to enable rapid resumption of translation elongation. This event required autophosphorylation of eEF2K on a canonical betaTrCP-binding domain. The inability to degrade eEF2K during checkpoint silencing caused sustained phosphorylation of eEF2 on Thr(5)(6) and delayed the resumption of translation elongation. Our study therefore establishes a link between DNA damage signaling and translation elongation.