Faculty Bibliography

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.

F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.

E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and differentiation. E2F3 has recently attracted particular attention, because it is amplified in various human tumors. Here we show that E2F3 becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/C (Cdh1) ). E2F3 interacts with Cdh1 but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the overexpression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/C (Cdh1) ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation.

RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/C(Cdc20) is the master switch and regulator of mitosis. The activity of APC/C(Cdc20) is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/C(Cdc20) during mitosis. Our study showed that RASSF1A and APC/C(Cdc20) form a molecular relay that regulates the APC/C(Cdc20) activity at early mitosis. We found that RASSF1A inhibits APC/C(Cdc20) function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/C(Cdc20) in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/C(Cdc20). Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/C(Cdc20) during the course of mitotic progression. As a result of RASSF1A degradation, APC/C(Cdc20) can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A-APC/C(Cdc20) interaction in ensuring the sequential progression of mitosis.

The rho guanine nucleotide exchange factor H1 (GEF-H1) is a critical regulator of cytokinesis, but the requirement and mechanism for its timely destruction for proper execution of cytokinesis are unknown. Here we show that GEF-H1 is regulated by the evolutionarily conserved E3 ligase SCFFbxo45. Fbxo45 promotes GEF-H1 ubiquitylation upon PLK1-mediated phosphorylation of GEF-H1 on serine 644, and this event is necessary for GEF-H1 degradation during mitosis. Fbxo45 silencing causes stabilization and abnormal cellular distribution of GEF-H1 with diffuse RhoA hyperactivation. A GEF-H1 mutant that is unable to bind Fbxo45 is stabilized in mitosis, and results in an uncoordinated hyperactive membrane phenotype, with abnormal aberrant cytokinesis leading to cell death or multinucleation. Our studies provide evidence that limiting activation of the RhoAGTPase via Fbxo45-mediated degradation of the GEF-H1 activator is essential for proper execution of cytokinesis

Fbxw7alpha is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-kappaB signalling, as an interactor of Fbxw7alpha. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7alpha, which recognizes a conserved motif phosphorylated by GSK3. Efficient activation of non-canonical NF-kappaB signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7alpha or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7alpha silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-kappaB activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7alpha and GSK3 function as pro-survival factors through the control of p100 degradation.

BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.

In response to genotoxic stress, eukaryotic cells activate the DNA damage response (DDR), a series of pathways that coordinate cell cycle arrest and DNA repair to prevent deleterious mutations. In addition, cells possess checkpoint mechanisms that prevent aneuploidy by regulating the number of centrosomes and spindle assembly. Among these mechanisms, ubiquitin-mediated degradation of key proteins has an important role in the regulation of the DDR, centrosome duplication and chromosome segregation. This review discusses the functions of a group of ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) family, in the maintenance of genome stability. Given that general proteasome inhibitors are currently used as anticancer agents, a better understanding of the ubiquitylation of specific targets by specific ubiquitin ligases may result in improved cancer therapeutics.

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 (refs 5, 6). 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