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Specific Small Molecule Inhibitors of Skp2-Mediated p27 Degradation

Wu, Lily; Grigoryan, Arsen V; Li, Yunfeng; Hao, Bing; Pagano, Michele; Cardozo, Timothy J
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.
PMCID:3530153
PMID: 23261596
ISSN: 1074-5521
CID: 207422

Centrosome homeostasis is controlled by ubiquitylation and deubiquitylation cycles [Meeting Abstract]

Li, J; D'Angiolella, V; Seeley, E; Kobayashi, T; Kim, S; Pagano, M; Dynlacht, B
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
EMBASE:71414289
ISSN: 1059-1524
CID: 884432

FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas [Meeting Abstract]

Duan, S; Cermak, L; Pagan, J K; Rossi, M; Martinengo, C; Francia, Di Celle P; Chapuy, B; Shipp, M; Chiarle, R; Pagano, M
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
EMBASE:71415220
ISSN: 1059-1524
CID: 884402

SCF-Mediated Degradation of p100 (NF-kappaB2): Mechanisms and Relevance in Multiple Myeloma

Busino, Luca; Millman, Scott E; Pagano, Michele
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.
PMCID:3871187
PMID: 23211527
ISSN: 1937-9145
CID: 197502

Fbw7gamma-mediated degradation of KLF13 prevents RANTES expression in resting human but not murine T lymphocytes

Kim, Dong Seok; Zhang, Wei; Millman, Scott E; Hwang, Byung Joon; Kwon, Seok Joo; Clayberger, Carol; Pagano, Michele; Krensky, Alan M
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.
PMCID:3429307
PMID: 22797700
ISSN: 0006-4971
CID: 177229

Coupled activation and degradation of eEF2K regulates protein synthesis in response to genotoxic stress

Kruiswijk, Flore; Yuniati, Laurensia; Magliozzi, Roberto; Low, Teck Yew; Lim, Ratna; Bolder, Renske; Mohammed, Shabaz; Proud, Christopher G; Heck, Albert J R; Pagano, Michele; Guardavaccaro, Daniele
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.
PMCID:3812825
PMID: 22669845
ISSN: 1937-9145
CID: 539832

Cyclin F-Mediated Degradation of Ribonucleotide Reductase M2 Controls Genome Integrity and DNA Repair

D'Angiolella, Vincenzo; Donato, Valerio; Forrester, Frances M; Jeong, Yeon-Tae; Pellacani, Claudia; Kudo, Yasusei; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Pagano, Michele
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.
PMCID:3616325
PMID: 22632967
ISSN: 0092-8674
CID: 167802

APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit

Ping, Zhen; Lim, Ratna; Bashir, Tarig; Pagano, Michele; Guardavaccaro, Daniele
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.
PMCID:3359123
PMID: 22580460
ISSN: 1551-4005
CID: 169025

Regulation of APC/C(Cdc20) activity by RASSF1A-APC/C(Cdc20) circuitry

Chow, C; Wong, N; Pagano, M; Lun, S W-M; Nakayama, K-I; Nakayama, K; Lo, K-W
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.
PMCID:3325600
PMID: 21874044
ISSN: 0950-9232
CID: 165670

SCFFbxo45 controls cytokinesis through ubiquitin-mediated proteolysis of GEF-H1 [Meeting Abstract]

Chen, X; Ahn, J -Y; Szankasi, P; Chung, F; Basrur, E; Lim, M S; Miller, A L; Pagano, M; Elenitoba-Johnson, K S J
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
EMBASE:70852966
ISSN: 0892-6638
CID: 178106