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Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2
Dankert, John F; Rona, Gergely; Clijsters, Linda; Geter, Phillip; Skaar, Jeffrey R; Bermudez-Hernandez, Keria; Sassani, Elizabeth; Fenyo, David; Ueberheide, Beatrix; Schneider, Robert; Pagano, Michele
SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent gammaH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.
PMCID:5097008
PMID: 27773672
ISSN: 1097-4164
CID: 2288562
TIMELESS Forms a Complex with PARP1 Distinct from Its Complex with TIPIN and Plays a Role in the DNA Damage Response
Young, Lauren M; Marzio, Antonio; Perez-Duran, Pablo; Reid, Dylan A; Meredith, Daniel N; Roberti, Domenico; Star, Ayelet; Rothenberg, Eli; Ueberheide, Beatrix; Pagano, Michele
PARP1 is the main sensor of single- and double-strand breaks in DNA and, in building chains of poly(ADP-ribose), promotes the recruitment of many downstream signaling and effector proteins involved in the DNA damage response (DDR). We show a robust physical interaction between PARP1 and the replication fork protein TIMELESS, distinct from the known TIMELESS-TIPIN complex, which activates the intra-S phase checkpoint. TIMELESS recruitment to laser-induced sites of DNA damage is dependent on its binding to PARP1, but not PARP1 activity. We also find that the PARP1-TIMELESS complex contains a number of established PARP1 substrates, and TIMELESS mutants unable to bind PARP1 are impaired in their ability to bind PARP1 substrates. Further, PARP1 binding to certain substrates and their recruitment to DNA damage lesions is impaired by TIMELESS knockdown, and TIMELESS silencing significantly impairs DNA double-strand break repair. We hypothesize that TIMELESS cooperates in the PARP1-mediated DDR.
PMCID:4618055
PMID: 26456830
ISSN: 2211-1247
CID: 1803582
SPOP Mutations or ERG Rearrangements Result in Enhanced Levels of ERG to Promote Cell Invasion in Prostate Cancer
Duan, Shanshan; Pagano, Michele
In this issue, An et al. (2015) and Gan et al. (2015) reveal that the CRL3(SPOP) ubiquitin ligase mediates the degradation of the transcription factor ERG and that translocations of ERG or mutations in SPOP prevent CRL3(SPOP)-dependent degradation of ERG in prostate cancer cells.
PMCID:4948578
PMID: 26384661
ISSN: 1097-4164
CID: 1779442
The Integrator complex controls the termination of transcription at diverse classes of gene targets
Skaar, Jeffrey R; Ferris, Andrea L; Wu, Xiaolin; Saraf, Anita; Khanna, Kum Kum; Florens, Laurence; Washburn, Michael P; Hughes, Stephen H; Pagano, Michele
Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration targeting-sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3' end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.
PMCID:4349240
PMID: 25675981
ISSN: 1001-0602
CID: 1481242
Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing
Pagan, Julia K; Marzio, Antonio; Jones, Mathew J K; Saraf, Anita; Jallepalli, Prasad V; Florens, Laurence; Washburn, Michael P; Pagano, Michele
An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(betaTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by betaTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.
PMCID:4415623
PMID: 25503564
ISSN: 1465-7392
CID: 1437062
SCF ubiquitin ligase-targeted therapies
Skaar, Jeffrey R; Pagan, Julia K; Pagano, Michele
The clinical successes of proteasome inhibitors for the treatment of cancer have highlighted the therapeutic potential of targeting this protein degradation system. However, proteasome inhibitors prevent the degradation of numerous proteins, which may cause adverse effects. Increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a multitude of processes at the cellular and organismal levels, and their dysregulation is implicated in many pathologies. SCF ubiquitin ligases are characterized by their high specificity for substrates, and these ligases therefore represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This Review explores and discusses potential strategies to target SCF-mediated biological processes to treat human diseases.
PMCID:4410837
PMID: 25394868
ISSN: 1474-1776
CID: 1369022
Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival
Chen, X; Sahasrabuddhe, A A; Szankasi, P; Chung, F; Basrur, V; Rangnekar, V M; Pagano, M; Lim, M S; Elenitoba-Johnson, K S J
Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.
PMCID:4158693
PMID: 24992930
ISSN: 1350-9047
CID: 1186632
Cdh1, a substrate recruiting component of APC/C ubiquitin E3 ligase, specifically interacts with PTEN and promotes its removal from chromatin
Choi, Byeong Hyeok; Pagano, Michele; Huang, Chuanshu; Dai, Wei
A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on S380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on S380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, The interaction of PTEN with Cdh1 was phosphatase-independent and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.
PMCID:4067225
PMID: 24811168
ISSN: 0021-9258
CID: 988602
Plk1 phosphorylates PTEN and regulates its mitotic activity during the cell cycle
Choi, Byeong; Pagano, Michele; Dai, Wei
PTEN is a well-known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pull-down analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated S380, T382 and T383, but not S385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only S380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.
PMCID:4022876
PMID: 24706748
ISSN: 0021-9258
CID: 988612
DRE-1/FBXO11-Dependent Degradation of BLMP-1/BLIMP-1 Governs C. elegans Developmental Timing and Maturation
Horn, Moritz; Geisen, Christoph; Cermak, Lukas; Becker, Ben; Nakamura, Shuhei; Klein, Corinna; Pagano, Michele; Antebi, Adam
Developmental timing genes catalyze stem cell progression and animal maturation programs across taxa. Caenorhabditis elegans DRE-1/FBXO11 functions in an SCF E3-ubiquitin ligase complex to regulate the transition to adult programs, but its cognate proteolytic substrates are unknown. Here, we identify the conserved transcription factor BLMP-1 as a substrate of the SCF(DRE-1/FBXO11) complex. blmp-1 deletion suppressed dre-1 mutant phenotypes and exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits, including entry into the dauer diapause and longevity. BLMP-1 protein was strikingly elevated upon dre-1 depletion and dysregulated in a stage- and tissue-specific manner. The role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as we observed direct protein interaction and degradation function for worm and human counterparts. Taken together, posttranslational regulation of BLMP-1/BLIMP-1 by DRE-1/FBXO11 coordinates C. elegans developmental timing and other life history traits, suggesting that this two-protein module mediates metazoan maturation processes.
PMCID:4040421
PMID: 24613396
ISSN: 1534-5807
CID: 883552