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256


SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins

Busino, Luca; Bassermann, Florian; Maiolica, Alessio; Lee, Choogon; Nolan, Patrick M; Godinho, Sofia I H; Draetta, Giulio F; Pagano, Michele
One component of the circadian clock in mammals is the Clock-Bmal1 heterodimeric transcription factor. Among its downstream targets, two genes, Cry1 and Cry2, encode inhibitors of the Clock-Bmal1 complex that establish a negative-feedback loop. We found that both Cry1 and Cry2 proteins are ubiquitinated and degraded via the SCF(Fbxl3) ubiquitin ligase complex. This regulation by SCF(Fbxl3) is a prerequisite for the efficient and timely reactivation of Clock-Bmal1 and the consequent expression of Per1 and Per2, two regulators of the circadian clock that display tumor suppressor activity. Silencing of Fbxl3 produced no effect in Cry1-/-;Cry2-/- cells, which shows that Fbxl3 controls clock oscillations by mediating the degradation of CRY proteins
PMID: 17463251
ISSN: 1095-9203
CID: 72420

DRE-1: an evolutionarily conserved F box protein that regulates C. elegans developmental age

Fielenbach, Nicole; Guardavaccaro, Daniele; Neubert, Kerstin; Chan, Tammy; Li, Dongling; Feng, Qin; Hutter, Harald; Pagano, Michele; Antebi, Adam
During metazoan development, cells acquire both positional and temporal identities. The Caenorhabditis elegans heterochronic loci are global regulators of larval temporal fates. Most encode conserved transcriptional and translational factors, which affect stage-appropriate programs in various tissues. Here, we describe dre-1, a heterochronic gene, whose mutant phenotypes include precocious terminal differentiation of epidermal stem cells and altered temporal patterning of gonadal outgrowth. Genetic interactions with other heterochronic loci place dre-1 in the larval-to-adult switch. dre-1 encodes a highly conserved F box protein, suggesting a role in an SCF ubiquitin ligase complex. Accordingly, RNAi knockdown of the C. elegans SKP1-like homolog SKR-1, the cullin CUL-1, and ring finger RBX homologs yielded similar heterochronic phenotypes. DRE-1 and SKR-1 form a complex, as do the human orthologs, hFBXO11 and SKP1, revealing a phyletically ancient interaction. The identification of core components involved in SCF-mediated modification and/or proteolysis suggests an important level of regulation in the heterochronic hierarchy
PMID: 17336909
ISSN: 1534-5807
CID: 79365

The pRb-Cdh1-p27 autoamplifying network [Comment]

Santamaria, Patricia G; Pagano, Michele
PMID: 17268477
ISSN: 1465-7392
CID: 72423

Response: More money, and less time! [Letter]

Pagano, M
ISI:000242330600010
ISSN: 0092-8674
CID: 69456

S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth

Dorrello, N Valerio; Peschiaroli, Angelo; Guardavaccaro, Daniele; Colburn, Nancy H; Sherman, Nicholas E; Pagano, Michele
The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth
PMID: 17053147
ISSN: 1095-9203
CID: 69030

American Idol and NIH Grant Review [Letter]

Pagano, Michele
PMID: 16923379
ISSN: 0092-8674
CID: 72424

Skp2 Contains a Novel Cyclin A Binding Domain That Directly Protects Cyclin A from Inhibition by p27Kip1

Ji, Peng; Goldin, Luba; Ren, Hao; Sun, Daqian; Guardavaccaro, Daniele; Pagano, Michele; Zhu, Liang
Skp2 is well known as the F-box protein of the SCF(Skp2).Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interaction. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 compromises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mechanism of Skp2 and a new regulatory mechanism of cyclin A
PMID: 16774918
ISSN: 0021-9258
CID: 66921

SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response

Peschiaroli, Angelo; Dorrello, N Valerio; Guardavaccaro, Daniele; Venere, Monica; Halazonetis, Thanos; Sherman, Nicholas E; Pagano, Michele
During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint
PMID: 16885022
ISSN: 1097-2765
CID: 66918

Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth

Lasorella, Anna; Stegmuller, Judith; Guardavaccaro, Daniele; Liu, Guangchao; Carro, Maria S; Rothschild, Gerson; de la Torre-Ubieta, Luis; Pagano, Michele; Bonni, Azad; Iavarone, Antonio
In the developing nervous system, Id2 (inhibitor of DNA binding 2, also known as inhibitor of differentiation 2) enhances cell proliferation, promotes tumour progression and inhibits the activity of neurogenic basic helix-loop-helix (bHLH) transcription factors. The anaphase promoting complex/cyclosome and its activator Cdh1 (APC/C(Cdh1)) restrains axonal growth but the targets of APC/C(Cdh1) in neurons are unknown. Id2 and other members of the Id family are very unstable proteins that are eliminated as cells enter the quiescent state, but how they are targeted for degradation has remained elusive. Here we show that Id2 interacts with the core subunits of APC/C and Cdh1 in primary neurons. APC/C(Cdh1) targets Id2 for degradation through a destruction box motif (D box) that is conserved in Id1 and Id4. Depletion of Cdh1 stabilizes Id proteins in neurons, whereas Id2 D-box mutants are impaired for Cdh1 binding and remain stable in cells that exit from the cell cycle and contain active APC/C(Cdh1). Mutants of the Id2 D box enhance axonal growth in cerebellar granule neurons in vitro and in the context of the cerebellar cortex, and overcome the myelin inhibitory signals for growth. Conversely, activation of bHLH transcription factors induces a cluster of genes with potent axonal inhibitory functions including the gene coding for the Nogo receptor, a key transducer of myelin inhibition. Degradation of Id2 in neurons permits the accumulation of the Nogo receptor, thereby linking APC/C(Cdh1) activity with bHLH target genes for the inhibition of axonal growth. These findings indicate that deregulated Id activity might be useful to reprogramme quiescent neurons into the axonal growth mode
PMID: 16810178
ISSN: 1476-4687
CID: 66920

A peptidomimetic siRNA transfection reagent for highly effective gene silencing

Utku, Yeliz; Dehan, Elinor; Ouerfelli, Ouathek; Piano, Fabio; Zuckermann, Ronald N; Pagano, Michele; Kirshenbaum, Kent
RNA interference (RNAi) techniques hold forth great promise for therapeutic silencing of deleterious genes. However, clinical applications of RNAi require the development of safe and efficient methods for intracellular delivery of small interfering RNA (siRNA) oligonucleotides specific to targeted genes. We describe the use of a lipitoid, a cationic oligopeptoid-phospholipid conjugate, for non-viral transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. We compare the lipitoid reagent to a standard commercial transfection reagent. The lipitoid is highly efficient even in primary IMR-90 human lung fibroblasts in which other commercial reagents are typically ineffective
PMID: 16880950
ISSN: 1742-206x
CID: 66919