Biomedical subjects

Michele Pagano

Publications and source records attributed to Michele Pagano.

At least 37 records · Page 2Linked to original sources

PubMed2004

Wagging the dogma; tissue-specific cell cycle control in the mouse embryo.

The family of cyclin-dependent kinases (Cdks) lies at the core of the machinery that drives the cell division cycle. Studies in cultured mammalian cells have provided insight into the cellular functions of many Cdks. Recent Cdk and cyclin knockouts in the mouse show that the functions of G1 cell cycle regulatory genes are often essential only in specific cell types, pointing to our limited understanding of tissue-specific expression, redundancy, and compensating mechanisms in the Cdk network.

Animals↗

PubMed2004

Don't skip the G1 phase: how APC/CCdh1 keeps SCFSKP2 in check.

By keeping the levels of Skp2 and Cks1 low during G(1) progression, APC/C(Cdh1) prevents unscheduled degradation of SCF(Skp2) substrates and premature entry into S phase. Thus, APC/C(Cdh1), a ubiquitin ligase involved in mitotic exit and maintenance of G(0)/G(1) phase, directly controls SCF(Skp2), a ubiquitin ligase involved in the regulation of S phase entry.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Ubiquitin-dependent degradation of p73 is inhibited by PML.

p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression.

Acetylation↗

PubMed2004

Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/2 axis.

A new study reveals a novel role for p27 in inhibiting Cdk1 activity at G2/M and shows that p27 deficiency almost completely rescues the aberrations observed in Skp2(-/-) mice, demonstrating that p27 is the principal downstream effector of the SCF(Skp2) ubiquitin ligase.

Animals↗

PubMed2004

Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome.

Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis.

Amino Acid Sequence↗

PubMed2004

Alterations in the expression of the cell cycle regulatory protein cyclin kinase subunit 1 in colorectal carcinoma.

BACKGROUND: Low levels of p27(Kip1) are associated with high aggressiveness and poor prognosis in various malignancies, including colorectal carcinoma. The authors showed that S phase kinase protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27(Kip1) for degradation, was overexpressed and was inversely related to p27(Kip1) levels in patients with colorectal carcinoma. The essential role of cyclin kinase subunit 1 (Cks1) in Skp2-dependent p27 degradation was recently discovered, but its role in human malignancies is unknown. METHODS: Quick-frozen colorectal tumor samples from 30 patients were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Cks1, Skp2, and p27(Kip1). The expression of Cks1 was also examined by immunohistochemistry using formalin-fixed, paraffin-embedded tissue sections from the same patients. RESULTS: A strong correlation was found between Cks1 levels and Skp2 expression and loss of tumor differentiation. A significant inverse relation was also observed between levels of Cks1 and p27(Kip1) and overall survival. CONCLUSIONS: The results of the current study suggest that increased expression of Cks1 may have an important causative role in decreasing levels of p27 in patients with aggressive colorectal carcinoma.

Aged↗

PubMed2004

Oncogenic aberrations of cullin-dependent ubiquitin ligases.

Accumulating evidence points to a key role of the ubiquitin-proteasome pathway in oncogenesis. Aberrant proteolysis of substrates involved in cellular processes such as the cell division cycle, gene transcription, the DNA damage response and apoptosis has been reported to contribute significantly to neoplastic transformation. Cullin-dependent ubiquitin ligases (CDLs) form a class of structurally related multisubunit enzymes central to the ubiquitin-mediated proteolysis of many important biological substrates. In this review, we describe the role of CDLs in the ubiquitinylation of cancer-related substrates and discuss how altered ubiquitinylation by CDLs may contribute to tumor development.

Animals↗

PubMed2004

Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase.

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Cell cycle, proteolysis and cancer.

Research in the past 15 years has shown that the mammalian cell cycle is controlled by the action of cyclin-dependent kinases (CDKs). A crucial substrate of the CDKs in G1-phase is the retinoblastoma tumor suppressor (pRB), which restrains proliferation largely by repressing the activity of the E2F transcription factors. More recent work has shown that the cell cycle is also a tale of two classes of ubiquitin ligases, referred to as SCF and APC/C ligases. CDKs, E2F and ubiquitin ligases reciprocally regulate each other, resulting in complex feedback loops. Perturbation of this network of molecular machines is associated with proliferative diseases, including cancer.

Animals↗

PubMed2004

Fenton's pre-treatment of mature landfill leachate.

The aim of this study was to check the effectiveness of the Fenton's reagent (Fe2+ + H2O2 + H+) for the pre-treatment of a municipal landfill leachate with the objective of improving its overall biodegradability, evaluated in terms of BOD5/COD ratio, up to a value compatible with biological treatment. The leachate came from a municipal sanitary landfill located in southern Italy and the average values of its main parameters were: pH=8.2; COD=10,540 mgl(-1); BOD5=2,300 mgl(-1); TOC=3,900 mgl(-1); NH4-N=5210 mgl(-1); conductivity=45,350 microScm(-1); alkalinity=21,470 mgl(-1) CaCO3. The effect of initial pH value on the pre-treatment effectiveness was evaluated by titrating the amount of acidic by-products formed. The extent of leachate oxidation was monitored and controlled by both pH and redox potential measurements. The best operational conditions for achieving the desired goal (i.e., BOD5/COD> or =0.5) resulted: Fe2+=275 mgl(-1); H2O2=3,300 mgl(-1); initial pH=3; reaction time=2 h. At the end of the Fenton's pre-treatment, in order to permit a subsequent biological treatment, residual ferric ions were removed increasing the pH up to 8.5 by adding 3 gl(-1) of Ca(OH)2 and 3 mgl(-1) of a cationic polyelectrolyte, the latter as an aid to coagulation. This final step also resulted in a further modest removal of residual COD due to co-precipitation phenomena.

Hydrogen Peroxide↗

PubMed2004

Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation.

Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy.

Adult↗

PubMed2004

The SCF ubiquitin ligase: insights into a molecular machine.

Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.

Animals↗

PubMed2004

Role of F-box protein betaTrcp1 in mammary gland development and tumorigenesis.

The F-box protein betaTrcp1 controls the stability of several crucial regulators of proliferation and apoptosis, including certain inhibitors of the NF-kappaB family of transcription factors. Here we show that mammary glands of betaTrcp1(-/-) female mice display a hypoplastic phenotype, whereas no effects on cell proliferation are observed in other somatic cells. To investigate further the role of betaTrcp1 in mammary gland development, we generated transgenic mice expressing human betaTrcp1 targeted to epithelial cells under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. Compared to controls, MMTV betaTrcp1 mammary glands display an increase in lateral ductal branching and extensive arrays of alveolus-like protuberances. The mammary epithelia of MMTV betaTrcp1 mice proliferate more and show increased NF-kappaB DNA binding activity and higher levels of nuclear NF-kappaB p65/RelA. In addition, 38% of transgenic mice develop tumors, including mammary, ovarian, and uterine carcinomas. The targeting of betaTrcp1 to lymphoid organs produces no effects on these tissues. In summary, our results support the notion that betaTrcp1 positively controls the proliferation of breast epithelium and indicate that alteration of betaTrcp1 function and expression may contribute to malignant behavior of breast tumors, at least in part through NF-kappaB transactivation.

Animals↗

PubMed2004

To be or not to be ubiquitinated?

Levels of p21, a cyclin-dependent kinase (CDK) inhibitor, are controlled in part at the post-translational level by protein degradation. Although the signaling pathways leading to p21 degradation have not yet been fully elucidated, it is evident that p21 ubiquitination is an essential factor in its degradation. We discuss that, with the only notable exception of ornithine decarboxylase, ubiquitination appears to be a prerequisite for proteasomal degradation rather than an unnecessary byproduct of such proteolysis.

Animals↗

PubMed2003

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

Amino Acid Motifs↗

PubMed2003

Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation.

We examined the mechanism responsible for the degradation of p21, a negative regulator of the cell division cycle. We found that p21 proteolysis requires functional ubiquitin and Nedd8 systems. Ubiquitinylated forms of p21 and p21(K0), a p21 mutant missing all lysines, are detected in vivo and in vitro, showing that the presence of lysines is dispensable for p21 ubiquitinylation. Instead, the free amino group of the N-terminal methionine of p21 is a site for ubiquitinylation in vivo. Although wild-type p21 is more abundantly ubiquitinylated than p21(K0) mutant due to the presence of internal lysine residues, their rates of proteolysis are indistinguishable. These results demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome.

Animals↗

PubMed2003

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase.

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.

Amino Acid Sequence↗

PubMed2003

Aberrant ubiquitin-mediated proteolysis of cell cycle regulatory proteins and oncogenesis.

The ubiquitin pathway plays a central role in the regulation of cell growth and cell proliferation by controlling the abundance of key cell cycle proteins. Increasing evidence indicates that unscheduled proteolysis of many cell cycle regulators contributes significantly to tumorigenesis and is indeed found in many types of human cancers. Aberrant proteolysis with oncogenic potential is elicited by two major mechanisms: defective degradation of positive cell cycle regulators (i.e., proto-oncoproteins) and enhanced degradation of negative cell cycle regulators (i.e., tumor suppressor proteins). In many cases, increased protein stability is a result of mutations in the substrate that prevent the recognition of the protein by the ubiquitin-mediated degradation machinery. Alternatively, the specific recognition proteins mediating ubiquitination (ubiquitin ligases) are not expressed or harbor mutations rendering them inactive. In contrast, the overexpression of a ubiquitin ligase may result in the enhanced degradation of a negative cell cycle regulator. This chapter aims to review the involvement of the ubiquitin pathway in the scheduled destruction of some important cell cycle regulators and to discuss the implications of their aberrant degradation for the development of cancer.

Animals↗