Faculty Bibliography

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option

Treatment, removal and recovery of lead (3 mg/L) from battery industry wastewaters have been investigated utilising a chemical precipitation process with soluble starch xanthate (SX) at pH 5-6. A reactant ratio, i.e., SX/Pb(II) = 6 mol/mol, a reaction time of 15 min., the addition of 15 mg/L of a cationic polyelectrolyte and a final filtration gave residual lead concentrations in the liquid phase less than 0.2 mg/L, well below the maximum limit established by the EU Directive. Lead was extracted from the obtained sludge by oxidation with sodium hypochlorite or hydrogen peroxide solutions. The amounts of oxidant needed were quantified as 13.5 mol NaClO/mol Pb and one order of magnitude larger, for H2O2, the latter due to the competitive disproportion reaction of the oxidant. The metal extraction was quantitative using sodium hypochlorite; when hydrogen peroxide was used, the formation of insoluble PbSO4 (Anglesite) gave a 80% metal extraction. In both cases molar ratios between sulphate and lead ions in the extracted solutions were in the range 2.1-2.2, in agreement with the stoichiometries of the reactions. Lead can be quantitatively recovered from the extracted (NaClO) solutions, for reuse, after a chemical precipitation process with 1M NaOH at pH 9-9.5, in the form of hydrocerussite [Pb3(CO3)2(OH)2]

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold

The protein expression of the cyclin-dependent kinase inhibitor p27 is often deregulated in human tumors. In lymphomas the inactivation of p27 is achieved through either increased degradation(1) or sequestration via D cyclins,(2) and p27 protein levels have been shown to have a prognostic significance.(1,3) Recently, S-phase kinase-associated protein 2 (Skp2) has been proved to mediate p27 degradation in normal cells(4-7) and to have oncogenetic properties.(8,9) In this study, B-, T-, and myeloid hematopoietic cell lines and a well-characterized panel of human lymphomas (n = 244) were studied for the expression of Skp2. In human lymphomas, the expression of Skp2 strongly related to the grade of malignancy, being low in indolent tumors and very high in aggressive lymphomas. Moreover, the percentages of Skp2- and S-phase-positive cells, as measured by DNA content or BrdU labeling, strictly matched and closely parallel that of Ki-67 and cyclin A. An inverse correlation between Skp2 and p27 was found in the majority of lymphoma subtypes. Nonetheless, most mantle cell lymphomas and a subset of diffuse large cell lymphomas failed to show this correlation, suggesting that alternative pathway(s) for the regulation of p27 might exist. The detection of Skp2 protein either by flow cytometry or by immunohistochemistry represents a simple method to precisely assess the S phase of lymphomas. The potential diagnostic and prognostic value of Skp2 is discussed

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic 'hot spot' structure, a subcellular distribution that is characteristic of Tax in HEK cells