Regulation of the DLG tumor suppressor by ß-catenin.

The discs-large (DLG) tumor suppressor plays essential roles in regulating cell polarity and proliferation. It localizes at sites of cell-cell contact where it acts as a scaffold for multiple protein interactions, including with the adenomatous polyposis coli (APC) tumor suppressor, which in turn regulates ß-catenin. Furthermore, many tumor types including breast and colon have increased levels of ß-catenin activity with correspondingly low levels of DLG expression. Here we provide evidence of a direct functional link between these apparently separate phenomena. We show that overexpressed ß-catenin can enhance the turnover of DLG in a proteosome dependent manner. This effect is specific to DLG and is not seen with two other PDZ domain-containing targets of ß-catenin, MAGI-1 and Scribble. Furthermore, siRNA-mediated ablation of endogenous ß-catenin expression also enhances DLG stability. ß-catenin-induced degradation of DLG appears to be a consequence of a direct association between the two proteins and requires ß-catenin PDZ binding potential. In contrast, the enhanced turnover of DLG requires the unique N-terminal sequences and its PDZ domains. Finally, we also show that the capacity of DLG to inhibit transformed cell growth in an oncogene cooperation assay is inhibited by ß-catenin. Taken together these studies suggest that one mechanism by which deregulated ß-catenin can contribute to tumorigenesis is through enhancing DLG degradation.

PDZ domains: the building blocks regulating tumorigenesis.

Over 250 PDZ (PSD95/Dlg/ZO-1) domain-containing proteins have been described in the human proteome. As many of these possess multiple PDZ domains, the potential combinations of associations with proteins that possess PBMs (PDZ-binding motifs) are vast. However, PDZ domain recognition is a highly specific process, and much less promiscuous than originally thought. Furthermore, a large number of PDZ domain-containing proteins have been linked directly to the control of processes whose loss, or inappropriate activation, contribute to the development of human malignancies. These regulate processes as diverse as cytoskeletal organization, cell polarity, cell proliferation and many signal transduction pathways. In the present review, we discuss how PBM-PDZ recognition and imbalances therein can perturb cellular homoeostasis and ultimately contribute to malignant progression.

Free haem levels in gingival crevicular fluid and their relationship to periodontal clinical parameters, smoking and subgingival microbial composition.

BACKGROUND AND OBJECTIVES: Periodontitis caused by multifactorial polymicrobial infection results in a destructive inflammatory process and loss of tooth supporting tissues. Many putative bacterial virulence factors that cause host destruction are regulated by iron and haem. Therefore, this study investigated the free haem levels in the gingival crevicular fluid (GCF) at periodontitis sites in smokers and nonsmokers and their relationship to subgingival microbial composition. MATERIALS AND METHODS: A cross-sectional study was carried out on 78 patients with a split-mouth design who were divided into Group I A - periodontally healthy sites and Group I B - periodontally diseased sites in nonsmokers with chronic periodontitis and Group II A - periodontally healthy sites and Group II B - periodontally diseased sites in smokers. Clinical parameters recorded included a plaque and gingival index, papillary bleeding index, pocket probing depth, and clinical attachment level. The collected GCF samples were subjected to Biovision™ Hemin Colorimetric Assay Kit and subgingival plaque samples to BANA™ test. RESULTS: Increased GCF free haem concentration and positive BANA sites were seen at periodontitis sites compared to healthy sites, in both smokers and nonsmokers group. However, no difference was found in GCF free haem levels between smokers and nonsmokers, but it was statistically significant with respect to BANA-positive sites. CONCLUSION: Thus, this study concludes that the higher concentration of GCF free haem at diseased sites indicates that it could be used as a potential biomarker to determine active periodontal sites, also smoking and BANA results did not influence the biomarker levels.

TIP60 inhibits metastasis by ablating DNMT1-SNAIL2-driven epithelial-mesenchymal transition program.

HIV-Tat-interacting protein of 60 kDa (TIP60) is a lysine acetyltransferase and known to be downregulated in multiple cancers. Among various signalling pathways, TIP60 is implicated in regulating epithelial-mesenchymal transition (EMT). Here, we show that TIP60 expression abrogates cell migration and metastatic potential of breast cancer cells using in vitro and in vivo models. Mechanistically, we show that this is through its ability to destabilize DNMT1 and inhibit SNAIL2 function (SNAIL2-mediated EMT/cell migration). Depletion of TIP60 stabilizes DNMT1 and increases SNAIL2 levels, resulting in EMT. Recruitment of DNMT1 to the SNAIL2 targets in the absence of TIP60 increases DNA methylation on their promoter region and further represses the expression of epithelial markers. In pathophysiological scenario, we find TIP60 to be significantly downregulated in breast cancer patients with poor overall survival and disease-free survival prognoses. These data suggest that levels of TIP60 can be a prognostic marker of breast cancer progression and stabilization of TIP60 could be a promising strategy to treat cancers.

A novel interaction between hScrib and PP1γ downregulates ERK signaling and suppresses oncogene-induced cell transformation.

Previous studies have shown that the cell polarity regulator hScrib interacts with, and consequently controls, the ERK signaling pathway. This interaction occurs through two well-conserved Kinase Interacting Motifs, which allow hScrib to bind ERK1 directly, resulting in a reduction in the levels of phospho-ERK. This suggests that hScrib might recruit a phosphatase to regulate this signaling pathway. Using a proteomic approach we now show that Protein Phosphatase 1γ (PP1γ) is a major interacting partner of hScrib. This interaction is direct and occurs through a conserved PP1γ interaction motif on the hScrib protein, and this interaction appears to be required for hScrib's ability to downregulate ERK phosphorylation. In addition, hScrib also controls the pattern of PP1γ localization, where loss of hScrib enhances the nuclear translocation of PP1γ. Furthermore, we also show that the ability of hScrib to interact with PP1γ is important for the ability of hScrib to suppress oncogene-induced transformation of primary rodent cells. Taken together, these results demonstrate that hScrib acts as a scaffold to integrate the control of the PP1γ and ERK signaling pathways and explains how disruption of hScrib localisation can contribute towards the development of human malignancy.

The mechanism and implications of hScrib regulation of ERK.

Scribble is a potential tumor suppressor protein, whose loss is a frequent event in late stage cancer development. In both Drosophila and mammalian model systems, Scribble has been shown capable of regulating cell polarity, cell proliferation and apoptosis. Although several interacting partners, including ßPiX, have been identified that help to explain how Scribble can regulate cell polarity and migration, little is known about how Scribble can control cell proliferation. Recent work from our laboratory has shown that Scribble can directly regulate the ERK signaling pathway. This is mediated by a direct protein-protein interaction between Scribble and ERK, which has two components. In the first, Scribble appears to anchor ERK at membrane-bound sites, with the loss of Scribble enhancing ERK nuclear translocation. In the second, Scribble can decrease the levels of active phosphorylated ERK, a function that is dependent upon the ability of Scribble to bind ERK directly. One of the consequences of this activity of Scribble is the inhibition of EJ-ras induced cell transformation. These results provide some of the first direct mechanistic information on how Scribble can regulate cell proliferation and, furthermore, they provide indications as to the identity of other signaling intermediates that may be recruited by Scribble to directly regulate mitogenic signaling pathways.

The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg.

High-risk mucosal HPV E6 oncoproteins target a number of PDZ domain-containing substrates for proteasome mediated degradation. One of these, Discs Large (Dlg), is involved in the regulation of cell polarity and proliferation control. Previous studies had suggested that Dlg when hyperphosphorylated by osmotic shock, or when present in the nucleus could be preferentially targeted by E6. In this study we use phospho-specific antibodies directed against Dlg phosphorylated at residues S158 and S442 to show that these two observations are, in fact, linked. Dlg, when phosphorylated on S158 and S442 by CDK1 or CDK2, shows a preferential nuclear accumulation. However, these forms of Dlg are absent in cells derived from HPV-induced cervical cancers. Upon either proteasome inhibition or siRNA ablation of E6 expression, we see specific rescue of these phosphorylated forms of Dlg. These results demonstrate that nuclear forms of Dlg phosphorylated on its CDK phospho-acceptor sites has enhanced susceptibility to E6-induced degradation and place previous studies on the stress-induced phosphorylation of Dlg into a relevant biological context.