Dlg5 has been reported to participate in cancer progression; however, its role in prostate cancer still remains poorly understood. In this study, we demonstrate that Dlg5 is frequently downregulated in prostate cancer. We show here that Dlg5 is involved in the regulation of cell migration and cancer cell invasion. Knockdown of endogenous Dlg5 markedly increased prostate cancer cell migration and invasion. Our studies, for the first time, demonstrate the interaction between Dlg5 and Girdin, an actin-binding Akt substrate. Importantly, we found that levels of Akt-mediated Girdin phosphorylation (p-Girdin-Ser1416) are increased in Dlg5-depleted cells. Small interfering RNA directed against Girdin and wortmannin treatment, which was found to reduce Girdin phosphorylation, impaired the effect of Dlg5 depletion on cell migration. Taken together, our findings demonstrate that Dlg5 interacts with and inhibits the activity of Girdin, thereby suppressing the migration of prostate cancer cells.
Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT.
Edited by Gianni CesareniKeywords: Discs large homolog 5 TGF-b receptor Protein degradation Crohn's disease Epithelial to mesenchymal transition a b s t r a c t Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins and is involved in epithelial-to-mesenchymal transition via transforming growth factor-b (TGF-b) signaling. However, the mechanism underlying the regulation of TGF-b signaling is unclear. We show here that Dlg5 interacts and colocalizes with both TGF-b type I (TbRI) and type II (TbRII) receptors at the plasma membrane. TbRI activation is not required for this interaction. Furthermore, the overexpression of Dlg5 enhances the degradation of TbRI. Proteasome inhibitors inhibited this enhanced degradation. These results suggest that Dlg5 interacts with TbRs and promotes their degradation. Structured summary of protein interactions:DLG5 physically interacts with TbRII and TbRI by anti tag coimmunoprecipitation (View interaction) DLG5 physically interacts with TbRII by anti tag coimmunoprecipitation (View interaction) TbRI physically interacts with DLG5 by anti bait coimmunoprecipitation (View interaction) DLG5, TbRI and TbRII colocalize by fluorescence microscopy (View interaction) DLG5 physically interacts with TbRI by anti tag coimmunoprecipitation (View interaction)
Vimentin is a type III intermediate filament protein that is typically expressed in mesenchymal cells. Overexpression of vimentin is frequently observed in several types of cancer and is often associated with epithelial-to-mesenchymal transition. It was recently reported that the serum vimentin level is significantly elevated in colon and liver tumors. Therefore, a more sensitive vimentin detection system may be useful for cancer screening and early detection. The V9 mouse monoclonal antibody (mAb), which recognizes the human vimentin protein, is widely used in routine pathology to identify mesenchymal cells using immunohistochemical analysis. Although it has been suggested that the epitope of the V9 mAb is located within the C-terminal region of vimentin, the precise amino acid sequence that it recognizes has not yet been identified. In the present study, we constructed several deletion mutants of the vimentin protein and examined their reactivity with the V9 mAb to accurately map its epitope. We confirmed that its epitope resides in the C-terminal region of vimentin, between amino acids 392–466. Additionally, cross-species comparison of amino acid sequence alignment of vimentin, as well as site-directed mutagenesis, revealed that one residue, the asparagine at position 417, is critical for antibody binding. Using smaller vimentin fragments ranging in length from 9 to 13 residues, each containing this critical asparagine, we determined that the minimal residues required for V9 mAb recognition of human vimentin are the thirteen amino acid residues at positions 411–423 (411ISLPLPNFSSLNL423).
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