Actinin-4-mediated inhibition of cell proliferation and mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells. Free

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Alpha-actinin-4 (ACTN4) was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 makes complexes with a large number of different partner proteins and is speculated to exert several distinct functions according to the partners. The expression level of actinin-4 was significantly decreased (p<0.001, Wilcoxon rank sum test) in prostate cancer cells (average±SEM:high grade cancer of 25±7% and low grade cancer of 24±4%) compared to that of non-cancerous basal cells (average±SEM:88±5%) in surgical prostate tissue by immunohistochemistry. In vitro, actinin-4 expression level of prostate cancer cell lines (22RV1, PC-3 and LNCaP) were all decreased compared to that of normal prostate epithelial cells (PrEC) by Western blotting. There were no differences about actinin-4 expression levels between hormone dependent and independent cancer cell lines. Restoration of actinin-4 expression significantly inhibited its cell proliferation of prostate cancer cell line 22RV1 in colony formation assay. Immunoprecipitation and mass spectrometric analysis revealed that actinin-4 makes native complexes with several partner proteins in 22RV1 cells, including β/γ-actin, calmodulin, clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, and ras-GTPase-activating protein SH3-domain-binding protein (G3BP). Clathrin is a coat protein that covers the internalized membrane pit and plays a central role in early endocytosis. We found the other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-γ, β-NAP, and p47A, interacted with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin were co-localized with actinin-4 at dorsal sites of membrane ruffling, and the transfection of actinin-4 cDNA facilitated the transport of transferrin into peri-nuclear endosomes. Endocytosis enforces the termination of signaling evoked by the cell surface receptors and regulates the recycling of receptors and ligands. We identified a panel of proteins whose expression and/or subcellular localization was regulated by actinin-4 using organelle fractionation and Isotope-Coded Affinity Tagging and Mass Spectrometry (ICAT-LC-MS). Disturbance of actinin-4-mediated endocytosis may be involved in prostate carcinogenesis.