Mass Spectrometry Analysis of the Native Protein Complex Containing Actinin-4 in Prostate Cancer Cells

Abstract

Actinin-4 was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 forms complexes with a large number of different partner proteins and is speculated to have several distinct functions depending on its partner. The level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited cell proliferation by prostate cancer cell line 22RV1. Immunoprecipitation and mass spectrometry analysis revealed that actinin-4 forms native complexes with several partner proteins in 22RV1 cells, including with ␤/␥-actin, calmodulin, the clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein A1, and Ras-GTPase-activating protein SH3 domain-binding protein. Clathrin is a coat protein that covers the internalized membrane pit that forms during early endocytosis. We found that other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-␦, ␤ subunit of neuronal adaptin-like protein, and p47A, also interact with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin co-localized with actinin-4 at the sites of membrane ruffling, and transfection of actinin-4 cDNA facilitated the transport of transferrin into perinuclear endosomes. Endocytosis terminates signaling evoked by 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 by performing organelle fractionation and ICAT-LC-MS/MS. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.

Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions

“…In addition to the kidney-specific phenotype of actinin-4-deficient mice, the tissue-specific regulation of actinin-4 function is suggested by several cancers. While elevated actinin-4 expression mediates tumor progression of colorectal and non-small lung cell cancers, the tumor suppressive effect of actinin-4 is observed with neuroblastoma and prostate cancer cells (12,14,33,53). Although the molecular mechanism for the lack of widespread epithelial dysfunction outside the kidneys remains elusive, highly homologous actinin-1 and/or many actinin-4-binding proteins are expected to play an important role in compensating for the actinin-4 deficiency outside of the kidneys in vivo.…”

ACTN4 (actinin, alpha 4)

“…In human prostate cancer cell lines (22RV1, PC-3, LNCaP) ACTN4 protein product was less abundant than in normal human prostate epithelial cells, an observation that is conflicting with the findings in the most reports investigating relationship ACTN4-cancer development. Increasing the level of α-actinin 4 in those prostate cancer cells markedly suppressed cell-growth (Hara et al, 2007). In the same report the authors propose disturbance in clathrin-mediated endocytosis as the main mechanism associated with α-actinin 4 mediated cellular effects.…”

“…α-Actinin has been found in isolated plasma membranes containing clathrin (Burridge et al, 1980) and association of α-actinin 4 and clathrin heavy chain has been identified in prostate cancer cells. Overexpression of α-actinin 4 facilitated transferrin endocytosis (Hara et al, 2007). v/ Function in glomerular podocytes.…”

Functions of nuclear actin-binding proteins in human cancer (Review)

“…ACTN4 interacts with HDAC7 by its C-terminal calmodulin (CaM) -like domain, and activates ERα transcription, which contributes to tumorigenesis in breast cancer (42). In addition, elevated levels of ACTN4 are widely identified in other malignancies, including colorectal, pancreatic, prostate and ovarian cancer, salivary gland carcinoma and esophageal cancer (38,(42)(43)(44)(45)(46).…”