Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells.
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 beta/gamma-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-delta, beta 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.
Hara
Tomohiko
T
Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Tokyo 104-0045, Japan.
Honda
Kazufumi
K
Shitashige
Miki
M
Ono
Masaya
M
Matsuyama
Hideyasu
H
Naito
Katsusuke
K
Hirohashi
Setsuo
S
Yamada
Tesshi
T
D000185:Actinin / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism*; D025941:Protein Interaction Mapping; D053719:Tandem Mass Spectrometry
C111453:ACTN4 protein, human; D008840:Microfilament Proteins; D000185:Actinin