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Originally published In Press as doi:10.1074/mcp.M600129-MCP200 on December 6, 2006.
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Molecular & Cellular Proteomics 6:479-491, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

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

Tomohiko Hara{ddagger},§, Kazufumi Honda{ddagger}, Miki Shitashige{ddagger}, Masaya Ono{ddagger}, Hideyasu Matsuyama§, Katsusuke Naito§, Setsuo Hirohashi{ddagger} and Tesshi Yamada{ddagger},

From the {ddagger} Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 and § Department of Urology, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ß/{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}, ß 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.

Prostate cancer is the most common malignancy, and 234,460 men are estimated to have been diagnosed with prostate cancer in the United States in 2006.1 The incidence of prostate cancer varies widely among ethnic populations and countries, and although the age-adjusted incidence of prostate cancer in Japan is one-tenth that in the United States, the number of patients has dramatically increased (1). The difference in incidence between the United States and Japan is caused by a combination of differences in genetic susceptibility and unidentified environmental factors. Prostate cancer is believed to arise as a result of changes in the balance between cell proliferation and differentiation (2), but the exact mechanism is still unknown. Molecular genetic changes, including promoter hypermethylation (GSTP1, etc.), loss of heterozygosity (PTEN, TP53, NKX3-1, CDKN1B, etc.), mutation (MS, RNASEL, ELAC2, COPEB, AR, etc.), amplification (AR, etc.), and polymorphisms (AR, CYP17, SRD5A2, etc.), are associated with prostate carcinogenesis (3), but post-translational mechanisms, such as protein modifications (glycosylation, ubiquitination, phosphorylation, etc.), proteolysis, protein-protein interaction, and intra/intercellular protein transport, no doubt also play important roles in carcinogenesis. The proteomics of prostate cancer is still being developed in comparison with the genetics approaches.

Actinin-4 was originally identified in our laboratory as an actin-binding protein associated with cell motility and cancer invasion and metastasis, and two non-muscle actinin isoforms, actinin-1 and actinin-4, have been identified thus far. Despite their high degree of sequence identity (~86% similarity at the amino acid level) the two actinin isoforms seem to have distinct localizations and functions. Actinin-4 is localized in moving structures, such as dorsal ruffles, lamellipodia, and filopodia, but not in the adherens junction where actinin-1 is concentrated (4). We recently reported finding that actinin-4 expression was increased in the majority of colorectal cancers examined (5). The level of actinin-4 expression is significantly increased in cells exhibiting greater motility (4), and the increased expression of actinin-4 dramatically changed the morphology and motility of colorectal cancer cells (5). The non-nuclear localization of actinin-4 has been found to be closely associated with the invasive phenotype of breast cancer and the poor outcome of breast cancer patients (4). The increased expression of actinin-4 mRNA has been reported to be the most significant predictor of a poor outcome of non-small cell lung cancer patients (6), and actinin-4 has been identified as one of the genes up-regulated by Rac1 and Cdc42 (7). Mutant actinin-4 has been found to promote tumorigenicity and increase the motility of lung cancer cells (8).

All of the above findings indicate that actinin-4 promotes carcinogenesis, whereas the results of the present study unexpectedly showed that actinin-4 expression was down-regulated in prostate cancers and that restoration of actinin-4 expression had a suppressive effect on prostate cancer cell proliferation, suggesting that the role of actinin-4 varies with the cell or tissue lineage. In a search for the molecular mechanism underlying the actinin-4-mediated cell growth inhibition we discovered that actinin-4 interacts with various endocytosis-related proteins in prostate cancer cells. The decreased expression of actinin-4 may cause aberrations in the intracellular trafficking of various cell surface molecules, such as growth factors and receptors. In the present study we used ICAT and LC-MS/MS and succeeded in identifying a panel of proteins in prostate cancer cells whose subcellular positioning and expression level are regulated by actinin-4.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—
Human androgen-independent prostate cancer cell lines 22RV1 (9) and PC-3, androgen-dependent prostate cancer cell line LNCaP, and cervical cancer cell line HeLa were purchased from the American Type Culture Collection (Manassas, VA). The 22RV1 and LNCaP cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS).2 PC-3 cells were cultured in Kaighn's modification of Ham's F-12 medium (Invitrogen) supplemented with 7% FBS, and the HeLa cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS. Normal human prostate epithelial cells (PrEC) were purchased from Cambrex (East Rutherford, NJ) and cultured as instructed by the supplier.

Antibodies—
Murine monoclonal anti-G3BP (clone 23), anti-78-kDa-glucose-regulated protein (GRP78) (clone 40), anti-nucleoporin p62 (clone 63), anti-adaptin {delta} (clone 18), anti-p47A (clone 26), anti-ß-NAP (clone 18), anti-dynamin (clone 41), and anti-HSP60 (clone 24) antibodies were purchased from BD Transduction Laboratories. Anti-alcohol dehydrogenase rabbit (sc-22750), anti-myosin heavy chain rabbit (sc-20641), anti-heterogeneous nuclear ribonucleoprotein (hnRNP) A1 goat (sc-10030), and anti-hnRNP A2/B1 goat (sc-10035) polyclonal antibodies and normal rabbit IgG (sc-2027) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-{gamma}-tubulin rabbit polyclonal antibody (T3559) was purchased from Sigma. Anti-galectin-1 goat polyclonal antibody (AF1152) was purchased from R&D Systems (Minneapolis, MN). Anti-T-complex protein 1[cepsilon] (TCP-1[cepsilon]) rat monoclonal antibody (MCA2178) was purchased from Serotec (Kidlington, UK). Anti-ß-actin mouse monoclonal antibody (AC-15) was purchased from Abcam (Cambridgeshire, UK). Anti-clathrin heavy chain mouse monoclonal antibody (clone TD1) was purchased from Lab Vision (Fremont, CA). Anti-calmodulin mouse monoclonal antibody (05-173) was purchased from Upstate (Charlottesville, VA). Anti-Bcl-2 mouse monoclonal antibody (clone 124) was purchased from Dako (Glostrup, Denmark). Anti-clathrin heavy chain mouse monoclonal antibody (clone X22) was purchased from Affinity BioReagents (Golden, CO). Anti-hemagglutinin (HA) rat monoclonal antibody (clone 3F10) was purchased from Roche Diagnostics GmbH. Two anti-actinin-4 rabbit polyclonal antibodies, designated Ab-1 and Ab-2, were raised against keyhole limpet hemocyanin-conjugated synthetic peptides GDYMAQEDDWC and NQSYQYGPSSAGNGAGC, respectively (4, 10).

Plasmid Construction—
N-terminally HA-tagged human actinin-4 cDNA and its truncated forms were subcloned into pcDNA3 (Invitrogen). Domains and amino acid sequences encoded by the truncated forms are depicted in Supplemental Fig. S1A. The composition of all constructs in this study was confirmed by restriction endonuclease digestion and sequencing.

Immunohistochemistry—
Prostate specimens from 29 untreated cases of sporadic prostate cancer were selected from the surgical pathology panel of the Yamaguchi University Hospital (Ube, Japan). Formalin-fixed, paraffin-embedded sections (5 µm thick) containing the maximum diameter of the tumors were deparaffinized with a graded series of ethanol and xylene, treated with 0.3% hydrogen peroxide in methanol, and immersed in 10 mmol/liter citrate buffer (pH 6.0). After autoclaving at 121 °C for 10 min the sections were sequentially incubated with anti-actinin-4 antibody (Ab-2), biotinylated horse anti-rabbit immunoglobulin G, and avidin-biotinyl-peroxidase complex generated using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine tetrahydrochloride was used as chromogen in the peroxidase reaction, and nuclear counterstaining with hematoxylin was performed as described previously (11).

Colony Formation Assay—
Cells (2 x 105/well) were plated on 24-well plates, and 18 h later the cells were transfected with Lipofectamine 2000 reagent (Invitrogen). At 24 h after transfection 5 x 104 22RV1 cells or 1 x 105 HeLa cells were seeded on 6-well plates, and the transfection medium was replaced with culture medium containing 0.6 mg/ml and 0.4 mg/ml G418 (Invitrogen), respectively. Cells were stained with the Giemsa solution (Wako, Tokyo, Japan) after G418 selection for 7 days (12).

Immunoprecipitation—
22RV1 cells were extracted with lysis buffer (10 mmol/liter HEPES (pH 7.4), 150 mmol/liter NaCl, 1 mmol/liter EDTA, 1% Triton X-100, 1% Nonidet P-40, 1 mg/ml NaN3) containing a protease inhibitor mixture (Sigma). Cell lysates were incubated overnight at 4 °C with anti-actinin-4 rabbit polyclonal antibody (Ab-2) or normal rabbit IgG and precipitated with 50 µl of Dynabeads Protein G (Dynal Biotech, Oslo, Norway). After washing with wash buffer (50 mmol/liter Tris-HCl (pH 7.4), 150 mmol/liter NaCl, 1 mmol/liter EDTA), immobilized immunocomplexes were eluted by competition with peptide solution (NQSYQYGPSSAGNGAGC) for 15 min at room temperature or by boiling in 2x Laemmli Sample Buffer (Bio-Rad) with 2-mercaptoethanol for 5 min at 95 °C. Eluted proteins were fractionated by SDS-PAGE and detected with the silver stain MS kit (Wako) or by Western blotting.

Protein Identification by Mass Spectrometry—
Silver-stained SDS-PAGE gels were cut into 1- mm cubic sections, reduced with NH4HCO3, and alkylated with iodoacetamide. The gel sections were washed with acetonitrile and hydrolyzed with modified trypsin (Promega, Madison, WI), and peptides were extracted as described previously (13, 14). The peptides were subjected to the de novo sequencing method by LC-MS/MS. Alternatively the immunocomplex was directly injected into LC-MS/MS. Mass spectra were obtained in the refractor mode with a Q-star Pulsar-i mass spectrometer (Applied Biosystems, Foster City, CA). The MS/MS data were used to search the NCBInr protein sequence database downloaded from the National Center for Biotechnology Information (Bethesda, MD) under the species restriction of Homo sapiens (March 19, 2004; 2,707,913 sequences; 755,632,936 residues) by using the in-house Mascot searching program (Version 2.0) (Matrix Science, London, UK) (15).

Western Blot Analysis—
Protein samples were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). After incubation at 4 °C overnight with the primary antibodies the blots were detected with the corresponding horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-rat, or anti-goat IgG antibody and enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences) (16). Blot intensity was quantified with an LAS-3000 image analyzer and Multi Gauze software (Fuji Film, Tokyo, Japan) (12).

Immunofluorescence Cytochemistry—
Cells cultured on poly-L-lysine-coated glass coverslips (BD Biosciences) were fixed with 4% paraformaldehyde for 30 min at room temperature and made permeable with 0.2% Triton X-100. After blocking with 10% normal swine serum (Vector Laboratories) for 60 min at room temperature, the cells were incubated at 4 °C overnight with anti-clathrin (clone X22) or anti-dynamin (clone 41) and anti-HA (clone 3F10) antibodies. Following incubation with Alexa Fluor 488/594 anti-rat/mouse IgG antibodies (Molecular Probes, Inc., Eugene, OR), the specimens were examined under a laser scanning microscope (LSM5 Pascal, Carl Zeiss, Jene, Germany).

Transferrin Trafficking Analysis—
HeLa cells were transfected with pcDNA3 carrying HA-tagged actinin-4 cDNA (hereafter, HA-ACTN4) or control plasmid (pcDNA3), starved with serum-free medium (Dulbecco's modified Eagle's medium containing 0.1% albumin) for 60 min at 37 °C, and then incubated with 5 µg/ml Alexa 594-transferrin (Molecular Probes) for 10 min at 37 °C. Cells were fixed, and immunofluorescence cytochemistry was performed as described above.

Cell Fractionation and ICAT Analysis—
9 x 106 22RV1 cells were passaged into 10-cm dishes 18 h before the transfection. Lipofectamine 2000 reagent was used to transfect 22RV1 cells with HA-ACTN4 or empty vector. Forty-eight hours after the transfection the cells were washed with ice-cold PBS and allowed to swell for 15 min in hypotonic buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 5 mM CaCl2, 1 mM PMSF, 2 mM TCEP) supplemented with protease inhibitor mixture (Roche Applied Science). The soluble fraction (Fraction 1) was separated by centrifugation for 60 min at 105,000 x g, and the pellet was resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 1 mM PMSF, 2 mM TCEP, and protease inhibitor mixture) and homogenized. The cell homogenates were then subfractionated into Fractions 2–5 by sequential differential centrifugation as depicted in Supplemental Fig. S2, and each fraction was dissolved in 50 mM Tris-HCl (pH 8.3), 0.4% SDS, 1% octyl glucoside, 8 M urea, 2 mM TCEP. After adjusting the concentration of urea to 2 M and SDS to 0.1%, 160-µg protein samples were reduced for 60 min at 37 °C and differentially labeled with the isotopically light (12C0) or heavy (13C9) acid-cleavable ICAT reagent (Applied Biosystems, Foster City, CA).

The 12C- and 13C-labeled samples were combined, digested with modified trypsin (Promega), fractionated by cation-exchange chromatography, and purified by avidin affinity chromatography. The ICAT-labeled peptides were concentrated and desalted on a 500-µm-inner diameter x 1-mm HiQ sil C18-3 trapping column (KYA Technologies, Tokyo, Japan) before loading onto a 150-µm-inner diameter x 5-cm C18W-3 separation column (KYA Technologies). Peptides were then fractionated on an acetonitrile gradient (0–80%, 200 nl/min for 3 h) and analyzed with a Q-Star Pulsar-i mass spectrometer equipped with the nanospray ionization source (Applied Biosystems). Data were processed with ProICAT software (Version 1.0 Service Pack 3, Applied Biosystems), and a search was conducted against the human protein database (human_KBMS3.0.20040121, Celera Discovery System). Peptides identified with a confidence score of less than 70 were excluded. Peptides identified with confidence between 70 and 90 were additively checked by visual inspection, and only qualified peptides were further analyzed. Singletons were generally peaks of low intensity and were not used for the differential analysis.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Decreased Expression of Actinin-4 in Prostate Cancer—
We first performed immunohistochemistry to evaluate actinin-4 expression in normal prostate tissue and prostate cancer tissue (Fig. 1). The basal cells of normal human prostate glands expressed actinin-4 (Fig. 1, B and C), but differentiated luminal cells lacked expression. The cytoplasmic actinin-4 expression level was significantly reduced in prostate cancers with low (Fig. 1, E, F, H, and I) and high Gleason grades (17) (Fig. 1, K and L). Staining intensity was classified as "positive" when the actinin-4 expression level was equal to or higher than that in vascular endothelial cells, and as "negative" when it was less than that in vascular endothelial cells (Fig. 2A). The proportion of positive cells was determined by randomly counting 100 cells/specimen (Fig. 2B). The proportion of basal cells that were positive in normal acini was 88 ± 5% (average ± S.E.), whereas only 11 ± 1% of luminal cells were positive. The percentage of positive cancer cells was significantly lower than the percentage of positive normal basal cells (p < 0.001, Wilcoxon rank sum test). There was no statistically significant difference in the percentage of positive cells between low (Grade G2-G3, 24 ± 4%) and high grade (Grade G4-G5, 25 ± 7%) cancer cells. Consistent with immunohistochemical analysis, Western blot revealed the expression level of actinin-4 protein to be reduced in prostate cancer cell lines as compared with that in normal prostate epithelial cells (Fig. 2C).


Figure 1
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  		FIG. 1. Expression of actinin-4 protein in human prostate tissue. Hematoxylin and eosin staining (A, D, G, and J) and immunoperoxidase staining with actinin-4 polyclonal antibody (AP-2) (B, C, E, F, H, I, K, and L) of representative human normal prostate (A–C), Gleason Grade 2 prostate cancer (D–I), and Gleason Grade 4 prostate cancer (J–L) glands are shown. A and B, D and E, G and H and J and K are serial sections of the same specimens. The areas in the red squares in B, E, H, and K have been enlarged and are shown in C, F, I, and L, respectively. Normal prostate glands morphologically consist of two layers (C). The outer layer (basal cells) expresses cytoplasmic actinin-4 at high levels, whereas the inner layer (luminal cells) lacks actinin-4 expression. The expression of actinin-4 in cancer glands is significantly weaker than that in normal or hyperplastic glands (F, I, and L). N, non-cancerous glands; V, vascular endothelium; C, cancer. Bars, 100 µm.

 

Figure 2
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  		FIG. 2. Quantitative evaluation of actinin-4 protein expression in prostate tissue. A, immunoperoxidase staining of a representative sample from prostate cancer case classified as negative. Actinin-4 expression levels were classified as positive and negative in comparison with vascular endothelial cells in the same specimens (internal control). The arrow indicates vascular endothelium. B, the average percentages of positive cells among 100 randomly selected normal luminal epithelial cells (Luminal), normal basal epithelial cells (Basal), low Gleason grade (1–3) cancer cells (Ca_G2G3), and high Gleason grade (4 and 5) cancer cells (Ca_G4G5) are indicated by columns. The bars represent the S.E., and the asterisks indicate a significant difference (p < 0.001, Wilcoxon rank sum test). C, protein samples (22.5 µg) from normal prostate epithelial cells (PrEC) and three prostate cancer cells (22RV1, LNCaP, and PC-3) were separated by SDS-PAGE and blotted with anti-actinin-4 and anti-ß-actin (loading control) antibodies.

 
Actinin-4 Inhibits Cell Proliferation—
To reveal the biological significance of the reduced actinin-4 expression, prostate cancer 22RV1 cells (9) and HeLa cells were transfected with HA-ACTN4, and their colony-forming activity was measured (12). The transfection of actinin-4 cDNA significantly suppressed the colony formation of 22RV1 cells (Fig. 3A) as well as HeLa cells (Fig. 3B). And a similar effect was observed in another prostate cell line, PC-3 (data not shown). Transfection with serial deletion mutants derived from HA-ACTN4 indicated that the actin-binding site (amino acids 28–269) and the spectrin repeat (amino acids 270–752) were responsible for the cell growth inhibition (Supplemental Fig. S1).


Figure 3
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  		FIG. 3. Inhibition of cell growth by actinin-4. Shown are macroscopic (top) and microscopic (bottom) views of 22RV1 (A) and HeLa (B) cells transfected with HA-ACTN4 (right) or the vector control (left), cultured with G418 for 7 days, and stained with the Giemsa solution. Bars, 200 µm.

 
Identification of Actinin-4 Partner Proteins in Prostate Cancer Cells—
To determine the precise mechanism underlying the growth-suppressing activity of actinin-4, we searched for proteins that interact with actinin-4. 22RV1 cell lysate was immunoprecipitated with anti-actinin-4 (Ab-2) antibody raised against a keyhole limpet hemocyanin-conjugated NQSYQYGPSSAGNGAGC peptide, and the immunocomplex was eluted by competition with the same peptide solution. SDS-PAGE and silver stain revealed that several proteins were selectively co-immunoprecipitated with actinin-4 but not with the control (Fig. 4A). Five proteins (Fig. 4A, a–e) were subjected to protein identification by mass spectrometry (15), and MS/MS and a database search identified them as myosin heavy chain nonmuscle form A, clathrin heavy chain 1 (Supplemental Fig. S3), actinin-4, tubulin-{alpha}2/ß-1, and cytoskeletal actin-{gamma}1 (Table I). To increase sensitivity the immunoprecipitate with actinin-4 antibody was directly injected into the LC-MS/MS system, and several additional proteins were identified as a result (Table I). The protein identification was confirmed by Western blotting with available antibodies (Fig. 4B).


Figure 4
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  		FIG. 4. Identification of actinin-4-associated proteins. A, 22RV1 cell lysates were immunoprecipitated with anti-actinin-4 rabbit polyclonal antibody (Ab-2) or normal rabbit IgG, fractionated by SDS-PAGE, and detected with silver staining. MS/MS and a database search revealed that bands a, b, c, d, and e were derived from myosin heavy chain nonmuscle form A (gi|625305), clathrin heavy chain 1 (gi|4758012), actinin-4 (gi|12025678), tubulin-{alpha}2/ß-1 (gi|135448 and gi|34740335), and cytoskeletal actin-{gamma}1 (gi|4501887), respectively. B, 22RV1 cell lysates were immunoprecipitated with anti-actinin-4 rabbit polyclonal antibody (Ab-2) or normal rabbit IgG. The immunoprecipitates and whole cell lysate (Whole lysate) were fractionated by SDS-PAGE and detected by blotting with anti-actinin-4, anti-actin, anti-calmodulin, anti-clathrin (TD1), anti-myosin, anti-hnRNA A1, or anti-G3BP antibody. C, 22RV1 cell lysates were immunoprecipitated with anti-actinin-4 rabbit polyclonal antibody (Ab-2) or normal rabbit IgG. The immunoprecipitates and whole cell lysate (Whole lysate) were fractionated by SDS-PAGE and detected by blotting with anti-clathrin (TD1), anti-adaptin-{delta}, anti-ß-NAP, anti-p47A, or anti-dynamin antibody. IP, immunoprecipitation.

 

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  		TABLE I List of proteins present in the immunoprecipitate with anti-actinin-4 antibody identified by direct injection into LC-MS/MS

 
Clathrin is a coat protein that covers the internalized membrane pit, and it plays a central role in early endocytosis (18, 19). The immunoprecipitates with anti-actinin-4 antibody were analyzed by Western blotting with antibodies against other endocytosis-related proteins. Adaptin-{delta}, ß-NAP, p47A, and dynamin co-immunoprecipitated with anti-actinin-4 antibody (Fig. 4C), and confocal immunofluorescence microscopy revealed that the clathrin heavy chain and actinin-4 proteins were partially co-localized at the sites of membrane ruffling (Fig. 5, A–C), and dynamin and actinin-4 were co-localized in membrane ruffles and protrusions (Fig. 5, D–I, arrows).


Figure 5
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  		FIG. 5. Co-localization of actinin-4 with endocytosis-related proteins. A–C, 22RV1 cells transfected with HA-ACTN4 were stained with anti-HA antibody (A and green in C) and anti-clathrin heavy chain (X22) (B and red in C) antibody. D–I, 22RV1 cells transfected with HA-ACTN4 were stained by anti-HA (D, G, and green in F and I) and anti-dynamin (E, H, and red in F and I). The areas in the red boxes of D–F are enlarged in G–I. Actinin-4 and dynamin co-localized at the sites of membrane ruffling and protrusions (arrows).

 
Endocytosis regulates intracellular transport of various endogenous and exogenous molecules (20, 21). We examined the effect of actinin-4 on the transport of exogenous fluorescence-labeled transferrin. About 30–40% of HeLa cells were readily transfected using Lipofectamine (Fig. 6, B and E). Expression of HA-ACTN4 in HeLa cells induced the transport of fluorescence-labeled transferrin to perinuclear endosomes (Fig. 6, A–F). In untransfected HeLa cells (unstained cells in Fig. 6, B and E) as well as HeLa cells transfected with the control plasmid (Fig. 6, G and H) transferrin transport was rarely observed, confirming the functional involvement of actinin-4 in endocytosis.


Figure 6
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  		FIG. 6. Transferrin trafficking of actinin-4-expressing cells. HeLa cells were transfected with HA-ACTN4 (A–F) or control plasmid (G and H), serum-starved, and then incubated with Alexa 594-transferrin (5 µg/ml) for 10 min at 37 °C. Alexa Fluor 594-transferrin (red in A, C, D, and F) was preferentially transported to perinuclear endosomes (arrows) in cells transfected with HA-ACTN4 (green in B, C, E, and F) but not in untransfected cells (unstained in B, C, E, and F). HeLa cells transfected with control plasmid did not show such translocation (G and H). The areas in the blue squares in A, B, C, and G have been enlarged and are shown in D, E, F, and H, respectively. Bars, 20 µm. Tf, transferrin.

 
Actinin-4 Regulates Protein Localization—
Actinin-4 appears to function via the endocytic pathway. Endocytosis controls the ligand-induced internalization, degradation, and recycling of various cell surface receptors (22). Aberrant actinin-4 expression may affect intracellular molecule trafficking. We used organelle separation and ICAT-MS to search for proteins that were differentially expressed and/or differentially localized in 22RV1 cells transfected with HA-ACTN4 and the vector control (Fig. 7 and Supplemental Figs. S4 and S5). We confirmed that more than 50% of 22RV1 cells were readily transfected (data not shown). Whole protein homogenates were fractionated by a differential ultracentrifugation technique (Supplemental Fig. S2), and the equivalence of protein concentration and organelle separation were validated by Bradford protein assay (data not shown) and Western blot analysis with antibodies against known organelle marker proteins (Fig. 7A). A total of 589 peptides were detected (with a confidence MS/MS score of >70 determined by ProICAT) in one ICAT experiment. MS/MS and a database search revealed that these 589 peptides were derived from 369 different proteins (Table II). The TOF-MS spectra of all peptide pairs identified were checked visually to exclude errors by the software. The results showed that 48 proteins were up-regulated more than 2-fold and five proteins were down-regulated more than 2-fold by transfection with HA-ACTN4 (Tables II and III). Only five of the 53 (=48 + 5) proteins were detected in Fractions 1–3, and the most differentially expressed proteins were detected in Fraction 4 (rich in microsomes and Golgi bodies).


Figure 7
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  		FIG. 7. Actinin-4 regulates protein localization. A, each fraction (Fr. 1–5) of 22RV1 cells transfected with HA-ACTN4 (ACTN (+)) and control plasmid (Vector (–)) was separated by SDS-PAGE and blotted with anti-HA, anti-ß-actin, anti-alcohol dehydrogenase (ADH), anti-nucleoporin p62, anti-Bcl-2, and anti-GRP78 antibodies. B, each fraction (Fr. 1–5) of 22RV1 cells transfected with HA-ACTN4 (+) and control plasmid (–) was separated by SDS-PAGE and blotted with anti-actinin-4 (Ab-1), anti-hnRNP A2/B1, anti-TCP-1[cepsilon], anti-HSP60, and anti-galectin-1 antibodies. Red boxes indicate differential expression by 2-fold or more detected by ICAT-MS. C, functional classification of the actinin-4-regulated proteins based on the Gene Ontology Consortium annotation (www.geneontology.org/).

 

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  		TABLE II Numbers of proteins (peptides) detected and differentially expressed following the transfection of actinin-4 cDNA

Number in parentheses indicate number of peptides.

 

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  		TABLE III List of proteins whose expression level was changed by transfection of prostate 22RV1 cells with actinin-4 cDNA

 
The differential expression and/or localization of representative proteins identified by ICAT-MS/MS was confirmed by Western blotting with commercially available antibodies, including hnRNP A2/B1, TCP-1[cepsilon], HSP60, and galectin-1 (Fig. 7B). Expression of actinin-4 (Fraction 1), hnRNP A2/B1 (Fraction 4), TCP-1[cepsilon] (Fraction 4), HSP60 (Fraction 5), galectin-1 (Fraction 4), and galectin-1 (Fraction 5) was increased 3.99-, 3.34-, 2.69-, 3.50-, 3.35-, and 2.75-fold according to the results of ICAT (Table III) and 3.37-, 2.17-, 1.48-, 1.64-, 2.26-, and 1.20-fold according to the results of Western blotting (Fig. 7B). Supplemental Figs. S4 and S5 show representative ICAT-TOF-MS and MS/MS spectra. Based on the functional annotation provided by the Gene Ontology Consortium (www.geneontology.org/), 14 (30%) proteins were classified as being involved in protein biosynthesis, five (11%) proteins were classified as being involved in mRNA processing, four (9%) proteins were classified as being involved in glycolysis, four (9%) proteins were classified as being involved in protein folding, and other proteins were classified as having other functions (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The epithelium of prostate glands morphologically consists of al least two layers: inner and outer (Fig. 1A). Cell proliferation is observed only in the basal cell layer (outer layer), and prostate cancer is believed to arise from certain undifferentiated progenitor cells (or stem cells) residing in the basal cell layer (2, 23). In this study, we observed the expression level of actinin-4 in prostate cancer cells to be significantly down-regulated in comparison with that of cells in the basal layer (Fig. 1). The ACTN4 gene, which encodes actinin-4, has been mapped to chromosomal locus 19q13.2, and loss of heterozygosity at 19q13.2 is uncommon in prostate cancer (24). Although an alternative splice variant of actinin-4 (25) and mutant actinin-4 (8) have been discovered in lung cancer, no genetic abnormalities of ACTN4 have been reported in prostate cancer. The molecular mechanisms underlying the decreased expression of actinin-4 protein (Fig. 1) were not identified in this study.

The loss of actinin-4 expression may have a certain role in prostate carcinogenesis. Restoration of actinin-4 expression had a marked cell growth-suppressing effect (Fig. 3 and Supplemental Fig. S1). Actinin-4 seems to have several distinct functions depending on its interacting partner protein. Overcoming several technical difficulties, we searched for partner proteins that formed native complexes with endogenous actinin-4 in prostate cancer cells (Fig. 4) because we wanted to avoid the discovery of artificial interactions, such as those often seen in transfection experiments (26). Competitive elution as well as extensive washing of the immunocomplex was necessary to reduce the background and achieve optimal specificity. A large variety of proteins, including actin (27), ß-catenin (26), bullous pemphigoid antigen-2 (28), CLP-36 (29), RN-tre (30), Na+/H+ exchanger 3 (31), densin-180 (32), inducible nitric-oxide synthase (33), and brain-expressed RING finger protein (BERP) (34, 35), had been identified as interacting with actinin-4. However, none were identified in the current study probably because of their tissue type specificity and/or protein expression levels.

One interesting protein identified as interacting with actinin-4 was clathrin heavy chain. The clathrin-dependent endocytotic pathway mediates the internalization of molecules from the cell surface to inverted membrane compartments (22). The clathrin triskelia assemble into a polygonal lattice at the plasma membrane to form coated pits that bud and pinch off from the membrane and give rise to clathrin-coated vesicles (36). Clathrin-coated vesicles are uncoated after internalization and then fuse with the early endosome where molecules are sorted for either recycling or degradation in the late endosome/lysosome (22). Endocytosis controls the ligand-induced internalization and degradation of various cell surface receptors, and failure of proper endocytosis may result in deregulated activation of receptor-evoked signaling pathways and carcinogenesis (37). Involvement of actinin-4 in endocytosis seems clear based on the following evidence. The actinin-4-Hrs-BERP-myosin V complex has been reported to be associated with transferrin receptor recycling (35), and actinin-4 transfection affects transferrin trafficking in prostate cancer cells (Fig. 6). Actinin-4 is associated with other endocytosis-related molecules, i.e. adaptin-{delta}, ß-NAP, p47A, and dynamin (Fig. 4C). Dynamin assembles around the neck of clathrin-coated pits and assists in pinching vesicles from the plasma membrane. Adaptin-{delta}, ß-NAP, and p47A are main components of adaptor-related protein complex 3 (36, 38). Although adaptor-related protein complexes 1 and 2 bind to clathrin, the adaptor-related protein complex 3 seems to be involved in clathrin-independent endocytosis (39). Actinin-4 is associated with an endocytosis-regulating GTPase Rab5 (30, 40), and in a cDNA microarray analysis actinin-4 was identified as one of the genes up-regulated by Rac1 and Cdc42 (7). Cdc42 is necessary for actin polymerization during compensatory endocytosis (41), and endocytosis is closely related to exocytosis, phagocytosis, and pinocytosis. Actinin-4 is preferentially concentrated at the sites of circular ruffling and macropinocytosis of mouse macrophages (42). Actinin-4 is expressed at the apical surface of intestinal epithelial cells, suggesting that it is involved in pinocytic absorption of nutrients (43).

Endocytosis regulates the transport of various molecules (20, 21). Aberrant actinin-4 expression may affect proper intracellular molecule trafficking. We searched for proteins that were differentially expressed and/or localized in 22RV1 cells transfected with HA-ACTN4 and the vector control (Fig. 7 and Supplemental Figs. S4 and S5). Organelle separation and ICAT-MS revealed shifts in a large number of proteins into the microsome and Golgi-rich fraction (Fraction 4) (Table II). Actin microfilaments contribute to clathrin-coated vesicle assembly not only at the plasma membrane but also at Golgi sites (44) and mediate transport from the Golgi complex to the endoplasmic reticulum in mammalian cells (45, 46). It is reasonable to assume that actinin-4 transfection might affect the transport of a certain set of proteins from the Golgi complex. One of the proteins that accumulated in Fraction 4 after transfection was galectin-1 (Table III and Supplemental Fig. S5), a ß-galactoside-binding protein implicated in modulating cell-cell and cell-matrix interactions. Galectin-3 induces endocytosis of ß1 integrins in breast carcinoma cells (47) and impedes the endocytosis of epidermal growth factor and transforming growth factor-ß receptors by binding to their receptors (48).

Investigating how the endocytic pathway is involved in cell growth inhibition will be the task of a future study. The decreased expression of actinin-4 in prostate cancer cells may cause aberrations in intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.


  FOOTNOTES
 
Received, April 11, 2006, and in revised form, August 23, 2006.

Published, MCP Papers in Press, December 6, 2006, DOI 10.1074/mcp.M600129-MCP200

1 American Cancer Society statistics for 2006. Back

2 The abbreviations used are: FBS, fetal bovine serum; ACTN4, actinin-4; G3BP, Ras-GTPase-activating protein SH3 domain-binding protein; SH3, Src homology 3; GRP, glucose-regulated protein; HA, hemagglutinin; hnRNP, heterogeneous nuclear ribonucleoprotein; HSP60, heat shock protein 60; TCP-1[cepsilon], T-complex protein-1[cepsilon]; TCEP, tris(2-carboxyethyl)phosphine; ß-NAP, ß subunit of neuronal adaptin-like protein; BERP, brain-expressed RING finger protein. Back

* This work was supported by the "Program for Promotion of Fundamental Studies in Health Sciences" conducted by the National Institute of Biomedical Innovation of Japan and the "Third-Term Comprehensive Control Research for Cancer" conducted by the Ministry of Health, Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed. Tel.: 81-3-3542-2511; Fax: 81-3-3547-6045; E-mail: tyamada{at}gan2.res.ncc.go.jp


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