Cancer Cell Biology
The human Y-encoded testis-specific protein interacts functionally with eukaryotic translation elongation factor eEF1A, a putative oncoprotein†
Tatsuo Kido,
Yun-Fai Chris Lau,
Tatsuo Kido
Division of Cell and Developmental Genetics, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, CA
Search for more papers by this authorCorresponding Author
Yun-Fai Chris Lau
Division of Cell and Developmental Genetics, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, CA
Fax: +1-415-750-6959.
Division of Cell and Developmental Genetics, Department of Medicine, VA Medical Center 111C5, University of California, 4150 Clement Street, San Francisco, CA 94121, USASearch for more papers by this authorTatsuo Kido,
Yun-Fai Chris Lau,
Tatsuo Kido
Division of Cell and Developmental Genetics, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, CA
Search for more papers by this authorCorresponding Author
Yun-Fai Chris Lau
Division of Cell and Developmental Genetics, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, CA
Fax: +1-415-750-6959.
Division of Cell and Developmental Genetics, Department of Medicine, VA Medical Center 111C5, University of California, 4150 Clement Street, San Francisco, CA 94121, USASearch for more papers by this author†
This article is a US Government work and, as such, is in the public domain in the United States of America.
Abstract
Testis-specific protein Y-encoded (TSPY) is the putative gene for the gonadoblastoma locus on the Y chromosome. TSPY is expressed in normal germ cells of fetal and adult testis and ectopically in tumor germ cells, including gonadoblastoma in intersex patients, testicular germ cell tumors, prostate cancer and other somatic cancers. It is a member of the TSPY/SET/NAP1 superfamily and harbors a highly conserved domain, termed SET/NAP domain. To explore its possible role(s) in tumorigenesis, we had performed a yeast two-hybrid screen of a fetal gonadal cDNA library and identified the translation elongation factor eEF1A as a binding partner for TSPY at the SET/NAP domain. TSPY and eEF1A were highly expressed and colocalized in tumor germ cells of human seminoma specimens, suggesting their possible interaction in germ cell tumors. They were colocalized in the cytoplasm and could be co-immunoprecipitated from transfected COS7 cells. Significantly, both eEF1A1 and eEF1A2 have postulated to be involved in various types of human cancer, including breast and prostate cancers. TSPY enhanced protein synthesis of a reporter gene, which was augmented by an overexpression of eEF1A. TSPY also increased the nuclear redistribution of eEF1A, resulting in a parallel increase in reporter gene transcripts. Our results suggest that TSPY could exert its oncogenic function(s) by interacting with eEF1As and stimulating gene expression via its enhancements in protein synthesis and gene transcription. Published 2008 Wiley-Liss, Inc.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
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| IJC_23697_sm_suppfig1.tif9.7 MB | Figure S1. Alignment between SET and TSPY amino acid sequences. (a) Amino acid sequences of SET[1-223] and TSPY [1-291]. The ?-helices (orange columns) and ?-strands (green arrows) in SET are indicated on top lines (modified from Muto et al.). The secondary structure of TSPY[1-150] was predicted by ‘Phyre’ program (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html). Two long ?-helices were predicted in TSPY[1-150] as drown by gray columns in bottom lines (?1 and ?2). TSPY[151-291] was aligned with SET[74-223], the conserved amino acids are indicated by ‘*’, and the chemically identical amino acids are indicated by ‘⁁’. (b) Three dimensional structure of SET. A screen-shot on ‘PDB SimpleViewer’ program derived from the Protein Data Bank (PDB ID code 2E50; www.pdb.org) is shown. NAP-domain and ?-helices; ?2, ?3, ?4, ?5, ?7, and ?8 are indicated. |
| IJC_23697_sm_suppfig2.tif10 MB | Figure S2. TSPY is co-expressed with eEF1A in human germ cell tumor cells in seminoma sample SE#2-5. Tissue sections were stained by immunofluorescence technique as described in Materials and Methods. (a and b) Representative example of immunofluorescence of TSPY or eEF1A in human seminoma (Se) and CIS/ITGCNU (CI). Merged images shows the signals of anti-TSPY (green), anti-eEF1A (red) and DAPI for DNA staining (blue). The elongation factor eEF1A was detected in the TSPY-positive cells of seminoma and CIS/ITGCNU. b showed the highly magnified images of the boxed area in a, respectively. TSPY and eEF1A were well co-localized in cytoplasm of germ cell tumor cells, resulting yellowish color in merged images. (c) Immunofluorescence obtained with a control (negative) mouse IgG on adjacent sections, showing no specific staining. (d) Immunofluorescence of seminoma sample SE#3 to 5 using anti-TSPY and anti-eEF1A, respectively. Scale bar= 200 ?m in b, c and d. |
| IJC_23697_sm_suppfig3.tif7.4 MB | Figure S3. Cellular distribution of FLAG-TSPY and FLAG-NLS-TSPY in COS7 cells. Cells were transiently transfected with FLAG-TSPY or FLAG-NLS-TSPY expression vector, and examined by indirect immunofluorescence using anti-FLAG antibody. The FLAG-TSPY was distributed in both nuclei and cytoplasm similarly with non-tagged TSPY (a, top), and FLAG-NLS-TSPY showed a preferential nuclear distribution (b, top). DNA was visualized by DAPI. Some cells transfected with FLAG-TSPY displayed the nuclear distribution of FLAG-TSPY at high level (arrows in a). (c) Stimulation of the reporter protein synthesis by FLAG-TSPY and FLAG-NLS-TSPY. HEK293 cells seeded on 24-well plate were cotransfected with pRL-TK (5 ng/well) with pFLAG-TSPY (0.1 or 0.2 ?g/well) or pFLAG-NLS-TSPY (0.2 ?g/well). Total DNA was adjusted with the empty vector (p3XFLAG-CMV-7). Luciferase activities were measured 2 days after transfection. Top panel indicates the relative activity based on the luciferase activity of control (i.e. without cotransfection of pFLAG-TSPY and pFLAG-NLS-TSPY). The data represent the means ± SE of three individual experiments. Middle panels show the representative Western blots of FLAG-TSPY, FLAG-NLS-TSPY and ?-actin in whole cell lysates. Bottom panel show the relative protein level of FLAG-TSPY and FLAG-NLS-TSPY quantified in silico using ImageJ. The data are means ± S.E. for three individual experiments. The rate of FLAG-NLS-TSPY protein degradation was significantly higher than FLAG-TSPY (data not shown). (d) The relative luciferase activity per amount of TSPY protein was calculated. The data are means ± S.E. for three individual experiments. FLAG-NLS-TSPY is more effective in stimulating the reporter protein synthesis than FLAG-TSPY. |
| IJC_23697_sm_suppfig4.tif1.1 MB | Figure S4. Estimation of luciferase RNA by semiquantitative RT-PCR. HEK293 cells were cotransfected with pSV-Luc (0.1 or 0.5 ?g/well) with or without pCS-TSPY (0.2 ?g/well). Two days after transfection, RNA was isolated from cells by using RNeasy Mini Kit (QIAGEN), and subjected to semiquantitative RT-PCR (32 cycles). Reverse-transcription PCR (RT-PCR) was performed as described previously. (a) Signals represented by negative printing of ethidium bromide staining of the RT-PCR products fractionated by agarose gel electrophoresis. ?-actin was used as an internal control (28th cycle). +RT indicates the results with reverse transcription reaction; -RT, without reverse transcription reaction. (b) Estimation of relative amounts of RT-PCR products determined in a. The electrophoresis image was quantified in silico, using ImageJ program, and the relative amounts of respective RT-PCR products were calculated as fold changes with reference to signals from cells transfected with the luciferase reporter gene alone (0.1??g/well). The data are means ± S.E. for three individual experiments. The data were statistically analyzed by one way analysis of variance (ANOVA) followed by student-Newman-Keuls test. ‘*’ indicates significant differences (p <0.05); ND indicates no difference between two groups. The PCR primers for luciferase gene were Luc-F4 (5′-ACG GAT TAC CAG GGA TTT CAG TC-3′) and Luc-B5 (5′-TGG AAG ATG GAA GCG TTT TGC-3′). |
| IJC_23697_sm_suppfig5.doc679 KB | Supplementary Figure 5. |
| IJC_23697_sm_supptable1.doc66 KB | Supplementary Table 1. |
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