From the Division of Endocrinology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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Pituitary tumor-transforming gene (PTTG) is a
recently characterized proto-oncogene that is expressed specifically in
adult testis. In this study, we have used in situ
hybridization and developmental Northern blot assays to demonstrate
that PTTG mRNA is expressed stage-specifically in spermatocytes and
spermatids during rat spermatogenic cycle. We have used the yeast
two-hybrid system to identify proteins that interact with PTTG in
testicular cells. Two positive clones were characterized. One of the
clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that
testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat
testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ
cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be
co-immunoprecipitated with S10 and HSJ2 from transfected cells.
Moreover, the binding sites for both proteins were located within the
C-terminal 75 amino acids of the PTTG protein. These results suggest
that PTTG may play a role in spermatogenesis.
Pituitary tumor-transforming gene
(PTTG)1 was originally
isolated from a rat pituitary tumor cell line by RNA differential display (1). Overexpression of PTTG resulted in cell transformation in
fibroblasts and tumor formation in nude mice (1). PTTG mRNA was
found to be highly expressed in a variety of tumor cell lines, suggesting a possible role of PTTG in tumorigenesis outside the pituitary gland.2 Testis is
one of the few normal adult tissues where a shorter form of PTTG
mRNA is expressed (1). The genomic structure of the rat PTTG has
recently been characterized (2). A transcriptional enhancer element was
identified in the PTTG 5'-flanking region that was required for
transcriptional activation of PTTG in a testicular germ cell line, GC2
(2). This enhancer element also contains the binding sites for multiple
nuclear proteins, two of which are germ cell-specific (2).
Although PTTG mRNA is expressed in various testicular cell lines,
including Sertoli, Leydig, and germ cells (2), the expression pattern
of PTTG in normal testis is unknown. In this study, we have used
in situ hybridization and developmental Northern blot assays
to determine the cell type and the differentiation stage in which PTTG
mRNA is expressed during the spermatogenic cycle. Our results
showed that PTTG mRNA is expressed predominantly in spermatocytes
and spermatids in a stage-specific manner, suggesting that PTTG may
play a role in spermatogenesis. Because the amino acid sequence of PTTG
does not reveal any identifiable structural or functional motifs, it is
difficult to deduce its possible functions. To further our
understanding of the role of PTTG in spermatogenesis, an in
vivo strategy was employed to identify proteins capable of
physically associating with PTTG. The yeast two-hybrid system developed
by Fields and Song (3) was used to provide a physiological environment
in which to detect potential interactions involving the PTTG protein.
This system has been used to screen cDNA libraries for clones
encoding proteins capable of binding a protein of interest (4-6).
Using this approach, we have identified several cDNA clones encoding proteins that interact with PTTG. One clone isolated in this
screen encodes a novel human homologue of the bacterial heat-shock
protein DnaJ (designated HSJ2). Eukaryotic DnaJ proteins have been
implicated in various cellular processes, including correct protein
folding (7-9), intracellular vesicle traffic (10, 11), activity
control of regulatory proteins (12), promotion of translation
initiation (13), and tumor suppression (14). Another cDNA clone
isolated encodes a previously characterized protein, ribosomal protein
S10. The cloning and characterization of HSJ2 and S10 is described, as
well as their association with PTTG and the possible functional
implications of this interaction.
In Situ Hybridization--
Paraffin sections of rat testis were
obtained from Novagen. 35S-Labeled antisense and sense
riboprobes were synthesized with T7 and T3 polymerase, respectively,
and hydrolyzed in mild alkali to a length of 100-200 nucleotides. The
slides were hydrated, post-fixed in paraformaldehyde followed by
treatment with proteinase K and acetic anhydride. Slides were
hybridized at 50 °C overnight in a buffer containing 0.3 M NaCl and 60% formamide and a probe concentration of
107 cpm/slide. Washing was performed as follows. Wash 1:
2× SSC (1× SSC = 0.15 M NaCl and 0.015 M
sodium citrate) at 50 °C for 30 min. RNase A digestion: 20 µg/ml
RNaseA in 2× SSC at 37 °C for 30 min. Wash 2: 2× SSC, 50%
formamide at 50 °C for 30 min. Wash 3: 0.2× SSC, 0.05% sodium
pyrophosphate at 55 °C for 30 min. Slides were exposed to Cloning PTTG into pAS2 DNA Binding Domain Vector--
The coding
region of PTTG was synthesized by polymerase chain reaction using the
following primers: P1, 5'-CAGGATGGCTACTCTGATCTTTGTTGAT-3' and P2,
5'-CTGTTAAATATCTGCATCGTAACAAACAGG-3'.
The polymerase chain reaction product was cloned into TA vector
(Invitrogen), and the insert was excised from EcoRI site and cloned in frame into pAS2 vector (CLONTECH). The
resulting plasmid was designated pAS2-PTTG.
Yeast Two-hybrid Library Screen--
A human testis cDNA
library cloned in yeast pACT2 transactivation domain vector
(CLONTECH) was screened according to
manufacturer's instructions. Plasmid DNA prepared from the library and
pAS2-PTTG were co-transformed into yeast strain CG-1945
(CLONTECH) using the lithium acetate method (15,
16). Approximately 106 independent clones were screened.
Positive clones were identified by growth on
The blue colonies were characterized further by quantitative liquid
Characterization of Positive Clones--
Yeast plasmid DNA was
isolated using Yeastmaker yeast plasmid isolation kit
(CLONTECH) according to manufacturer's protocol. Plasmid isolated from yeast was used to transform HB101
Escherichia coli cell using electroporation. Cells were grow
on M9 agar plates containing 50 µg/ml ampicillin, 40 mg/ml proline,
and 1 mM thiamine hydrochloride. Plasmid DNA was isolated
from Leu+, AmprHB101 transformants using a
standard plasmid mini-prep procedure. Positive clones were divided into
groups based on restriction enzyme digestion patterns. The insert of
each independent positive clone was sequenced using Gal4 AD sequencing
primer (CLONTECH) and Sequenase
(U. S. Biochemical Corp.). A homology search was performed using
BLAST system. The inserts of several positive clones containing
cDNAs of interest were excised from pAC2 vector by BglII
digest and were cloned at the BamHI site into the pBCMV eukaryotic expression vector (Stratagene) for further analysis.
In Vitro Transcription and Translation--
The
above-characterized cDNAs were transcribed from T3 promoter and
translated in reticulocyte lysate using TNT-coupled reticulocyte lysate
system (Promega). A typical reaction contains 25 µl of rabbit
reticulocyte lysate, 2 µl of reaction buffer, 20 µM
amino acid mixture, 1 µg of DNA template, 40 units of ribonuclease
inhibitor, 10 units of T7 RNA polymerase, and 1 µl of
TranscentTM Biotin-lysyl-tRNA (Promega) in a total volume
of 50 µl. The reactions were carried out at 30 °C for 1 h.
Construction of Glutathione S-Transferase (GST)-PTTG Fusion
Protein and in Vitro Binding--
To construct GST-PTTG, plasmid
TA-PTTG was digested with EcoRI, and the insert was
subcloned in frame at the EcoRI site of pGEX-4T-1 (Amersham
Pharmacia Biotech). Expression of GST fusion protein was induced with
0.5 mM isopropyl-
The in vitro binding assay was performed as follows. 20 µl
of the in vitro translated protein was incubated with beads
containing 200 ng of GST or GST-PTTG fusion protein in the sonication
buffer for 90 min at 4 °C. Complexes were washed extensively with
the sonication buffer, boiled in loading buffer, and separated on 10%
SDS-polyacrylamide gels. Gels were transferred to nylon membranes and
blocked by incubation with Tris-buffered saline containing 0.5% Tween
20 (TBSB). The membranes were incubated with Streptavidin-horseradish peroxidase conjugate in TBST for 45 min, washed 4 times with TBST and 3 times with TBS. The membranes were then incubated with the chemiluminescent substrate mixture for 1 min and exposed to Kodak x-ray
film for 20 min.
Immunoprecipitation--
S10 and HSJ2 were subcloned into pCMV
eukaryotic expression vector with HA tag. pCMV-S10 and pCMV-HSJ2 were
transiently transfected into COS-7 cells either alone or together with
pCMV-PTTG. 48 h post transfection, whole cell extracts were made
from transfected cells. Cells were lysed in NET-N buffer containing 50 mM Tris, pH 7.6, 5 mM EDTA, 0.3 M
NaCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin, pepstatin, and aprotinin, and centrifuged at 30,000 × g for 1 h at 4 °C. The supernatant was stored at
Mutagenesis--
The N-terminal deletion mutant of pAS2-PTTG
were generated using ExSite polymerase chain reaction-based
site-directed mutagenesis kit (Stratagene) following manufacturer's
instructions. The upper strand primers were
(1) 5'-TTAGATGGGAAATTGCAGGTTTCAACGCCA-3',
(2) 5'-GTCAACAGAGTTACTGAAAAGCCAGTGAAG-3',
(3) 5'-TCTACTAAGACACAAGGCTCTGCTCCTGCT-3',
(4) 5'-AGTTTTGACCTGCCTGAAGAGCACCAGATC-3', and
(5) 5'-AAGCTGCTGCACCTGGACCCCCCTTCCCCT-3'.
The lower strand primer was: 5'-CATCCTGAAGCCGAATCCGGCCTCCATGGCAT-5'.
Northern Blot Analysis--
The human RNA Master Blot and
Multi-tissue Blot were purchased from CLONTECH.
Total RNA was isolated from the testes of rats of various ages using
RNAeasy RNA isolation kit (Qiagen). The full-length cDNAs of PTTG,
S10, and HSJ2 were labeled with [32P]dCTP using Prime-It
II Random Primer labeling kit (Stratagene). Hybridization was performed
in ExpressHyb hybridization solution (CLONTECH) at
65 °C overnight for RNA Master Blot and total RNA blots or for
1 h for Multi-tissue blot. The membrane was washed three times in
2× SSC and 0.05% SDS at 65 °C, followed by two washes in 0.1× SSC
and 0.1% SDS at 55 °C. Each wash was 20 min. The membrane was
exposed to x-ray film for 3 to 24 h. It was then stripped by
boiling for 10 min in 0.5% SDS and was hybridized to the second probe
as described above.
Southern Blotting--
DNA was extracted from human lymphocytes.
Cells were incubated in lysis buffer (100 mM NaCl, 25 mM EDTA, 1% SDS, and 100 µg/ml proteinase K) at 50 °C
overnight. Protein contaminants were removed by phenol/chloroform
extractions, and DNA was precipitated with ethanol and resuspended in
Tris-EDTA. 10 µg of DNA was digested with either BamHI,
EcoRI, HindIII, or PstI at 37 °C
overnight. The DNA was separated on 0.8% agarose gel. The gel was
denatured and neutralized, and the DNA was transferred to nylon
membrane. The membrane was probed with the HSJ2 cDNA using standard
Southern blot hybridization and washing conditions. The membrane was
exposed to x-ray film for 48 h before developing.
Cellular Localization of PTTG mRNA in Testis--
Because
testis is composed of multiple cell types, including somatic Sertoli
and Leydig cells, as well as proliferating and differentiating germ
cells, it is important to determine in what cell type and in which
differentiation stage during the spermotogenic cycle PTTG is expressed.
In situ hybridization was performed on paraffin sections of
adult rat testis using 35S-labeled riboprobe. As shown in
Fig. 1A, when the antisense
PTTG probe was hybridized to adult testis sections, a whole range of signal strengths could be seen between tubule cross-sections, indicative of spermatogenic stage-specific expression. Fig.
1B shows two cross-sections probed with the sense probe. No
hybridization signal was detected. Fig. 1C shows a group of
seminiferous tubules at various stages of the cycle at higher
magnification. Identification of the cycle of the seminiferous
epithelium was determined by parameters such as the number of layers of
spermatids, their position in the epithelium, and their nuclear shape.
Fig. 1D compares two tubules at early (stage 3 or 4, left) or late (stage 12 or 13, right) stage of
the cycle. The tubule in the later stage expressed PTTG mRNA to
much higher levels than the tubules at the early stage (Fig.
1D). The hybridization signals were strongest over the cell
layers representing spermatocytes and spermatids (Fig. 1D).
Fig. 1E shows a stage 14 tubule hybridized to PTTG antisense probe. Strong hybridization signals were again detected in
spermatocytes and spermatids. In contrast, there was no hybridization
signal to the sense probe in a tubule of the same differentiation stage (Fig. 1F).
To confirm these results, developmental Northern blot analysis was
performed using testis RNA from rat of various ages. As shown in Fig.
2, PTTG mRNA was barely detectable in
the testis of 7-day-old rat. At this time, testis contained somatic
cells and spermatogonia cells only (17). By day 14, when leptotene spermatocytes appeared (17), low level PTTG expression was detected. Increasing amount of PTTG mRNA was detected on day 21 when zygotene spermatocytes are present (17). By day 28, PTTG mRMA levels increased
dramatically and reached the expression levels in adult animals. At
this stage, both pachytene spermatocytes and spermatids are present in
rat testis. These results indicate that PTTG mRNA is expressed in
both spermatocytes and spermatids.
Screening PTTG Interactive Proteins Using Yeast Two-hybrid
System--
Stage and cell type-specific expression of PTTG suggests
that it may play a role in spermatogenesis. Because the amino acid sequence of PTTG does not provide any functional information, we sought
to identify cDNAs that encode proteins able to interact with PTTG
using a yeast two-hybrid screening strategy. The entire coding region
of the rat PTTG was cloned into a Gal4 DNA binding domain vector pAS2
(18). This hybrid plasmid and plasmid from a human testis cDNA
library constructed in the activation domain vector were cotransformed
into the yeast host strand CG-1945. The transformants were plated on
minimal medium lacking Leu, Trp, and His to select for those containing
both types of plasmids (i.e. Leu+,
Trp+) and that also express interacting hybrid proteins
(His+). Primary His+ transformants were then
tested for expression of the second reporter gene, LacZ, using a filter
assay for
Approximately 106 transformants were placed under
selection. Of the transformants plated on SD/
To further test whether the phenotype observed in the original screen
was reproducible and dependent on the pAS2-PTTG hybrid, plasmid DNA
from each of the seventy clones was used to transform CG-1945 either
alone or with pAS2-PTTG. Transformants were assayed for Characterization of Positive Clones Dependent on PTTG Hybrid
Expression--
Sequence analysis of the 10 positive clones revealed
that eight clones showed no significant homology to known protein
sequences reported to GenBank. One cDNA insert (y9) encoded a
protein with a predicted molecular mass of 19 kDa and was identical to
human S10 ribosomal protein (20). Another positive clone (y3) contained an open reading frame of 217 amino acids (Fig.
3A) and was 62 and 47%
identical to MSJ-1 (mouse DnaJ) (21) and HSJ1 (homosapiens DnaJ 1)
(22), respectively. The highest identity shared by the three proteins
was at the N termini, where the 82 amino acids were 70% identical
(Fig. 3B). This similarity was shared by several other DnaJ
proteins, including the E. coli DnaJ (23, 24) and yeast,
homologous of DnaJ, SCJ1(25), YDJ1 (26), and SIS1 (27). The similarity
between y3 and other DnaJ proteins suggested that y3 is a new member of
the human DnaJ family of proteins, and therefore was designated HSJ2
(homosapiens DnaJ 2).
Tissue Distribution of S10 and HSJ2--
A human RNA master blot
containing mRNA from 50 human tissues was probed with the S10 or
HSJ2 cDNA probe. Both S10 and HSJ2 mRNAs were detected in all
the tissues examined, although the expression level of HSJ2 mRNA
varies among different tissues (data not shown). To determine the
transcript size of S10 and HSJ2, Northern blot analysis was performed
on selected tissues. The HSJ2 cDNA detected two bands of about 2.8 and 1.7 kb (Fig. 4A). The
highest level of expression was detected in testis for the 1.7-kb
transcript (normalized to loading control, Fig. 4C). Other tissues, including spleen, intestine, ovary, thymus, prostate, and
leukocytes showed much lower level of HSJ2 mRNA expression (Fig.
4A). The S10 probe detected a single transcript of about 600 base pairs, and the expression levels of this transcript were similar
in the tissues examined (Fig. 4B).
Developmental Regulation of HSJ2 and S10 Expression--
To
determine whether HSJ2 and S10 expression undergo developmental
regulation, we have used postnatal rat testis as a model. RNAs were
isolated from testes of postnatal rats of various ages and used in
Northern blot analysis. As shown in Fig.
5A, very low level HSJ2
mRNA was detected at day 7. Increasing levels of HSJ2 mRNA were
detected on day 14 and 21. By day 28, there was a more than 10-fold
increase in HSJ2 expression (Fig. 5A) (after normalizing to
actin control, Fig. 5C). There was no further increase in
HSJ2 mRNA levels in adult rat testis (Fig. 5A). S10
mRNA, on the other hand, was expressed to similar levels from
postnatal day 7 to adult (Fig. 5B). These results indicate
that although HSJ2 expression undergoes developmental regulation, S10
is constitutively expressed.
Genomic Analysis of HSJ2--
As an initial step to characterize
HSJ2 gene structure, human genomic DNA analysis was performed. As shown
in Fig. 6, the HSJ2 probe detected two
bands in human genomic DNA digested with BamHI,
EcoRI, HindIII, and PstI. Because
these enzymes do not cut within the cDNA, these results suggest
that either the sites for these enzymes are present within the introns
of the gene or there are two different HSJ2 genes if the enzymes do not
cut within the genes.
Both S10 Ribosomal Protein and HSJ2 Bind PTTG in Vitro--
To
confirm and extend the protein interaction data obtained in yeast, PTTG
was expressed as a GST fusion protein in E. coli (28). To
test the ability of S10 and HSJ2 proteins to bind PTTG in
vitro, S10 and HSJ2 was in vitro transcribed and
translated in the presence of TranscentTM Biotin-lysyl-tRNA
(Promega). After translation, GST-PTTG immobilized on
glutathione-Sepharose was added. The beads were washed extensively, and
the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis. After electroblotting, the bound, biotinylated protein
was visualized by binding to streptavidine-horseradish peroxidase,
followed by chemiluminescent detection. As shown in Fig.
7, both S10 and HSJ2 were retained on the
glutathione beads when they were incubated with GST-PTTG, whereas
neither protein was retained when incubated with GST alone. These
results indicated that S10 and HSJ2 protein were retained on the matrix
through the PTTG segment of the fusion protein.
Both S10 and HSJ2 Proteins Can Be Coimmunoprecipitated with PTTG
Protein--
To further characterize the association of PTTG protein
with S10 and HSJ2 proteins, we examined whether PTTG protein could be
coimmunoprecipitated from transfected cells. Cos-7 cells were transiently transfected with HA-tagged HSJ2 and S10 expression vectors,
either alone or together with PTTG expression plasmid. Cells were
lysed, and the resulting extracts were immunoprecipitated with anti-HA
monoclonal antibody (29). The immunoprecipitates were then resolved by
SDS-polyacrylamide gel electrophoresis, and the gel was immunoblotted
and probed with an antibody specific for the N-terminal 17 amino acids
of PTTG (1). A band corresponding to PTTG was seen only in cells that
were cotransfected with both S10 and PTTG or HSJ2 and PTTG expression
vectors (Fig. 8). These results indicated
that PTTG could form a complex with either S10 or HSJ2.
The C Terminus of PTTG Protein Is Required for Binding of S10 and
HSJ2--
To determine the region of PTTG protein involved in
interaction with S10 and HSJ2, sequential N-terminal deletion mutants (Fig. 9A) of PTTG were
subcloned into pAS2 and used to co-transform yeast CG-1945 with the
Gal4 activation domain-S10 or HSJ2 fusion plasmid. The resulting
transformants were then assayed for Previous studies indicated that PTTG is specifically expressed in
adult rat testis (1). In this study, the precise cellular location of
PTTG mRNA has been determined. Our results show that PTTG messenger
is predominantly expressed in spermatocytes and spermatids and in a
stage-specific manner during the rat spermatogenic cycle. This
expression pattern of PTTG is similar to that of proto-oncogene c-kit,
in that both transcripts are present in germ cells. However, expression
of c-kit mRNA starts in spermatogonia and continues into
spermatocytes (30). c-kit is known to play a role in supporting spermatogonia proliferation and survival. Spermatogonia proliferate to
replace themselves and differentiate to give rise to primary spermatocytes in which DNA synthesis is carried out to double chromosomes, after which they enter the first meiotic prophase. Expression of PTTG mRNA in nonproliferating spermatocytes and spermatids suggests that it may play a role in survival and
differentiation of these germ cells.
Because the primary structure of PTTG protein does not provide
functional information, we have used the yeast two-hybrid system to
identify proteins that interact with PTTG in testicular cells. We have
used the rat PTTG to screen a human library, because the human PTTG
cDNA was not available. Because of the species difference, it is
possible that PTTG-interactive proteins that do not share high homology
between human and rat may not be detected using this screen. However,
using this method, we identified and characterized two proteins capable
of interacting with PTTG. We demonstrated that both proteins were able
to bind to PTTG in vitro and in vivo and that the
C-terminal 75 amino acids of PTTG protein were required for this interaction.
One of the cDNA clones isolated from this screen encodes a
novel human homologue of the bacterial heat-shock protein DnaJ (HSJ2).
HSJ2 shares highest sequence similarity with the previously characterized MSJ-1 (21) and HSJ1 protein (22). Like these proteins,
the similarity between HSJ2 and E. coli DnaJ is restricted to the N-terminal region of the molecule. MSJ-1 was shown to have two
transcripts of 1.2 and 1 kb that are specifically expressed in testis
(21). HSJ1 has two alternatively spliced transcripts of 2 and 3 kb,
which are preferentially expressed in neurons (22). In contrast, HSJ2
mRNA is more widely expressed, although testis shows the highest
level of expression. They are also two HSJ2 transcripts (1.7 and 2.8 kb) that may be the result of alternative splicing, use of alternative
promoters, or polyadenylation sites. Like MSJ-1, HSJ2 expression
undergoes developmental regulation in testis. However, although MSJ-1
expression is restricted in spermatids (21), HSJ2 is expressed in germ
cells in the earlier stages of differentiation pathway as well as in
spermatids. The developmental expression pattern of HSJ2 in postnatal
rat is similar to PTTG mRNA expression profile. Both transcripts
show very low levels expression on postnatal day 7, with increasing
levels of expression on days 14 and 21 and dramatic increase on day 28. It is likely that HSJ2 mRNA is expressed in similar germs cells where PTTG messenger is found. Future in situ hybridization
studies of HSJ2 in rat testis will confirm these results.
HSJ2 also shows high sequence similarity (59%) to the yeast DnaJ
homologue, SIS1 protein (27). The SIS1 gene is essential for viability.
In the absence of normal SIS1 function, the polysome levels decrease,
and 80S ribosomes accumulate to high levels (13). It is believed that
SIS1 functions to dissociate 80S ribosome into 40S and 60S subunits.
SIS1 was shown to be associated with 40S ribosomal subunits and the
smaller polysomes (13). This evidence indicates that SIS1 is required
for the normal initiation of translation (13).
Another protein that interacts with PTTG protein is the human ribosomal
large subunit protein S10 (20). The mammalian ribosomal is a complex
structure assembled from approximately 80 proteins and four ribosomal
RNA molecules (31). The ribosomal proteins are thought to facilitate
the folding and maintenance of an optimal configuration of the rRNAs
(32), perhaps in this way conferring speed and accuracy on protein
synthesis. Recently, the involvement of the ribosomal proteins in cell
proliferation (33-37), differentiation (38), and apoptosis (39) has
been demonstrated. It has been show that S10 protein can be
cross-linked to eukaryotic initiation factor 3 (40), an observation
suggesting that S10 protein forms part of the domain involved in
binding of the initiation factor to the 40S subunit at the start of the
translation (40). The binding of PTTG protein to S10 protein suggests
that PTTG protein may associate with ribosomes through this interaction.
What is the significance of the association of PTTG with HSJ2 and S10
proteins? Both S10 and SIS1 proteins have been shown to be associated
with 40S ribosomal subunit and are required for the initiation of
translation (13, 40). Although the association of HSJ2 with ribosome is
unknown at the present, because DnaJ has been shown to mediate the
dissociation of several protein complexes, it is possible that HSJ2
might be able to mediate the dissociation of a specific protein complex
of the translation. Interestingly, PTTG also interacts with S10, a
ribosome protein. One possibility is that PTTG is targeted to the
ribosome through interaction with S10 protein, and association with
HSJ2 results in dissociation of PTTG-S10 complex and separates it from
the ribosome. These hypotheses will be tested in future studies.
In summary, we demonstrated in this study that PTTG is expressed in a
stage-specific manner in spermatocytes and spermatids during the
spermatogenic cycle. Through interaction with ribosomal protein S10 and
a novel human homologue of DnaJ protein, PTTG may play a role in male
germ cell differentiation.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
-Max
hyperfilm for 2 days and were then dipped in NTB2 emulsion, exposed for
1 to 3 weeks, developed, and stained with methyl green.
Leu/
Trp/
His
synthetic dropout (SD) agar plates containing 5 mM
3-amino-1,2,4-triazole (3-AT).
-Galactosidase Assays--
Transformants grown on
Leu/
Trp/
His/+3AT SD plates were screened further by colony-lift
filter assay. For each plate of transformants, a Whatman No. 5 filter
was presoaked in 2.5 ml of Z buffer/5-bromo-4-chloro-3-indolyl
b-D-galactopyranoside (16.1 g/liter
Na2HPO4·7 H2O, 5.5 g/liter
NaH2PO4·H2O, 0.75 g/liter KCl, 0.246 g/liter
MgSO4·7 H2O, 0.34 mg/ml
5-bromo-4-chloro-3-indolyl b-D-galactopyranoside, 38 mM
-mercaptoethanol ). A dry filter was placed over the
surface of the plate of colonies, and after the filter was oriented to
the plate, it was lifted off and transferred to a pool of liquid
nitrogen for 10 s. The filter was thawed at room temperature and
placed on the presoaked filter. The filters were incubated at 30 °C
for 30 min to 4 h, and the blue colonies were identified.
-galactosidase assays. A single colony was used to inoculate a 5-ml
liquid medium, and the culture was incubated at 30 °C overnight. 2 ml of the overnight culture was inoculated into 8 ml
yeast/peptone/glucose medium and grown at 30 °C mid-log phase. The
exact A600 was recorded. 1.5 ml of the culture
was centrifuged, and the cell pellet was resuspended in 300 µl of Z
buffer. 100 µl of the cell suspension was transferred to a fresh
tube, frozen in liquid nitrogen, and thawed at 37 °C. 0.7 ml of Z
buffer was added to the tube. After recording the time, 0.16 ml of
O-nitrophenyl-
-D-galactopyranoside was added
and the tube was incubated at 30 °C. After the yellow color
developed, 0.4 ml of 1 M Na2CO3 was
added to the reaction, and the elapsed time was recorded. After
removing cell debris by centrifugation, the A420
was measured. The
-galactosidase units were calculated as the
following:
-gal units = 1,000 × A420/(t × V × A600), where t = elapsed time
(in min) of incubation, V = 0.1 ml × concentration factor, and A600 is of 1 ml of culture.
-thiogalactopyranoside at 37 °C for
90 min. Cells were centrifuged, and the resulting pellet was
resuspended in a sonication buffer containing 150 mM KCl,
40 mM HEPES, pH 7.9, 0.5 mM EDTA, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.05% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
aprotinin. Cells were lysed by sonication. Cell debris was removed by
centrifugation, and the supernatant was added to Sepharose 4B beads
(Amersham Pharmacia Biotech).
80 °C. 1 mg of cell extract was incubated with 2 µg of anti-HA
(clone 12CA5, Borhinger Mannheim) for 2 h at 4 °C. Immune
complexes were then precipitated with protein A/G-Sepharose at 4 °C
for 1 h. The precipitate was collected by centrifugation and
washed extensively in NET-N buffer. Protein complexes were separated by
SDS-polyacrylamide gel electrophoresis and Western blotted using 1:5000
dilution of the anti-PTTG polyclonal antibody.
RESULTS
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Fig. 1.
Localization of PTTG transcript in adult
testis by in situ hybridization. Panels A and
B show the entire sections of testis hybridized to antisense
(A) or sense (B) probe, respectively. The slides
were exposed to -Max hyperfilm for 2 days. Hybridization signals
appear as dark grain. Panel C, low power
bright-field view of testis cross-section. Leydig cells are indicated
by a bar. The hybridization signal is visible as dark
grains. Panel D, high magnification, bright-field view
of two tubules at earlier (left) or later (right)
stage of the spermatogenic cycle for comparison of PTTG mRNA
expression levels. Panel E, high magnification, bright-field
view of a stage 14 tubule; strong hybridization signals over
spermatocytes and spermatids appear as dark grains.
Panel F, high magnification, bright-field view of a stage 14 tubule, probe with sense control.
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Fig. 2.
PTTG mRNA expression in postnatal rat
testis. 20 µg of total RNA isolated from testes of rats of
various ages was separated on agarose gel, blotted, and probed with
PTTG (panel A) or actin (panel B). The age of the
rat is indicated on the top of each lane. Hybridization
signals are indicated by arrows.
-galactosidase activity (19).
His/
Leu/
Trp/+5
mM 3AT medium, about 2000 grew into colonies within 3 to 5 days. Seventy of the His+ colonies were also blue when
screened for their ability to produce
-galactosidase using the
filter lift assay. These His+ blue colonies were considered
positive in the initial screen and were used for additional studies.
-gal
activity, and those showing activity only in the presence of pAS2-PTTG
were considered positive. Of the 70 initial isolates, 10 of the
recovered plasmids induced the expression of lacZ only in the presence
of pAS2-PTTG, and their relative
-activity was shown in Table
I.
-Gal activity of the 10 PTTG-dependent positive clones
-gal activity was quantified using
O-nitrophenyl-
-D-galactoside as substrate.
The
-gal activity of control transformants is <0.1.
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Fig. 3.
Amino acid sequence of HSJ2.
A, alignment of HSJ2 to MSJ1, HSJ1, and SIS1. B,
amino acid sequence of HSJ2 is aligned to amino acids of MSJ1, HSJ1,
and SIS1. Identical amino acid sequences to HSJ2 are
highlighted.
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Fig. 4.
Transcript size of HSJ2 and S10: Northern
blot analysis. HSJ2 probe (A), S10 probe
(B), actin probe (C) poly(A)+ RNA
from eight different human tissues (indicated at the top of the figure)
were used for Northern blot analysis. Molecular mass markers are shown
on the side. The exposure time was 24 h.
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Fig. 5.
HSJ2 and S10 mRNA expression in postnatal
rat testis. 20 µg of total RNA isolated from testes of rats of
various ages was separated on agarose gel, blotted, and probed with
HJS2 (panel A), S10 (panel B), or actin
(panel C). The age of the rat is indicated on the top of
each lane. Hybridization signals are indicated by
arrows.
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Fig. 6.
Genomic Southern blot analysis of HSJ2.
10 µg of human genomic DNA was digested with the indicated
restriction enzyme, separated on agarose gel, blotted, and probed with
HSJ2 cDNA. The hybridization signals are indicated by
asterisks, and the molecular weight markers are indicated on
the left side of the gel.
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Fig. 7.
PTTG binds to HSJ2 and S10 in
vitro: GST pull-down assay. HSJ2 and S10 were transcribed
and translated in vitro and labeled with
TranscentTM Biotin-lysyl-tRNA. The proteins were allowed to
bind to either GST alone or GST-PTTG immobilized on Sepharose 4B beads.
The bound proteins were analyzed on SDS-polyacrylamide gels in the
lanes indicated, blotted, and visualized by chemiluminescent
detection.
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Fig. 8.
PTTG associates with HSJ2 and S10 proteins in
the cell: immunoprecipitation assay. Cos7 cells were transfected
with the indicated plasmids. 48 h post-transfection, whole cell
lysates were immunoprecipitated with anti-HA monoclonal antibody. The
immuno complexes were separated by SDS-polyacrylamide gel
electrophoresis and blotted onto a nylon membrane. The blot was probed
with anti-PTTG antibody and visualized by chemiluminescent.
-gal activity. As shown in Fig.
9B, transformants resulting from co-transformation of
Gal4-S10 or Gal4-HSJ2 with PTTG mutants containing deletions up to 123 N-terminal amino acids (i.e. mutant 1 to 4) grew on SD/
Trp/
Leu/
His/+3AT plates and showed
-gal activity similar to
transformants of the wild type PTTG. When the N-terminal deletion was
made to amino acid 153 (mutant 5), however, there was no growth on
SD/
Trp/
Leu/
His/+3AT plates, suggesting that this PTTG mutant was
unable to bind to S10 and HSJ2. These results suggested that both S10
and HSJ2 bind to PTTG within the 75 amino acids at the C terminus and
that amino acid residues 124-153 play an essential role for PTTG
interaction with these proteins.
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Fig. 9.
HSJ2 and S10 protein bind to similar regions
of the PTTG protein. A, schematics of various PTTG
mutants used for Gal4-PTTG fusion. B, detection of
interaction between HSJ2 or S10 and PTTG mutants in vivo.
CG-1945 was cotransformed with the indicated panel of
Gal4-PTTG mutants and either HSJ2- or S10-expressing plasmid. Colony
color was determined by the colony filter lift assay.
O-Nitrophenyl- -D-galactopyranoside
quantitation of the
-gal activity was performed in triplicate for
each transformation (see "Materials and Methods"). WT,
wild type.
DISCUSSION
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ACKNOWLEDGEMENTS |
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I would like to thank Dr. Amiya P. Sinha Hikim (Harbor-UCLA Medical Center) for advice on testis morphology and identifying stages of the rat spermatogenic cycle and Dr. John S. Adams (Cedars-Sinai Medical Center) for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK-02346 (to L. Pei.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF080569.
To whom correspondence should be addressed: Div. of Endocrinology,
Cedars-Sinai Medical Center, 8700 Beverly Blvd., D-3066, Los Angeles,
CA 90048. Tel.: 310-855-7682; Fax: 310-559-2357; E-mail:
pei{at}cshs.org.
The abbreviations used are:
PTTG, pituitary
tumor-transforming gene; SD, synthetic dropout; -gal,
-galactosidase; GST, glutathione S-transferase; TBST, Tris-buffered saline containing 0.5% Tween 20; HA, hemagglutinin; kbp, kilobase pair(s); HSJ2, homosapien DnaJ2 homologue.
2 L. Pei, unpublished data.
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REFERENCES |
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