Identification and Characterization of a Haploid Germ
Cell-specific Nuclear Protein
Kinase (Haspin) in Spermatid Nuclei and Its Effects on Somatic
Cells*
Hiromitsu
Tanaka
,
Yasuhide
Yoshimura
,
Masami
Nozaki
,
Kentaro
Yomogida
,
Junji
Tsuchida
,
Yasuhiro
Tosaka
,
Toshiyuki
Habu
,
Tomoko
Nakanishi§,
Masato
Okada¶,
Hiroshi
Nojima
, and
Yoshitake
Nishimune
**
From the Departments of
Science for Laboratory Animal
Experimentation and
Molecular Genetics, Research Institute for
Microbial Diseases, the ¶ Division of Protein Metabolism,
Institute for Protein Research, and the § Department of
Experimental Genome Research, Genome Information Research Center, Osaka
University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan
 |
ABSTRACT |
We have cloned the entire coding region of a
mouse germ cell-specific cDNA encoding a unique protein kinase
whose catalytic domain contains only three consensus subdomains
(I-III) instead of the normal 12. The protein possesses intrinsic
Ser/Thr kinase activity and is exclusively expressed in haploid germ
cells, localizing only in their nuclei, and was thus named Haspin (for
haploid germ cell-specific nuclear
protein kinase). Western blot analysis showed that specific antibodies recognized a protein of
Mr 83,000 in the testis. Ectopically expressed
Haspin was detected exclusively in the nuclei of cultured somatic
cells. Even in the absence of kinase activity, however, Haspin caused
cell cycle arrest at G1, resulting in growth arrest of the
transfected somatic cells. In a DNA binding experiment, approximately
one-half of wild-type Haspin was able to bind to a DNA-cellulose
column, whereas the other half was not. In contrast, all of the
deletion mutant Haspin that lacked autophosphorylation bound to the DNA
column. Thus, the DNA-binding activity of Haspin may, in some way, be
associated with its kinase activity. These observations suggest that
Haspin has some critical roles in cell cycle cessation and
differentiation of haploid germ cells.
 |
INTRODUCTION |
Maturation of male germ cells in mammals involves numerous
structural and functional changes that are precisely timed. These complex processes, known collectively as spermatogenesis, may be
represented by the following three major events: proliferation and
differentiation of spermatogonia, meiotic events at prophase of
spermatocytes, and drastic morphological change during differentiation from the haploid round spermatids to sperm (1). To uncover the
mechanism of spermatogenesis, many germ cell-specific molecules have
been studied using various strategies (2-5).
During haploid germ cell differentiation, or spermiogenesis, the round
spermatid undergoes marked morphological change to become a sperm
without cell division; and the nucleus is shaped, mitochondria are
rearranged, the flagellum is developed, and the acrosome is generated
(6). Over this long period of time, ~2 weeks for the mouse, no
division of haploid germ cells occurs. Some of the regulatory proteins
localized in the nucleus must participate in this precise regulation.
It appears that there are at least two types of mechanisms at work in
haploid germ cell-specific gene regulation: one involves the
CRE,1 and the other, as yet
uncharacterized, does not. Several proteins are specifically expressed
in the nuclei of haploid germ cells: transition protein (7), protamine
(8), histone H1t (9), zinc finger proteins (10), testis-specific HMG
(11), lamin B3 (12), and CREM
(13). Through knockout of the
Crem
gene in mice, CREM
capable of binding to the
sequence of CRE has been shown to play important roles in the
regulation of spermiogenesis (14, 15). In contrast, some of the
proteins specifically expressed in haploid germ cells do not have any
CRE motifs in the promoter region (16), implying that some other
regulatory mechanism exists.
We have isolated many cDNA clones specifically expressed in germ
cells using a subtracted cDNA library prepared from supporting cells of mutant testes and the wild-type testis (4). Using a partial
cDNA clone previously obtained, we have cloned the entire coding
region and characterized a novel gene encoding a protein with various
well known motifs. The protein, which we named Haspin (haploid germ cell-specific nuclear
protein kinase), is specifically expressed in
haploid germ cells, localizes in nuclei of round spermatids, binds to
DNA, and has Ser/Thr protein kinase activity. Since transfection of
haspin cDNA into cultured somatic cells caused cessation
of cell proliferation, Haspin could be involved in regulation of
proliferative activity as well as specific gene expression in haploid
germ cells.
 |
EXPERIMENTAL PROCEDURES |
Construction of the Subtracted Library and Screening--
Total
RNAs were extracted by the guanidine thiocyanate/CsTFA method (17) from
the testes of adult wild-type C57BL/6 mice and 4-month-old
W/Wv mutant mice lacking germ cells (4). The corresponding
cDNA libraries were prepared as described by Gubler and Hoffmann
(18), with some modifications. Prepared cDNA fragments were
directionally inserted between NotI (dephosphorylated) and
BglII sites of the pAP3neo vector (4). The
ligated DNAs were electroporated into MC1061A cells as described
previously (19). The complexities of the cDNA libraries obtained
were ~6 × 106 colony-forming units in both cases. A
germ cell-specific cDNA library was generated by subtracting
cDNAs of mutant (W/WV) testis that contains no germ
cells from wild-type testis cDNAs (4). Plasmid DNA of each clone
randomly picked from the subtracted cDNA library was subjected to
cDNA dot-blot analysis. As a probe to select testicular germ
cell-specific cDNA clones, RNAs of testis cDNA libraries of
wild-type and mutant mice were generated with T7 RNA polymerase and
labeled with the hapten digoxigenin. To clone the complete cDNA of
gsg2, a testis-specific partial cDNA obtained previously
by screening the subtracted library (4), a library of Escherichia
coli MC1061A cells carrying the adult wild-type testis cDNAs
was diluted to seed at 1 × 105 colony-forming units
on a nitrocellulose filter placed on an LB plate. After incubation at
37 °C, grown colonies were transferred to two nylon replica filters
and lysed by sequential soakings in the following solutions at room
temperature: 5 min in 0.5 N NaOH and 1.5 M
NaCl, 5 min in 0.5 M Tris-HCl (pH 7.4) and 1.5 M NaCl, and 5 min in 2× SSC. After baking at 80 °C for
2 h, the filters were washed, and the bacterial debris was
removed. A 32P-labeled probe was prepared with a BcaBest
random primer kit (Takara, Osaka, Japan) using a 1.3-kilobase
EcoRI-NotI fragment of the partial
gsg2 cDNA fragment (4). The filters were then hybridized
with the partial clone gsg2 probe at 65 °C for 20 h (4× SSC, 10× Denhardt's solution, 0.1% SDS, and 100 µg/ml
denatured sonicated salmon sperm DNA). Several positive clones were
isolated by screening 2 × 106 colonies.
5'-RACE--
To isolate the 5'-end of the gsg2
cDNA, we performed 5'-RACE (20, 21) using a 5'-AmpliFINDER RACE kit
(CLONTECH). Synthesis of the single-stranded
cDNA was carried out using 10 pmol of a first antisense
oligonucleotide primer (20-mer) designed from the sequence data of the
gsg2 clone, which corresponded to nucleotides 913-932 of
the gsg2 cDNA (see Fig. 1), and 2 µg of mouse testis poly(A)+ RNA. After hydrolysis of RNA with NaOH,
single-stranded DNAs were purified by GENO-BIND
(CLONTECH) and ligated to AmpliFINDER Anchor
(5'-phosphorylated 3'-aminated oligonucleotide,
5'-CACGAATTCACTATCGATTCTGGAACCTTCAGAGG-NH2-3') with T4
ligase. Polymerase chain reaction amplification of the 5'-end region
was performed using a second antisense linker oligonucleotide primer
(19-mer corresponding to nucleotides 317-335 and a BamHI recognition sequence) and an anchor primer
(5'-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCTCGATAG-3'). Amplification
products were then digested with EcoRI and BamHI and ligated to EcoRI-BamHI-cut pBluescript II
SK+ plasmid DNAs. Three independent clones were selected
and sequenced.
Northern Blot Analysis and DNA Sequencing--
Freshly removed
organs of adult mice (C57BL/6 strain) were homogenized in
RNAzolTM B (Tel-Test, Inc., Tokyo, Japan). Germ and other
somatic cells of the testes were prepared as described in our previous
report (22). Cryptorchid testes and those of various mutants defective in germ cell differentiation such as jsd/jsd,
Sl17H/Sl17H, and W/Wv were also
used (5). Total RNAs were extracted according to the manufacturer's
recommendations (Tel-Test Inc., Tokyo, Japan) and quantified by optical
density measurement. RNA samples containing 2.2 M
formaldehyde were subjected to electrophoresis on a 1.1% agarose gel
containing 0.66 M formaldehyde (23). RNAs were transferred to a nitrocellulose filter in 20× SSC. Hybridization was performed with 32P-labeled cDNA prepared with the BcaBest random
primer kit at 42 °C for 16 h in a solution containing 4× SSC,
5× Denhardt's solution, 0.2% SDS, 12 µg/ml denatured salmon sperm
DNA, and 50% formamide. Filters were washed twice in 0.1× SSC and
0.1% SDS at 60 °C. Signals of the bands were detected with a Fuji
image analyzer.
Dideoxy chain termination sequencing reactions (24) were performed with
fluorescent dye-labeled primers and thermal cycle sequencing kits
purchased from Li-COR. The reaction products were analyzed by a Li-COR
model 4000 DNA Sequencer. GenBankTM, EBI, DDBJ, SWISS-PROT,
and PIR data banks were searched for homology to the haspin
cDNA or amino acid sequence. The deduced amino acid sequence of the
haspin cDNA was analyzed by the Fujitsu Bioresearch/PR system.
Antiserum Preparation--
Two kinds of synthetic peptides
(KKK-1, TPPRHYHQSKKKRKA at residues 311-325; and DDP-2, ASDDPDDPDFPG
at residues 59-70) designed from the deduced amino acid sequence of
Haspin were purified by HP-high pressure liquid chromatography and used
for immunization of rabbits (Peptide Institute, Inc., Osaka, Japan).
Furthermore, a partial haspin cDNA fragment (HAS-3,
amino acids 422-754) was subcloned into the pGEX-1 vector (25).
Glutathione S-transferase fusion proteins were produced in
Escherichia coli by
isopropyl-
-D-thiogalactopyranoside induction and
purified with glutathione-agarose beads. Polyclonal antisera were
obtained by injection of the above antigens followed by booster
injections at 3-week intervals, seven times in total, into New Zealand
White rabbits.
Each anti-Haspin rabbit antiserum (KKK-1, DDP-2, and HAS-3) reacted
with the same molecule when examined by Western blot analysis. The most
effective anti-Haspin antiserum was used for each biochemical assay.
Western Blot Analysis--
Freshly prepared organs or cell
fractions of adult C57BL/6 mice or cultured cells were homogenized on
ice with a lysis buffer containing 10 mM
Na2HPO4 (pH 7.2), 160 mM NaCl, 1%
Triton X-100, 1% deoxycholic acid, 0.3% SDS, and 2 mM
phenylmethylsulfonyl fluoride (PMSF) (Sigma). After centrifugation, the
protein concentration of each supernatant was estimated by the Bradford
protein assay (Bio-Rad). Each extract containing ~100 µg of protein
was subjected to SDS-PAGE as reported by Laemmli (26), followed by
electroblotting onto polyvinylidene difluoride membrane filters
(Millipore Corp., Bedford, MA). The filters were blocked with 5%
nonfat dry milk and washed for 15 min with TBS-T (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20). The
filters were then reacted with anti-Haspin rabbit antiserum (KKK-1,
1:300 dilution) in TBS at 25 °C for 1 h, washed with TBS-T for
3 min, washed again three times for 5 min each time, and incubated with
peroxidase-conjugated anti-rabbit Ig (1:500; Dako A/S, Glostrup,
Denmark) at 25 °C for 1 h. After further washing, reactive
bands were visualized by development with diaminobenzidine in 50 mM Tris-HCl (pH 7.5) plus 0.3%
H2O2.
Immunohistochemistry--
To prepare frozen tissue specimens,
testes were immersed in O.T.C. compound embedding medium (Tissue-Tek,
Sakura, Tokyo, Japan) and frozen at
20 °C. Sections of 10 µm in
thickness were prepared by using a cryomicrotome (Microm HM 500OM) and
were fixed with 80% methanol at
20 °C for 5 min. Each section was
treated with 0.3% hydrogen peroxide, incubated with 20% normal goat
serum in phosphate-buffered saline, and then reacted with anti-Haspin
rabbit antiserum (HAS-3) diluted at a ratio of 1:100 after blocking
with a blocking kit (Vector Labs, Inc., Burlingame CA). Sections were reacted with biotinylated sheep anti-rabbit Ig (Amersham- Pharmacia Biotech, Tokyo, Japan) diluted at 1:500 and subjected to a
peroxidase/avidin system (Vectastain ABC kit, Vector Labs), followed by
incubation with 0.05% diaminobenzidine in 50 mM Tris-HCl
buffer (pH 7.2). Some of the sections were counterstained with hematoxylin.
Construction of Expression Vectors of Haspin (pEGFP-HASP) and a
Deletion Mutant of the ATP-binding Site (pEGFP-dHASP)--
Polymerase
chain reaction amplification of the haspin cDNA coding
region was performed using an antisense primer for the 5'-region of the
haspin cDNA (5'-GTTTCTGTTTGAAACGCCGGG-3', nucleotides
1-21) and a linker (BamHI) oligonucleotide primer of the
3'-region (5'-CGCAATTCCTGGCTTACCTAAATAGAC-3', nucleotides 2285-2304).
Amplification products were then digested with KpnI, which
corresponded to nucleotides 58-63 of the haspin cDNA
(see Fig. 1), and with BamHI, whose recognition site had been included in the linker oligonucleotide primer, and ligated at the
KpnI-BamHI site of the mammalian expression
vector pEGFP-C1 (CLONTECH). The resultant clone was
capable of expressing the EGFP-Haspin fusion protein.
The expression vector with a deletion mutant of haspin was
also constructed by polymerase chain reaction amplification using the
haspin cDNA as a template. For this, the following
strategy was used. First, the haspin cDNA was cut into
two pieces in the region of the ATP-binding site; then, using the 5'-
and 3'-fragments, a deletion was introduced by polymerase chain
reaction; and the two DNA fragments were joined later to obtain a
complete deletion mutant. The linker (HindIII)-containing
oligonucleotide primer sequence corresponding to nucleotides 1354-1368
(5'-CCAAGCTTCTCACACCGTTC-3') and the antisense primer of the 5'-region
as described above were used for the 5'-region DNA fragment of the
deletion mutant haspin. The linker
(HindIII)-containing oligonucleotide primer sequence corresponding to nucleotides 1406-1421 (5'-AGAAGCTTAACGACCAAGCACC-3') and the linker (BamHI) oligonucleotide primer of the
3'-region as described above were used for the 3'-region DNA fragment
of the deletion mutant haspin. The two DNA fragments were
subcloned into polycloning sites of Bluescript II to obtained the
deletion mutant haspin cDNA (see Fig. 2A).
The cDNA fragment obtained, i.e. the deletion mutant of
haspin, was ligated to the KpnI-BamHI site of the mammalian expression vector pEGFP-C1.
Transfection of Cultured Cells with Expression
Vectors--
COS-7 (27) and HEK-293 (28) cells were transfected with
expression vectors pEGFP-HASP and pEGFP-dHASP using calcium phosphate (29) and LipofectAMINE Plus reagent (Life Technologies, Inc.), respectively. Sixteen hours after transfection, COS-7 cells were washed
sequentially with 20% Me2SO, 10% Me2SO, and
phosphate-buffered saline and then incubated in Dulbecco's modified
Eagle's medium. However, in the case of HEK-293 cells, transfected
cells were washed with the medium and incubated. After 2 days, for
in vitro protein kinase assay, the cells were washed with
phosphate-buffered saline and scraped with a plastic policeman under a
microscope. The expression vector pEGFP-C1 alone without the
haspin cDNA was used as a negative control.
In Vitro Kinase Assay--
Mouse testes and transfected cells
were lysed with a lysis buffer consisting of 10 mM Tris-HCl
(pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.03% SDS, 0.15 M NaCl, and 0.01 mM PMSF, and the lysates were
centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was
treated with protein A-Sepharose at 4 °C for 1 h to eliminate
nonspecific binding materials. Preimmune normal rabbit serum or
specific antiserum (DDP-2) was then added at a 1:500 dilution and
shaken at 4 °C for 2 h. The samples were incubated with protein
A-Sepharose beads at 4 °C for 1 h and centrifuged. The
precipitated protein A-Sepharose beads were washed three times with the
lysis buffer and then two times with kinase assay buffer (40 mM HEPES (pH 7.4), 10 mM MgCl2, 3 mM MnCl2, 5 mM CaCl2,
and 150 mM NaCl) and incubated at 37 °C for 10 min in 40 µl of kinase assay buffer with 10 µCi of [
-32P]ATP
(3000 Ci/mmol; Amersham Pharmacia Biotech). The samples mixed with
Laemmli sample buffer were subjected to SDS-PAGE and electrotransferred
to polyvinylidene difluoride membrane filters, and
32P-labeled proteins were visualized with a Fuji image
analyzer. Phosphoamino acid analysis was performed as described (30) in the protein kinase resource of the World Wide
Web.2
FACS Analysis--
After transfection with expression vector
DNAs, on days 2, 4, and 6, HEK-293 cells were harvested, suspended in
tubes, and fixed with 70% methanol. After being treated with 0.1 µg/ml RNase, cells were stained with propidium iodide (10 µg/ml).
The cell suspensions were analyzed with a fluorescence-activated cell
sorter (FACS-Calibur, Becton Dickinson Co., Ltd.).
DNA Binding Assay of Haspin--
The affinity between the Haspin
protein and DNA was examined by monitoring an elution profile of the
Haspin protein by DNA affinity column chromatography. Two days after
being transfected with pEGFP-HASP or pEGFP-dHASP, HEK-293 cells were
sonicated in 0.5× TBS-T (12.5 mM Tris-HCl (pH 7.5), 75 mM NaCl, 25 mM KCl, and 0.05% Tween 20)
containing 1 mM PMSF. Cell lysates were centrifuged at
10,000 rpm for 10 min at 4 °C and filtered through a 0.45-µm microfilter (Kurabo, Tokyo). Supernatants were applied to
chromatography columns (0.5-µl bed volume; Bio-Rad, Tokyo) with calf
thymus native DNA-cellulose (P-L Biochemicals). Materials were eluted
stepwise sequentially with 1-ml volumes of 0.1, 0.2, 0.3, 0.4, 0.6, and 1.0 M NaCl in 0.5× TBS-T containing 1 mM PMSF
after washing two times with 5-ml volumes of buffer consisting of 0.5×
TBS-T containing 1 mM PMSF. The eluates were immunoblotted
after SDS-PAGE and subjected to Western blot analysis with anti-Haspin
rabbit antiserum.
 |
RESULTS |
Structural Characterization of haspin cDNA and Its Deduced
Protein--
Twenty independent cDNA clones have been isolated by
screening 2 × 106 clones from a pAP3neo
mouse testis cDNA library with the 32P-labeled
gsg2 probe that had been previously cloned as a germ cell-specific cDNA (4). All nucleotide sequences of the five independent isolates with inserts of ~2.6 kilobase pairs revealed a
single long open reading frame. As no stop codon was found anywhere upstream of the 5'-coding region, we performed 5'-RACE using a gsg2-specific antisense primer prepared from the sequence
data and isolated five independent clones. Since a stop codon was
located 24 bases upstream of the ATG sequence in all five clones, we
assumed the ATG to be the translation initiation codon of the cDNA
(Fig. 1). The complete nucleotide
sequence and its deduced amino acid sequence are shown in Fig. 1. The
protein consisted of 754 amino acids beginning from the first
methionine at nucleotide 34. A computer-assisted homology search for
the nucleotide sequence revealed that no homologous sequence has ever
been reported. The cDNA sequence was without a poly(A) tail, but
contained a 3'-untranslated region of 532 nucleotides, including two
possible consensus AATAAA polyadenylation signals at nucleotides 2512 and 2788.

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Fig. 1.
Nucleotide and deduced amino acid sequences
of haspin cDNA. The complete open reading
frame was determined by sequencing overlapping cDNA clones and
5'-RACE clones. Underlining indicates the primer sequence
for 5'-RACE (at nucleotides 317-335) and a deleted region (amino acids
447-453) of deletion mutant haspin. The putative
polyadenylation signals are indicated by dots.
Shaded regions indicate a basic amino acid sequence
(residues 76-81), a region homologous to MEF2B (residues 144-162),
and parts of protein kinase consensus sequences (residues 440-498).
The gray box indicates a putative nuclear localization
signal (amino acids 320-324). Double underlining of amino
acids indicates the leucine zipper motif (residues 585-613). Each
series of asterisks indicates a potential target site for
some protein kinase, and one asterisk indicates a stop codon
in the haspin open reading frame.
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The deduced amino acid sequence of the cDNA showed the following
well known motifs (Figs. 1 and
2A): a nuclear localization signal (KKKRK) at amino acids 320-324 (31); a leucine zipper at
residues 585-613 (32); and parts of protein kinase consensus sequences
over the region of amino acids 440-498 (33, 34), which was in
particular homologous to CDC2 kinase (Fig. 2B). It also
contained many potential target sites for protein kinases: phosphorylation sites for protein kinase C (consensus
(S/T)X(R/K)) (35) located at residues 24-26, 338-340,
437-439, 480-482, and 705-707; a cAMP- and
cGMP-dependent protein kinase phosphorylation site
(consensus RKX(S/T)) (36) at residues 323-326; and 11 target sites for casein kinase II (consensus (S/T)XX(D/E))
(37) in Haspin (Fig. 1). It had also a region homologous to murine
MEF2B (myocyte-specific enhancer
factor 2B) (38) at residues 144-162 (Figs. 1
and 2). We named this protein Haspin.

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Fig. 2.
Schematic presentation of major motifs in the
Haspin protein and comparisons of Haspin with the ATP-binding site of
CDC2 kinase and with transcription factor MEF2B. A,
schematic presentation of locations of major motifs present in the
Haspin amino acid sequence. B, comparisons of Haspin and the
deletion mutant of Haspin with the mouse CDC2 kinase catalytic domain.
Roman numerals in A and B indicate
subdomains of the kinase catalytic domain. Underlining also
indicate subdomains of the kinase catalytic domain. C,
comparison of Haspin and mouse MEF2B. Numbers at both ends
indicate amino acids of each protein. A.A., amino
acids.
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Expression of Haspin mRNA and Protein--
Northern blot
analysis showed that haspin mRNA was specifically
detected in the testis as a major transcript of 2.8 kilobases, expressed exclusively in germ cells within the testis, but was not
detectable in somatic tissues such as the brain, heart, intestine, kidney, liver, lung, muscle, and spleen (Fig.
3A). No transcript was
observed in the ovary, either. In both the cryptorchid and mutant mouse
testes, in which there are no differentiated germ cells (5), no
transcript was observed. Western blot analysis with anti-Haspin rabbit
antiserum detected one positive signal with Mr
83,000 specifically in mouse testis extracts (Fig. 3B), consistent with the results of Northern blot analysis. A positive signal with a low molecular weight was also detected in the muscle with
this antiserum. However, the protein was believed to be a cross-reacting molecule since no haspin mRNA was
detected in the muscle by Northern blot analysis. During male germ cell
development, the transcript was not detected in neonatal mouse testis
before 24 days of age. Then, the signal strengthened up to adulthood (Fig. 4A). The chronological
change in the level of the haspin transcript was very
similar to the pattern for protamine mRNA, indicating that its
expression in the testis is haploid germ-cell specific. In the case of
Western blot analysis, Haspin was first detected in the 24-day-old
testis, and the signal gradually increased with age until adulthood.
This result was positively correlated with the mRNA expression
(Fig. 4B). Thus, these results indicate that both
transcription and translation of the haspin gene occur exclusively in haploid male germ cells and that the timing of gene
expression is precisely regulated during the development of male germ
cells.

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Fig. 3.
Expression of haspin
mRNA and protein in various tissues and mutant testes.
A, total RNAs were prepared from various tissues. Testicular
cells were separated into two fractions (Fr.): germ cell and
tubule fractions. The cryptorchid (cryp.) testes and mutant
testes of jsd/jsd, W/Wv, and
Sl17H/Sl17H mice do not have spermatocytes and
more differentiated germ cells after 12 weeks of age. Ten micrograms of
each total RNA sample was electrophoresed, transferred to a nylon
membrane, and hybridized with the haspin cDNA probe.
After exposure, the same filter was rehybridized with the -actin and
protamine cDNAs. Arrows indicate ribosomal RNA
positions. B, ~100 µg of protein of each tissue prepared
from an adult mouse was electrophoresed and electrotransferred to a
membrane filter. Western blot analysis was done with anti-Haspin
antiserum (KKK-1) as described under "Experimental Procedures."
Arrows indicate molecular weights (×103) of
marker proteins. The asterisk indicates the Haspin
protein.
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Fig. 4.
Developmental expression of haspin
mRNA and protein in prepubertal mice testes.
A, 10 µg of total RNA from prepubertal mouse testes was
used for Northern blot analysis. Arrows indicate the origin
(Ori) of electrophoresis and 28 S and 18 S ribosomal RNAs.
B, ~100 µg of testicular protein from mice of different
ages was electrophoresed and electrotransferred to a membrane filter.
Western blot analysis was done with anti-Haspin antiserum (KKK-1) as
described under "Experimental Procedures."
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Immunohistochemical Identification of the Haspin Protein in Male
Germ Cells--
Immunohistochemical examination of Haspin in adult
mouse testis showed that some germ cells stained positively, but
somatic cells such as Leydig and Sertoli cells were negative (Fig.
5, B-D). The expression of
the Haspin protein was first detected in haploid round spermatids at
step 1 (Fig. 5E). However, the accumulating rate gradually
decreased as differentiation proceeded further. In late spermatids at
steps 15 and 16, in which nuclear condensation was almost completed, no
signal was detected. No germ cells present in the first layer of the
seminiferous epithelium (spermatogonia and leptotene and zygotene
spermatocytes) were stained (Fig. 5, B-D). These
observations were in good agreement with the results of Western blot
analysis, indicating that Haspin is a novel differentiation-associated
protein whose expression level peaked at the appearance of haploid
spermatids. Subcellular localization of Haspin was limited to the
nuclei of round (Fig. 5E) and elongated (Fig. 5F)
spermatids. However, upon development of spermatids undergoing
remodeling of the nucleus and acrosome formation, localization of
Haspin appeared to change from the whole nucleus to a limited area of
the nucleus, in the vicinity of the acrosome (Fig. 5F).

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Fig. 5.
Immunohistochemical staining with anti-Haspin
rabbit antiserum. Frozen sections of an adult mouse testis were
stained with anti-Haspin antiserum (HAS-3) using the streptavidin
method as described under "Experimental Procedures." Testicular
sections were treated as follows: control (preimmune rabbit serum;
A) and stained with anti-Haspin antiserum (B-F).
Testicular sections of C and D include round and
elongated spermatids in seminiferous tubules, respectively.
E indicates a blow-up of early spermatids. F was
stained with hematoxylin after being immunostained with anti-Haspin
antiserum. Spermatids in this section can be classified into two types:
round spermatids at steps 2 and 3 and elongated spermatids at steps 14 and 15. Numbers at the top of F indicate
developmental steps of spermatids. Bars = 100 µm
(A-D) and 20 µm (E and F).
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In Vitro Kinase Assay--
Haspin has a core kinase catalytic
domain that includes consensus kinase subdomains I-III, but lacks the
C-terminal consensus subdomains IV-XII. To examine the biochemical
function of Haspin, we investigated its kinase activity. Adult testis
lysates were immunoprecipitated with DDP-2 antiserum. An aliquot of the
immunocomplexes was subjected to in vitro kinase assay in
the presence of Mg2+, Mn2+, and
Ca2+ together with [
-32P]ATP. The
kinase-assayed sample and the remaining immunocomplexes were both
subjected to SDS-PAGE followed by Western blot analysis with KKK-1
antiserum. By using the same polyvinylidene difluoride filter,
phosphorylated proteins labeled with 32P were detected by
autoradiography, and Haspin proteins were detected by immunostaining.
Two prominent bands of Mr 83,000 and 50,000 labeled with 32P were detected (Fig.
6A). These two bands
corresponded to phosphorylated Haspin and the heavy chain of
immunoglobulin, respectively. In addition, one weaker band was also
detected at Mr ~75,000. Because the band was
detected with all three different anti-Haspin rabbit antisera (see
"Experimental Procedures"), it might be a specific substrate
associated with Haspin kinase. Alternatively, it could be a degradation
product of Haspin. To demonstrate that the kinase activity was
intrinsic to Haspin, a deletion of 10 amino acids was introduced into
kinase subdomain I, where ATP is supposed to bind (Fig. 2, A
and B). The expression vectors were constructed to produce
the wild-type and mutant Haspin proteins, each fused with green
fluorescence protein (pEGFP-HASP and pEGFP-dHASP), and were transfected
into HEK-293 cells. The transiently expressed Haspin proteins were
immunoprecipitated with anti-Haspin antiserum (DDP-2). By Western blot
analysis with anti-Haspin antiserum (KKK-1), one major band with
Mr ~110,000 was detected for both the
wild-type and deletion mutant Haspin proteins (Fig. 6B). The
immunoprecipitates of the transfected cell lysates were also subjected
to in vitro kinase assay in the presence of
[
-32P]ATP, followed by Western blot analysis. The
autoradiography results showed no phosphorylation on the deletion
mutant lacking kinase subdomain I. Phosphoamino acid analysis revealed
that both serine and threonine were specifically phosphorylated. Taken
together, these results indicate that Haspin has intrinsic
serine/threonine kinase activity undergoing
autophosphorylation.

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Fig. 6.
In vitro phosphorylation of
immunocomplexes. Extracts of the testis (A) and
cultured HEK-293 cells transfected with the pEGFP-C1 vector carrying
haspin cDNA (B) were immunoprecipitated with
anti-Haspin antiserum (DDP-2). Aliquots of each immunoprecipitated
sample were subjected to SDS-PAGE followed by Western blot analysis
using anti-Haspin antiserum (KKK-1) (Western). The rest was
subjected to in vitro kinase assay followed by SDS-PAGE and
Western blotting and exposed to x-ray film (Kinase).
P, immunoprecipitated with preimmune serum; H,
immunoprecipitated with anti-Haspin antiserum; C, positive
control (testis extract, not immunoprecipitated); W,
wild-type haspin cDNA in the pEGFP-C1 vector;
D, the deletion mutant haspin cDNA in the
pEGFP-C1 vector; M, mock transfection with the pEGFP-C1
vector DNA alone without an insert; IgG, IgG heavy chain.
Arrows on the right indicate molecular weights
(×103) of marker proteins. The results from the
phosphoamino acid analysis of Haspin are shown (C). In
vitro phosphorylation was done as described under "Experimental
Procedures." After hydrolysis of Haspin, phosphorylated amino acids
were analyzed by thin-layer chromatography. The positions of the three
phosphoamino acids are represented schematically in panel a.
The profiles of amino acids reacted with ninhydrin solution in
panel b and of 32P-labeled phosphoamino acids in
panel c are shown.
|
|
Ectopic Expression of Haspin Inhibits Proliferation of HEK-293 and
COS-7 Cells--
To understand the function of Haspin, we have
examined its localization and the effects of its ectopic expression on
cultured cells. When HEK-293 and COS-7 cells were transfected with the cDNA of wild-type haspin (pEGFP-HASP) or deletion mutant
haspin (pEGFP-dHASP), both proteins were localized
exclusively in the nuclei of the cultured cells (Fig.
7), similar to the physiological localization of Haspin in haploid spermatids (Fig. 5). In the cultured
cells, however, localization in Haspin was restricted to some
subnuclear foci, discretely present in a punctate form within the
nucleus (Fig. 7, J and H). Because transfected
cells did not proliferate, we have examined the effect of Haspin on cell cycle progression. Cultured HEK-293 cells were transfected with
wild-type haspin or deletion mutant haspin
cDNA. After an appropriate period of culture, cells were harvested
and separated into two groups, i.e. fluorescence-positive
cells (Fig. 8, peak a) and
fluorescence-negative cells (peak b). The latter negative cells were supposedly the non-transfected cells; thereby, these cells
could be considered as the negative control for the former positive
cells capable of expressing exogenous Haspin. The control cells in both
mutant and wild-type haspin-transfected cultures showed the
normal log phase growth pattern (Fig. 8, A, peak
b; and B, peak b). In contrast, the
fluorescence-positive cells showed cell cycle arrest at G1
phase (Fig. 8, A, peak a; and B,
peak a). Thus, the transfected cells capable of expressing
exogenous Haspin were not able to proliferate, and furthermore, almost
all the cells had detached from the culture dishes 7 days after
transfection. The expression level of deletion mutant Haspin lacking
the kinase activity was similar to that of wild-type Haspin in
transfected HEK-293 cells judging from the fluorescence intensity of
EGFP-fused Haspin (data not shown). Nevertheless, the deletion mutant
induced a quicker response than did the wild type: 2 days after
transfection, all of the HEK-293 cells expressing mutant Haspin had
undergone cell cycle arrest at G1 phase, whereas those with
wild-type Haspin showed cell cycle arrest 6 days after transfection. It
should be pointed out that the negative control EGFP protein spread
over the whole cell (Fig. 7, E, F, K,
and L) and did not affect the cell cycle or growth of the
transfected cells (data not shown).

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Fig. 7.
Localization of the EGFP-Haspin fusion
protein in cultured cells transfected with the cDNAs.
Expression vectors pEGFP-HASP and pEGFP-dHASP were transfected into
COS-7 and HEK-293 cells using calcium phosphate and LipofectAMINE Plus
reagent, respectively. The expression vector pEGFP-C1 alone without
haspin cDNA was used as a control. COS-7 and HEK-293
cells were grown on plastic dishes for 48 h after transfection and
were observed for transient expression of Haspin (A-D,
G, and H), the deletion mutant of Haspin
(I and J), and control EGFP (E,
F, K, and L). The cells were observed
with a Leica inverted fluorescent microscope (DMIRB) under normal light
(A, C, E, G, I,
and K) using Leica fluorescein isothiocyanate filter sets
(B, D, F, H, J,
and L). Photographs of COS-7 (A-F) and HEK-293
(G-L) cells were taken with a PXL KAF1400-G2 digital camera
(Photometrics Ltd.; shutter speed of 1 s) with a Leica K3 blue
filter (original magnification × 400).
|
|

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Fig. 8.
FACS analysis of HEK-293 cells transfected
with the haspin and deletion mutant haspin
cDNAs in expression vector pEGFP-C1. The HEK-293 cells
in the culture dish transfected with cDNA were separated into
transfected and non-transfected cells according to intensities of EGFP
fluorescence on days 2, 4, and 6 after transfection. A, the
cells were subjected to FACS analysis. A gate was set with EGFP
fluorescence (488 nm) to separate transfected HEK-293 cells (peak
a) from non-transfected HEK-293 cells (peak b) in the
same culture dish. B, shown are changes in the DNA content
profiles of transfected (peak a) and non-transfected
(peak b) HEK-293 cells after transfection with the
haspin and deletion mutant haspin
(d-Haspin) cDNAs in the pEGFP-C1 vector.
|
|
DNA-binding Ability of Haspin--
Since both the physiological
localization and ectopic expression of Haspin were restricted to
nuclei, we examined the association of Haspin with nuclear DNA. The
binding affinities of Haspin and mutant Haspin for DNA were compared by
monitoring their elution profiles by DNA affinity column
chromatography. Extracts from the cells transfected with the wild-type
or mutant haspin cDNA were applied to calf thymus native
DNA-cellulose minicolumns, and proteins were eluted stepwise with
increasing concentrations of NaCl. After SDS-PAGE, the EGFP-Haspin
proteins were detected by Western blot analysis with anti-Haspin
antiserum. As shown in Fig. 9, more than
half the amount of the Haspin protein was recovered from the
flow-through fraction, and the rest was retained by the DNA column.
With 0.1 M NaCl, almost all the bound Haspin was eluted. In
contrast, all of the mutant Haspin defective in kinase activity was
able to bind to the DNA column and was eluted with 0.1 M
NaCl. Thus, these results indicate that the mutant Haspin lacking the
autophosphorylation activity has higher affinity for DNA than the wild
type and suggest that Haspin is present in at least two forms in terms
of DNA-binding ability.

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Fig. 9.
Elution of the EGFP-Haspin fusion protein
from a DNA-cellulose column. Supernatants from the HEK-293 cells
transfected with the pEGFP-HASP or pEGFP-dHASP expression vector were
applied to native DNA-cellulose minicolumns and then eluted stepwise
sequentially with 0.1, 0.2, 0.3, 0.4, 0.6, and 1.0 M NaCl.
T, F, and W indicate the applied
sample and the flow-through and washing fractions, respectively. Haspin
in each fraction was visualized by Western blot analysis with KKK-1
antiserum. d-Haspin; deletion mutant Haspin.
|
|
 |
DISCUSSION |
Recently, we have isolated various germ cell-specific cDNAs
from a subtracted cDNA library of the mouse testis (4) and investigated the roles of genes in spermatogenesis. The present study
demonstrated that Haspin is a haploid germ cell-specific nuclear
protein expressed exclusively in spermatids from steps 1 to 14 and
decreasing thereafter as the spermatids maturate. Western blot analysis
using three anti-Haspin rabbit antisera (KKK-1, DDP-2, and HAS-3)
capable of recognizing three different epitopes in the Haspin molecule
showed one positive band of Mr 83,000 exclusively in the testis. In addition, another positive band having a
smaller size was detected in the muscle with KKK-1 antiserum. However,
the protein must be a cross-reactive molecule other than Haspin since
no positive band was detected with the other two antisera (data not
shown) and also no haspin transcript was observed in the
muscle by Northern blot analysis.
Haspin contains several known motifs in the deduced amino acid
sequence. First, a core part of the protein kinase consensus sequence
was found. Eukaryotic protein kinases belong to a very extensive family
of proteins sharing a catalytic core consisting of 12 conserved motifs
common to both the serine/threonine and tyrosine protein kinases (33,
34). One of the motifs involved in ATP binding is conserved in the
middle of the Haspin protein (amino acids 440-498). However, Haspin
has only three consensus motifs out of the 12 conserved kinase motifs.
To examine whether Haspin really has kinase activity or not, we carried
out an in vitro kinase assay. The results suggest that
Haspin has kinase activity and phosphorylates its own serine and
threonine residues. When the ATP-binding site was lost by introducing a
deletion into subdomain I without changing any putative phosphorylation
sites (serine/threonine and tyrosine), Haspin showed no or little
autophosphorylation protein kinase activity, thus indicating that
Haspin has intrinsic protein kinase activity.
Second, Haspin contains many potential target sites for protein
kinases, i.e. potential phosphorylation sites for protein kinase C (consensus (S/T)X(R/K)) (35), casein kinase II
(consensus (S/T)XX(D/E)) (37), and cAMP- and
cGMP-dependent protein kinases (consensus
RKX(S/T)) (36). It is possible that the biological activity
of the phosphorylated form of Haspin is different from that of the
non-phosphorylated form. We have shown that Haspin association with DNA
was different between wild-type and kinase-less mutant Haspin proteins,
of which the former was capable of autophosphorylation and the latter
was not. The interaction with DNA may cause some biological activity,
and such phosphorylation might be involved in the interaction.
Third, a leucine zipper pattern could also be constructed within the
region of amino acids 585-613 using the consensus sequence LX6LX6LX6L
(32), suggesting that Haspin associates with some other proteins to
form protein complexes. Recently, many leucine zipper proteins have
been reported and revealed to play key roles in cell growth and
differentiation (39). In haploid germ cells, a specific cAMP response
element modulatory protein (CREM
) is believed to control some gene
expression (40, 41). Haspin may associate with such a factor to play a
regulatory role in haploid-specific gene expression. Haspin has a
nuclear localization signal (KKKRK) (31) at residues 320-324. However,
when the sequence was deleted, ectopically expressed Haspin was still
observed exclusively in nuclei of COS-7 or HEK-293 cells (data not
shown). Thus, the nuclear localization signal (KKKRK) is unlikely to
play an important role in specific localization of Haspin in spermatid
nuclei. It is possible that the basic amino acid sequence in the
N-terminal region of Haspin functions for the specific localization in
the nucleus. Within the nucleus, Haspin seems to localize in a punctate form, implying its involvement with some subnuclear foci such as nucleoli.
Ectopic expression of Haspin caused cell cycle arrest at G1
phase in transfected HEK-293 and COS-7 cells (Fig. 8) and ES cells (data not shown). The deleterious effect of Haspin, which was not due
to the toxicity of the EGFP tag (42) in these cultured cell lines, was
not due to the kinase activity either since the same or a rather
severer effect was observed with the kinase-negative deletion mutant
Haspin protein. However, when we examined the DNA-binding ability of
Haspin using the DNA-cellulose minicolumn, there was a clear difference
observed between the wild-type and mutant Haspin proteins.
Approximately one-half of EGFP-fused wild-type Haspin was bound to DNA,
but the rest was not and was recovered from the flow-through fraction.
In contrast, EGFP-fused kinase-negative mutant Haspin bound to the DNA
column completely. Thus, wild-type Haspin has heterogeneity for DNA
affinity, possibly by the modification of phosphorylation. The
N-terminal basic region of Haspin might serve as a DNA-binding domain,
and its binding ability might be negatively controlled by
phosphorylation. In this way, autophosphorylation may participate in
the regulation of cell cycle control. Since Haspin caused cell cycle
arrest in somatic cells, it may exert a somewhat similar function in
the testis. It should be noted, however, that Haspin expression was
observed only in the haploid germ cells that were no longer able to
replicate. In conclusion, Haspin may participate in the negative
control of the cell cycle progression of spermatids.
Haspin has a region homologous to MEF2B at residues 144-162 (38) and
similarly has a basic amino acid region at its N terminus. The
mef2B gene encodes a MADS box transcription factor,
which regulates the expression of many muscle-specific genes (43). As
Haspin does not have the MADS box and MEF2B does not have the protein
kinase domain, Haspin should differ in function from MEF2B. However,
Haspin has the basic domain at its N terminus and can bind to DNA.
Thus, it is possible that Haspin is another type of testis-specific
transcription factor or a cell cycle regulatory factor of haploid germ cells.
The haspin gene has been mapped to mouse chromosome 11 (44).
A known mouse mutant locus close to haspin is the ovum
mutant (om), which shows a notable phenotype related to
fertilization (45). Its corresponding gene acting in spermatozoa is the
S element. Haspin might be a kind of germ cell factor. Further studies are now in progress to elucidate the molecular function of Haspin in
testicular germ cell proliferation and differentiation.
 |
ACKNOWLEDGEMENT |
We are grateful to Dr. A. Tanaka for editorial assistance.
 |
FOOTNOTES |
*
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) D87326.
**
To whom correspondence should be addressed. Tel.: 81-6-6879-8338;
Fax: 81-6-6879-8339.
2
Available at http://www.sdsc.edu/.
 |
ABBREVIATIONS |
The abbreviations used are:
CRE, cAMP response
element;
RACE, rapid amplification of cDNA ends;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
TBS, Tris-buffered saline;
EGFP, enhanced green
fluorescent protein;
FACS, fluorescence-activated cell sorter.
 |
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