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J Biol Chem, Vol. 275, Issue 11, 7994-7999, March 17, 2000
From the Department of Pharmacology and Cancer Biology, Duke
University Medical Center, Durham, North Carolina 27710
Ca2+/calmodulin-dependent
protein kinase IV and calspermin are two proteins encoded by the
Camk4 gene. Both are highly expressed in the testis, where
in situ hybridization studies in rat testes have
demonstrated that CaMKIV mRNA is localized to pachytene
spermatocytes, while calspermin mRNA is restricted to spermatids.
We have examined the expression patterns of both CaMKIV and calspermin
in mouse testis and unexpectedly find that CaMKIV is expressed in
spermatogonia and spermatids but excluded from spermatocytes, while
calspermin is found only in spermatids. CaMKIV and calspermin
expression in the testis are stage-dependent and appear to
be coordinately regulated. In germ cells, we find that CaMKIV is
associated with the chromatin. We further demonstrate that a fraction
of CaMKIV in spermatids is hyperphosphorylated and specifically
localized to the nuclear matrix. These novel findings may implicate
CaMKIV in chromatin remodeling during nuclear condensation of spermatids.
Spermatogenesis consists of three phases: mitosis, meiosis, and
spermiogenesis. Spermatogonia comprise the stem cell population around
the periphery of the seminiferous tubules and maintain cell numbers by
mitotic divisions. Unknown signals lead to the commitment of some cells
to differentiate into spermatocytes, which spend the majority of time
in meiotic prophase before undergoing meiosis. Postmeiotic spermatids
are closest to the lumen and differentiate into spermatozoa during
spermiogenesis, a process that includes flagellar formation and nuclear
condensation. Within the seminiferous tubule, there are well
characterized cell associations that define the histological
classification of spermatogenesis in the mouse into 12 distinct stages
(1).
Ca2+/calmodulin-dependent protein kinase IV
(CaMKIV)1 and calspermin
(CaS) are two proteins expressed in the testis and encoded by a common
gene, Camk4. This gene has three promoters; the first two
regulate expression of the Little is currently known about the regulation of CaMKIV in the testis.
We have examined the expression and regulation of CaMKIV and calspermin
in mouse testis. We report that, contrary to predictions based on
studies in rat testis, mouse CaMKIV is expressed in spermatogonia and
spermatids and excluded from pachytene spermatocytes. In contrast,
calspermin is restricted to spermatids as expected. The expression of
CaMKIV and calspermin in seminiferous tubules is
stage-dependent and appears to be coordinately regulated. We further demonstrate that CaMKIV is associated with chromatin and the
nuclear matrix and may be targeted to the latter structure by
phosphorylation. These results may point to a novel function for CaMKIV
in posttranscriptional spermatids.
Antibody Production--
The cDNA for mouse calspermin was
cloned by RT-PCR from mouse testis RNA with the following primers:
sense 5'-TTCCCATGGACACTGCTCAGAAGA-3' and antisense
5'-CTGATCTAGAGGAAGCCAGCTTAG-3'. The sequence was then
subcloned into the NcoI and XbaI sites of the
pET-30c vector (Novagen, Madison, WI) to generate
His6-tagged calspermin. The fusion protein was expressed in
Escherichia coli and purified over a nickel resin according
to the manufacturer's protocol. Purified protein was mixed with
Freund's adjuvant and injected into rabbits to raise a polyclonal
antibody against calspermin (anti-CaS).
Western Blotting--
Testes were homogenized in a buffer with
25 mM Hepes, 1 mM EGTA, 1 mM EDTA,
0.5 mM dithiothreitol, 10% glycerol, 10 µg/ml aprotinin,
1 µg/ml leupeptin, 20 µg/ml trypsin inhibitor, and 0.1 µg/ml
Pefablock. 75 µg of protein were subjected to electrophoresis on a
12% gel, transferred to a polyvinylidene difluoride membrane, and
blocked with 5% milk in Tris-buffered saline with 0.05% Tween 20. Membranes were incubated with anti-CaS (1:5000), anti-CaMKIV Calmodulin Overlay--
75 µg of testis protein were subjected
to electrophoresis on a 12% gel, transferred to an Immobilon membrane
(Millipore Corp., Bedford, MA), and then incubated with 100 mM imidazole (pH 7.0) for 10 min followed by a 40-min
incubation in solution G (20 mM imidazole, pH 7.0, 200 mM KCl, 0.1% bovine serum albumin, 0.02% NaN3, 1 mM CaCl2, 0.05% Tween 20)
(14). The membrane was then incubated in solution G with
125I-calmodulin (specific activity 1 × 106 cpm/ml) for 2 h and washed twice with solution G
for 30 min. Calmodulin binding was detected by autoradiography.
Iodination of CaM was performed with Bolton-Hunter reagent (Amersham
Pharmacia Biotech) as described (15).
Histology/Immunohistochemistry--
Testes were fixed in
Bouin's fixative and paraffin-embedded. 7-µm sections were cut and
stained with periodic acid Schiff-hematoxylin (Poly Scientific, Bay
Shore, NY). For immunohistochemistry, sections were incubated in 3%
H2O2 to block endogenous peroxidase activity, subjected to antigen retrieval by microwaving in 10 mM
sodium citrate, pH 6.0, for 10 min, and incubated with anti-calspermin at 1:200 overnight at 4 °C. Following three washes in
phosphate-buffered saline, sections were incubated with biotinylated
secondary antibody and streptavidin-horseradish peroxidase (Vector
Laboratories, Burlingame, CA), detected with diaminobenzidine (Sigma)
or NovaRed substrate (Vector Laboratories), and counterstained with hematoxylin.
Hormonal Treatment--
23-Day-old male mice were injected
intraperitoneally with FSH administered in the form of 5 IU of
pregnant mare serum gonadotropin (Sigma) each day for 7 days and then
sacrificed. In a separate experiment, hypophysectomized 6-week-old male
mice were obtained from Taconic (Germantown, NY) and sacrificed 7 days postsurgery.
Nonidet P-40 Extraction of Testis Proteins--
Testis proteins
were fractionated by Nonidet P-40 solubility as described by Moroi
et al. (16). Briefly, testes were homogenized in 1 ml of
Nonidet P-40 buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1%
Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin), rotated at 4 °C for 30 min, and centrifuged at
10,000 × g for 30 min. The supernatant was designated
the Nonidet P-40-soluble fraction, and the pellet was resuspended in
100 µl of SDS buffer (25 mM HEPES, pH 7.4, 4 mM EDTA, 25 mM NaF, 1% SDS, 1 mM
Na3VO4, 10 µg/ml leupeptin, 10 µg/ml
aprotinin). Following the addition of 900 µl of Nonidet P-40 buffer,
samples were rotated at 4 °C for 30 min and then centrifuged at
10,000 × g for 30 min. The resulting supernatant was
designated the Nonidet P-40-insoluble fraction.
Dephosphorylation with Chromatin and Nuclear Matrix Preparation--
Isolation of
chromatin and nuclear matrix was performed as described (17) with
modifications. Testes were homogenized in cytoskeletal buffer (CSK; 10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.5% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 25 mM NaF). Following
incubation at 4 °C for 3 min, samples were centrifuged at 5000 × g for 3 min. Chromatin was released by digestion with 1000 units/ml RNase-free DNase I (Roche Molecular Biochemicals) in CSK
buffer for 15 min at 37 °C. Ammonium sulfate in CSK buffer was added
to a final concentration of 0.25 M. Samples were incubated at 4 °C for 5 min and centrifuged. The pellet was extracted with 2 M NaCl in CSK buffer for 5 min at 4 °C and then
centrifuged. The resulting nuclear matrix fraction was solubilized in
urea buffer (8 M urea, 0.1 M
NaH2PO4, 0.01 M Tris, pH 8).
Although the rat and mouse forms of CaMKIV are highly conserved in
their amino termini, they diverge significantly at the carboxyl termini
(18). To characterize the patterns of CaMKIV and calspermin expression
in the mouse, we cloned and expressed mouse calspermin as a His-tagged
fusion protein (his-CaS; Fig. 1A, lanes
1 and 2). Purified His-tagged calspermin is
recognized at high concentrations by an antibody raised against rat
calspermin (lane 3) and binds
125I-CaM on overlay (lane 4),
confirming the presence of a functional calmodulin binding domain in
the recombinant protein. This protein was used to raise a polyclonal
rabbit antibody that detects both CaMKIV and calspermin (Fig.
1B).
We used the polyclonal rabbit antibody to examine the
age-dependent expression patterns of CaMKIV and calspermin
in the mouse testis during postnatal development. The first wave of
spermatogenesis after birth proceeds synchronously. For the first two
postnatal weeks, seminiferous tubules are populated only by Sertoli
cells and spermatogonia, with pachytene spermatocytes appearing on day 14 and round spermatids on day 21 (19). We unexpectedly found that
CaMKIV protein is already expressed at day 9, although at low levels
(Fig. 1C, upper panel). The appearance
of CaMKIV in the testis occurs earlier than would be expected for
localization in pachytene spermatocytes and suggests that CaMKIV may in
fact be expressed instead in spermatogonia. CaMKIV protein levels first peak at day 15, with a second, more pronounced increase after day 25. In contrast, calspermin protein does not appear until day 25, as
predicted if expressed in postmeiotic spermatids (Fig. 1C,
lower panel). Western blotting of testis extracts
from germ cell-deficient W/Wv mice, which contain only
Sertoli cells, confirmed that CaMKIV is restricted to germ cells (Fig.
1D).
To examine the cell types expressing CaMKIV and calspermin, we
performed immunohistochemical analysis of testes from mice of varying
ages. Positive staining can be seen in numerous cells at the periphery
of the seminiferous tubule by day 8, when Western blot results indicate
that only CaMKIV is expressed (Fig.
2A). Since CaMKIV is not
expressed in Sertoli cells, these cells must be spermatogonia. Sections
incubated with preimmune serum or with antibody preabsorbed with
antigen show no positive staining of spermatogonia (Fig. 2B
and data not shown). By day 15, CaMKIV-positive cells are clearly
restricted to peripheral spermatogonia, with no staining visible in
newly emerging pachytene spermatocytes (Fig. 2C).
Immunostaining of testes at day 25, when calspermin is first expressed
as determined by Western blotting, revealed the appearance of positive
staining in round spermatids as they reached stages IV and V,
confirming that calspermin is produced in haploid postmeiotic germ
cells (Fig. 2D). The adult pattern of immunohistochemical
staining was established by day 30 (Fig. 2E). Interestingly,
there is a clear stage dependence in staining of both spermatogonia and
spermatids. Both of these cell types first stain positive at stages IV
and V and maintain expression through stages VIII and IX (Fig.
2F). These results establish a novel cellular localization
for CaMKIV as well as an apparently coordinated
stage-dependent pattern of expression for both CaMKIV and
calspermin.
Ca2+/Calmodulin-dependent Protein Kinase
IV Is Expressed in Spermatids and Targeted to Chromatin and the Nuclear
Matrix*
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and 
isoforms of CaMKIV, while the
third promoter lies within the 10th kinase intron and controls transcription of calspermin (2-4). CaMKIV is a multifunctional enzyme
dependent on both calcium and calmodulin for activity (2, 3), while
calspermin consists of the COOH-terminal 164 amino acids of CaMKIV,
including the calmodulin binding domain (5). CaMKIV is expressed in
several tissues in addition to the testis, including brain and thymus
(2), whereas calspermin is found only in the testis (5). Within the
testis, the mRNAs encoding CaMKIV and calspermin have distinct
expression patterns. In situ hybridization studies in rat
testis have demonstrated that Camk4 mRNA is expressed
within pachytene spermatocytes (2). On the other hand, calspermin
mRNA is localized to postmeiotic round spermatids (2, 5). The
physiological functions of neither protein are well understood. Several
reports have suggested that CaMKIV plays a role in transcriptional
regulation in lymphocytes and neurons (6-10). In the testis, CaMKIV
has been proposed to regulate calspermin expression by phosphorylation
and activation of the testis-specific transcription factor CREM
,
which in turn drives transcription of several germ cell genes,
including calspermin, whose promoters contain cyclic AMP-responsive
elements (4, 11, 12). Calspermin has no kinase activity and instead may be involved in regulating high levels of calmodulin found in germ cells
(13).
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(1:1000) (kindly provided by Dr. Hiroyuki Sakagami, Tohoku University School of Medicine, Sendai, Japan), anti-CaMKK (1:1000) (Transduction Laboratories, San Diego, CA), or anti-lamin B (1:1000) (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), detected with horseradish peroxidase-conjugated antibody, and developed with the ECL
chemiluminescence system (Amersham Pharmacia Biotech).
-Phosphatase--
50 µg of testis
protein were incubated with 100, 400, or 1000 units of phosphatase
(New England Biolabs, Beverly, MA) for 1 h at 30 °C.
Phosphatase activity was inhibited by the addition of 50 mM EDTA.
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View larger version (35K):
[in a new window]
Fig. 1.
Expression of mouse calspermin and production
of anti-calspermin antibody. A, purification and
detection of mouse calspermin. His-tagged mouse calspermin
(his-CaS) was cloned and expressed in E. coli as
described under "Experimental Procedures." Bacterial cell lysate
(lane 1) or purified His-tagged calspermin
(lanes 2-4) was resolved by SDS-PAGE and
subjected to Coomassie staining (lanes 1 and
2), Western blotting with an antibody against rat calspermin
(lane 3), or calmodulin overlay with
125I-CaM (lane 4). B,
His-tagged calspermin was injected into rabbits to generate a
polyclonal antibody that detects purified His-tagged calspermin protein
(37 kDa; lane 1), and both CaMKIV (65 kDa) and
calspermin (34 kDa) in testis lysates (lane 2).
Characterization of CaMKIV expression in the testis is shown.
C, age-dependent expression of CaMKIV
(upper panel) and calspermin (lower
panel) in mouse testes. Testis homogenates from mice of
varying ages were immunoblotted for the presence of CaMKIV and
calspermin. D, CaMKIV is expressed only in germ cells.
Testis lysates were prepared from wild-type (+/+) and germ
cell-deficient (W/Wv) mutant mice and immunoblotted for
expression of CaMKIV.
View larger version (168K):
[in a new window]
Fig. 2.
Developmental stage-specific expression of
CaMKIV and calspermin in mouse testis. Testes were isolated from
8-day-old (A and B), 15-day-old (C),
25-day-old (D), and 30-day-old (E) mice and
analyzed by immunohistochemistry as described under "Experimental
Procedures." Expression of CaMKIV is restricted to spermatogonia in
mice less then 25 days old (arrowheads in A and
C). No staining is seen in tubules incubated with antibody
preabsorbed with antigen (B). Expression of calspermin is
first detected in haploid stage IV and V round spermatids at day 25 (arrow in D). Stage-specific CaMKIV and
calspermin expression is evident in adult testes (E) and is
diagrammed in F (modified from Ref. 1). Striped
bar, CaMKIV expression; dotted bar,
calspermin expression. A-E, scale
bar, 50 µm.
There is a significant increase in CaMKIV protein levels after day 25 (Fig. 1C). This is coincident with the time when mice begin to attain sexual maturity and may simply reflect the increase in germ cell numbers at puberty. However, there is a surge in gonadotropin levels at this time as well, raising the possibility that CaMKIV might be hormonally regulated. Injections of a peptide with FSH action can induce early puberty in mice (20). To determine whether repeated administrations of FSH could increase CaMKIV levels in the testis, 23-day-old mice were injected with 5 IU of FSH every day for a week. Testis lysates from hormone-treated and control mice were then immunoblotted for CaMKIV, with no detectable change in CaMKIV levels (data not shown). Hypophysectomy has also been used to study hormonal effects on gene regulation in rat testis (21, 22). Postpubertal mice were hypophysectomized and sacrificed 1 week later. Again, no change in CaMKIV levels was evident (data not shown). These results indicate that CaMKIV is not acutely regulated by hormonal stimuli.
Another explanation for the observed increase in CaMKIV levels in
postpubertal mice would be the expression of CaMKIV in a new population
of cells. Although immunohistochemistry results confirm that CaMKIV is
expressed in spermatogonia while calspermin is found in spermatids,
they do not rule out the possibility that CaMKIV is also expressed in
spermatids, since the antibody cannot distinguish between the two
proteins. To address this question, we turned to germ cell
fractionation, in which relatively pure populations of germ cells can
be isolated by passing them through a density gradient (23). Western
blotting of isolated pachytene spermatocyte, round spermatid, and
condensing spermatid fractions demonstrated that CaMKIV is indeed
expressed at high levels in round and condensing spermatids but is
completely absent in pachytene spermatocytes (Fig.
3A). Therefore, contrary to
in situ hybridization studies to detect Camk4
mRNA in rat testis, our results establish that mouse CaMKIV protein
is excluded from pachytene spermatocytes but expressed in both
spermatogonia and elongating spermatids.
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Proteins expressed in elongating spermatids are frequently subjected to
translational regulation. For instance, the protamine genes are
transcribed in round spermatids and then stored for 1 week as
cytoplasmic ribonucleoprotein particles before being translated in late
elongating spermatids (24). More extensive analysis has demonstrated
that the increase in CaMKIV protein levels occurs at approximately day
27 (Fig. 4B and data not
shown). If CaMKIV were translationally regulated, one might predict an up-regulation of mRNA levels before the increase in protein levels at day 27. Northern blot analysis of testis RNA from mice of increasing ages revealed that there is a significant increase in mRNA levels at day 27, along with an apparent decrease in the length of the CaMKIV
message (Fig. 3B). The increase in mRNA levels occurs
concomitantly with the increase in CaMKIV protein, indicating that
CaMKIV expression in spermatids is not translationally regulated.
Likewise, calspermin mRNA appears on day 27, the same day at which
calspermin protein is first expressed.
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By immunohistochemical staining at higher magnifications, CaMKIV appears to be present in the nucleus of both spermatogonia and spermatids, where CaMKIV might be expected to localize if it is involved in transcriptional regulation. The acidic COOH terminus of CaMKIV may be characteristic of chromatin-associated proteins (25). To determine whether CaMKIV might be bound to chromatin, we first examined whether CaMKIV in a testis lysate is resistant to detergent extraction (16). CaMKIV is present in both Nonidet P-40-soluble and -insoluble fractions (Fig. 4A). Furthermore, it exists as a doublet in both, although the upper band runs with slower electrophoretic mobility in the Nonidet P-40-insoluble fraction. Western blotting of testis lysates from mice of varying ages revealed that the higher band in the insoluble fraction does not appear until day 27 (Fig. 4B), suggesting that this might represent a spermatid-specific pool of CaMKIV. In support of this possibility, the upper band comigrates with a band of higher electrophoretic mobility seen in purified elongating spermatids (Fig. 3A and data not shown). The appearance of the higher band accounts for the increased protein levels after day 25 seen in Fig. 1C.
The doublet bands seen in both Nonidet P-40-soluble and -insoluble
fractions might represent different CaMKIV isoforms or posttranslational modification. Two isoforms of CaMKIV have been identified in the cerebellum, with the larger -isoform distinguished by a 28-amino acid NH2-terminal extension (26, 27). A
Western blot was performed with an antibody specific for the CaMKIV
isoform to determine whether the two bands of CaMKIV seen in the testis correspond to the two different isoforms. Although CaMKIV is clearly
expressed in the cerebellum, there is no discernible expression in the
testis, suggesting that the upper band is not the CaMKIV isoform
(Fig. 4C). Activation of CaMKIV requires phosphorylation by
an upstream Ca2+/calmodulin-dependent protein
kinase kinase (28, 29). To establish whether the upper band represents
a posttranslational modification of CaMKIV, testis lysates were
treated with increasing concentrations of -phosphatase (Fig.
4D). This resulted in the collapse of the doublet into a
single band of faster electrophoretic mobility, indicating that the
electrophoretic mobility shift is due to phosphorylation. This shift
could be prevented by the addition of 50 mM EDTA to inhibit
the phosphatase (Fig. 4D, lane 5).
To determine whether CaMKIV is indeed associated with chromatin, the
detergent-insoluble pellet was treated with DNase I followed by 0.25 M ammonium sulfate to release chromatin-bound proteins (17). The remaining pellet was then washed with 2 M NaCl
and resuspended in urea buffer to obtain the nuclear matrix. Western blotting of these fractions demonstrated that CaMKIV is associated with
both chromatin and the nuclear matrix (Fig.
5A, upper
panel). The presence of nuclear matrix proteins was
confirmed by blotting for lamin B, a matrix-specific marker (Fig.
5A, lower panel) (27). Interestingly,
the CaMKIV present in each fraction appears to be differentially
phosphorylated, with the higher band present only in the nuclear
matrix. To examine whether this might be spermatid-specific hyperphosphorylated CaMKIV, testes from mice at different ages were
fractionated and blotted for CaMKIV. CaMKIV is not present in the
nuclear matrix until day 27 (Fig. 5B, lane
12), although it can be found in the chromatin at any age
(Fig. 5B, lanes 2, 6, and
10). These results suggest that at least a portion of CaMKIV expressed in elongating spermatids is hyperphosphorylated and targeted
to the nuclear matrix.
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These studies have focused on the characterization of CaMKIV regulation and expression patterns in mouse testis. They have led to the unexpected finding that CaMKIV is not expressed in pachytene spermatocytes as had been predicted based on previous studies in rat testis (2). CaMKIV is instead expressed initially in spermatogonia, and then again in elongating spermatids. There are several potential explanations for the observed differences. One reason is that conclusions about CaMKIV localization in rat testis were based only on in situ hybridization to detect mRNA, whereas our experiments have examined the localization of the protein in mouse. It is possible that the mRNA is synthesized early in spermatocytes, then stored until the spermatid stage for translation, as occurs with the protamines (24). However, although Camk4 mRNA is already present at day 9 in mouse testis, there is a significant increase in mRNA level at day 27 coincident with the appearance of CaMKIV in spermatids, suggesting that CaMKIV expression in spermatids is not translationally regulated. This increase is accompanied by a reduction in the length of the Camk4 mRNA. Human testis has been reported to have CaMKIV mRNAs of several different lengths (30). The decrease in length may be due to use of an alternate polyadenylation site, shortening of the poly(A) tail, or perhaps even alternative splicing. In the rat brain, use of alternate polyadenylation sites results in CaMKIV mRNAs of differing lengths (31). Another explanation for the discrepancy in expression patterns is that there may be some species differences in CaMKIV expression patterns. Further studies on the localization of CaMKIV protein in rat testis will be required to clarify this issue.
We have also found that CaMKIV expression is
stage-dependent in the adult mouse testis and appears to be
coordinated with that of calspermin. To our knowledge, this is the
first demonstration of coordinated stage-dependent
expression for two proteins encoded by the same genetic locus. In the
testis, germ cell-specific isoforms of proteins are often produced by
the use of alternative promoters and/or splicing (32, 33). However,
CaMKIV and calspermin are unusual in that the proteins are functionally
distinct and both are expressed in the testis. Intriguingly, several
examples of functionally unrelated gene products involve other
calmodulin-dependent protein kinases. These include three
proteins lacking kinase activity, telokin/KRP (34), KAP (35), and
CARP (36), derived from the loci encoding myosin light chain kinase,
CaM-dependent protein kinase II, and
CaM-dependent protein kinase VI, respectively. The
coordinated expression of CaMKIV and calspermin in stages IV through IX
suggests a common regulation, although the mechanism is not clear.
However, the expression of calspermin is not dependent on CaMKIV, since
calspermin production is unaffected in mice lacking CaMKIV.2
The appearance of CaMKIV in elongating spermatids is temporally
coincident with the increase in CaMKIV protein and mRNA levels at
day 27 as well as the appearance of a slower migrating band of CaMKIV
resistant to detergent extraction. This shift in electrophoretic mobility is due to phosphorylation, since the testis is devoid of the
CaMKIV isoform and phosphatase treatment of testis lysates results
in the presence of only a single band of faster electrophoretic migration. Taken together, these findings suggest the presence of a
previously unidentified fraction of hyperphosphorylated CaMKIV in
elongating spermatids. This hyperphosphorylated CaMKIV is exclusively targeted to the nuclear matrix.
The nature of the phosphorylation events targeting CaMKIV to the
nuclear matrix has not been elucidated. Activation of CaMKIV is thought
to require a unique three-step mechanism: 1) binding of
Ca2+/CaM, 2) phosphorylation of Thr196 by
Ca2+/calmodulin-dependent protein kinase
kinase, and 3) autophosphorylation of Ser12 and
Ser13 in the amino terminus (8). Expression of CaMKIV in
BJAB cells (18) or rabbit reticulocyte lysates (8) results in the
appearance of two closely migrating bands. Mutations that abrogate
kinase activity abolish the upper band (8), suggesting that
phosphorylation of one or more of these sites may be responsible for a
shift in electrophoretic mobility. There is also a phosphorylated band in the soluble CaMKIV fraction that migrates somewhat faster than phosphorylated detergent-insoluble CaMKIV and may reflect
phosphorylation on fewer or perhaps alternate sites. Identification of
the target residues in each case may offer further insights into the
regulatory mechanisms governing CaMKIV expression and localization in
germ cells.
The novel finding of CaMKIV expression in elongating spermatids raises
the question of the function of CaMKIV in these cells. In lymphocytes
and hippocampal neurons, there is evidence to suggest that CaMKIV plays
a role in cyclic AMP-responsive element-binding protein-mediated
transcription (6-10). This led to the proposal that in the testis
CaMKIV has a similar function in regulating CREM, another member of
the B-ZIP family of transcription factors (11). However, the results we
report here cast doubt on the role of CaMKIV as a physiologically
relevant activator of CREM. CaMKIV is not expressed in pachytene
spermatocytes or early round spermatids, where it might colocalize with
CREM. In addition, CaMKIV is not produced until spermatids begin to
elongate, after transcriptional activity has ceased (37). Indeed, we
have recently demonstrated that cyclic AMP-responsive
element-dependent gene transcription is intact in mice with
a targeted deletion of CaMKIV.2
Some clues to the function of CaMKIV in spermatids might be found in
its localization to the chromatin (an observation previously noted in
neurons by Jensen et al. (42)) and nuclear matrix. One of
the hallmarks of spermiogenesis is nuclear condensation, during which
DNA is compacted by the sequential replacement of histones by
transition proteins and protamines (38). DNA packaging in sperm differs
from that of other cell types in that sperm DNA is organized into
linear, side-by-side arrays rather than into nucleosomes as found in
somatic cells (39). These linear arrays allow for greater packing of
DNA, with the result that sperm DNA is 6-fold more condensed than DNA
in mitotic chromosomes (39, 40). Of particular note is that CaMKIV
appears to be targeted to the nuclear matrix only in elongating
spermatids. In mammalian sperm nuclei, the nuclear matrix is believed
to organize DNA further into loop domains (41). The localization of
CaMKIV to the nuclear matrix may indicate its involvement in chromatin
remodeling. This proposed role for CaMKIV is supported by the finding
that spermiogenesis is abrogated in CaMKIV-deficient mice, with
impaired chromatin condensation and disruption of protamine 2 in late
spermatids.2 These results point to a novel function for
CaMKIV in mammalian spermatids.
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ACKNOWLEDGEMENTS |
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We are grateful to D. O'Brien for providing
purified germ cell fractions and to H. Sakagami for the anti-CaMKIV
antibody. We thank E. M. Eddy, X. F. Wang, Y. Zhuang, E. Linney, and S. Wu for helpful discussions.
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FOOTNOTES |
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* This work was supported by a National Institutes of Health (NIH) Medical Scientist Training Program award (to J. Y. W.) and NIH Grant HD07503 (to A. R. M.).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.
To whom correspondence should be addressed. Tel.: 919-681-6209;
Fax: 919-681-8461; E-mail: means001@mc.duke.edu.
2 J. Y. Wu and A. R. Means, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CaMKIV, CaM-dependent protein kinase type IV; CaS, calspermin; CREM, cyclic AMP-responsive element modulator; FSH, follicle stimulating hormone; CSK, cytoskeletal buffer; Pipes, 1,4-piperazinediethanesulfonic acid.
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