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Departments of Genetics and Development (D.J.W.) and Obstetrics and Gynecology (D.J.W.), Center for Reproductive Sciences (Q.Z., X.W., D.J.W.), The Herbert Irving Comprehensive Cancer Center (D.J.W.), and Institute of Human Nutrition, Columbia University College of Physicians and Surgeons (D.J.W.), New York, New York 10032
Address all correspondence and requests for reprints to: Debra J. Wolgemuth, Ph.D., Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: djw3{at}columbia.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three D-type cyclins, designated D1, D2, and D3, have been identified to date. Mouse cyclins D1 and D2 were first identified in a screen for delayed early response genes induced by colony-stimulating factor-1 in late G1 phase of mouse macrophage cell lines (2), and all three D-type cyclins can be detected in fibroblast cell lines (3). The three mouse D-type cyclins are more highly related to their human counterparts than to one another (1, 4). The various D-type cyclins may have specialized functions in particular cells, as each accumulates with distinct kinetics during cell cycle progression after mitogen challenge (2, 5), and some distinct in vivo tissue-specific expression patterns have been shown (6, 7).
Recent molecular genetic studies are providing insight into the specific and overlapping in vivo functions of the D-type cyclins, particularly in reproductive tissues. Cyclin D1-deficient mice were viable and fertile; however, they were consistently smaller in size than wild-type littermates and showed defects in the retina and breast (8, 9). Cyclin D2-deficient female mice were sterile, due at least in part to the inability of ovarian granulosa cells to proliferate normally in response to FSH (10). The males were fertile, but displayed hypoplastic testes. Elevated levels of cyclin D2 messenger RNA (mRNA) have been demonstrated in some human ovarian and testicular tumors (10).
The interaction of a regulatory cyclin component with its catalytic Cdk subunit is required for the activation of the kinase. The D-type cyclins have been reported to form complexes, at least in vitro, with Cdk2, Cdk4, Cdk5, and Cdk6 (11, 12, 13). The D-type cyclins appear to exhibit some level of tissue specificity with regard to their associating Cdks and potentially other proteins as well. For example, Cdk4 is a prominent partner for cyclin D1 in macrophages and fibroblasts (14) and for cyclin D3 in differentiating myotubes (15). In one of the few studies to attempt to address the question of the partners of cyclin Ds in vivo, Cdk6 has been reported to be the preferred partner for all three members of D-type cyclins in human peripheral T cells (13).
Proteins termed CDK inhibitory proteins can also bind to cyclin-Cdk complexes. In mammals there are two known groups of CDK inhibitory proteins, the INK4 proteins and the Kip/Cip proteins, which are comprised of p27, p21, and p57 (16). Kip/Cip proteins can inhibit each of the cyclin-CDK complexes essential for G1 progression and S phase entry, although p27 interacts strongly with D-type cyclins and Cdk4 in vitro and more weakly with cyclin E and Cdk2 (17).
Like the other D-type cyclins, cyclin D3 is expressed during G1 phase in dividing cells, but much less is known regarding its expression, activity, and regulation. Cyclin D3 is located on human chromosome 6p21, a region known to be amplified in over 70% of retinoblastoma tumors (18). The 6p21 region has been reported to be rearranged in several lymphoproliferative disorders (18, 19). A role for cyclin D3 in maintaining cells in the terminally differentiated state has been suggested by the observation that it is highly expressed in differentiated myotubes (15).
In this study, we have pursued aspects of the expression and function of cyclin D3 in in vivo systems where lineage specificity and cell cycle status can be assessed histologically, namely murine oogenesis and spermatogenesis. Cyclin D3 was shown to be expressed in mitotically dividing cells at early stages of testicular development and subsequently to be expressed predominantly in terminally differentiated cells in the adult testis. In the ovary, the highest level of cyclin D3 was detected in the nuclei of growing oocytes of smaller follicles. The levels of nuclear localized cyclin D3 appeared to decrease during follicular development. These observations suggested that cyclin D3 can play a role in both cell proliferation and differentiation. We extended this analysis at the cellular level to identify proteins interacting with cyclin D3 in the gonads. We found that cyclin D3 coimmunoprecipitated with Cdk4 and p27 in both testicular and ovarian lysates. At the cellular level, cyclin D3 colocalized with Cdk4 and p27 in some, but not all, cell types in the testis and ovary. In vitro kinase activity of cyclin D3 on histone H1 was detected at the highest levels in day 7 postnatal (pn) testicular lysates and was observed to decrease during development. Cyclin D3 kinase activity was also found in TM3 cells and in immature ovaries, but was undetectable in lysates from adult testis and ovary or in TM4 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 15P-1 (20), TM3, and TM4 cell lines (21) were grown to 70–80% confluence in 100-mm plates in DMEM with 10% FCS in 7.5% CO2 at 32 C for 15P-1 and in 5% CO2 at 37 C for TM3 and TM4.
Sources of reagents
Affinity-purified rabbit polyclonal antibodies to Cdk4
(catalogue no. sc-260), Cdk5 (catalogue no. sc-173), and p27 (catalogue
no. sc-528) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used at 2–4 µg/ml. Human Cdk2
antibody (catalogue no. 06–148) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and used at 5 µg/ml.
Cyclin D3 antibody that had been raised against full-length recombinant
cyclin D3 fusion protein (22) was a gift from Dr. Aubrey Thompson and
was used at a 1:300–500 dilution.
Tissue preparation and immunohistochemistry
Tissue samples were fixed in 4% buffered paraformaldehyde,
embedded in paraffin wax, and processed by our standard methods (23).
The avidin-biotinylated-peroxidase complex detection system was used
for immunocytochemistry (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). Before incubation with the
primary antibody, rehydrated sections were first microwaved for 10 min
in 0.01 M citric acid and then washed twice with double
distilled water (24). Endogenous peroxidase activity was abolished by
incubating sections in methanol containing 0.3% peroxide for 20 min.
After washing with 1 x PBS with 0.01% Triton X-100 (PBST),
sections were preincubated with blocking solution (3% normal goat
serum in 1 x PBST) for 1 h at room temperature and then
incubated with primary antibody at 4 C overnight. The sections were
rinsed three times with 1 x PBST, followed by incubation with
biotinylated horse antimouse IgG (1:200) for 2 h at room
temperature. After washing, the sections were incubated in ABC reagent
in 1 x PBST for 2 h at room temperature, followed by washing
in 1 x PBST. Sections were then equilibrated in 0.1 M
Tris, pH 7.2, for 5 min. Immunostaining was visualized using 0.2 mg/ml
diaminobenzadine and 0.01% hydrogen peroxide in 0.1 M
Tris, pH 7.2. The sections were counterstained with hematoxylin and
coverslipped using Protexx mounting medium (Baxter Diagnostics, Inc.,
Deerfield, IL). For controls, the slides were incubated with normal
rabbit IgG or preimmune serum instead of primary antibody. The
specificity of the antiserum for detecting cyclin D3 was assessed by
competing the antiserum with the fusion protein against which it was
raised (22). Slides were viewed on a Leitz photomicroscope
(Rockleigh, NJ) under brightfield optics, and photomicrographs were
taken with Fuji 100 film (Fuji Photo Film Co., Ltd.,
Elmsford, NY).
Immunoprecipitation and immunoblotting
Fresh or frozen tissues or cultured cells were treated with
lysis buffer containing protease inhibitors [50 mM Tris
(pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.15% SDS, 0.1 mM sodium vanadate, 1
mM dithiothreitol, 20 mM ß-glycerophosphate,
2 mM EDTA, 1 µg/ml aprotinin, 100 µg/ml
phenylmethylsulfonylfluoride, and 10 µg/ml leupeptin). The lysates
were cleared by centrifugation at 14,000 rpm for 10 min at 4 C. The
protein concentrations of lysates were determined by the Bradford dye
assay (25). Equal amounts of proteins from each sample were used for
immunoprecipitation and in vitro histone H1 kinase
analyses. The lysates were incubated with antibodies to Cdk2, Cdk4,
Cdk5, and cyclin D3 for 2 h at 4 C. Controls were incubated with
normal rabbit IgG or preimmune serum. Immune complexes were then
collected by the addition of protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ). Immunoprecipitated proteins on beads
were washed three times with lysis buffer. For immunoprecipitation
assays, an equal amount of 1 x sample buffer (26) was added to
the beads of each sample. Samples were denatured at 100 C for 2 min
before SDS-PAGE. After electrophoresis, the gel was blotted onto
nitrocellulose membrane. Blots were blocked in 6% nonfat milk in
Tris-buffered saline, incubated with primary antibodies specific for
cyclin D3 or Cdk4 for 1–2 h, washed, and then incubated with a 1:3000
dilution of horseradish peroxidase-conjugated secondary antibody
(Boehringer Mannheim, Indianapolis, IN). An enhanced
chemiluminescense kit (ECL Kit, Amersham, Arlington
Heights, IL) and autoradiography were used to detect immune
complexes.
In vitro kinase assays
Histone H1 kinase activity was assayed essentially as described
by Matsushime (27) and Chapman and Wolgemuth (28). The
immunoprecipitated proteins on beads were equilibrated in kinase buffer
[50 mM Tris (pH 7.5), 10 mM MgCl, 1
mM dithiothreitol, 20 mM EGTA, 0.1
mM sodium vanadate, and 80 mM
ß-glycerophosphate] and collected by centrifugation. Histone H1
kinase reactions were performed in kinase buffer with the addition of
0.1 mCi/ml [
-32P]ATP (6000 Ci/mM), 10
µM ATP, 50 µg/ml calf thymus histone kinase H1
(Boehringer Mannheim, Indianapolis, IN), and 5
µM cAMP-dependent protein kinase inhibitor (Sigma Chemical Co., St. Louis, MO) at 30 C for 30 min. An equal amount
of 2 x sample buffer (26) was added to each sample. Samples were
denatured at 100 C for 2 min before SDS-PAGE. After electrophoresis,
gels were fixed, dried, and exposed to Kodak XAR film (Eastman Kodak Co., Rochester, NY).
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Results |
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Initially, immunoblot analysis was used to determine the presence of
cyclin D3 protein in testes at different stages of postnatal
development, since both the proportion of actively dividing to
nondividing cells as well as the presence of specific cell types change
during postnatal testicular differentiation. The 33-kDa cyclin D3
protein was expressed most abundantly in more immature (day 7) testis,
which contains mitotically dividing somatic cells and spermatogonia,
and was shown to decrease during development (Fig. 1A
).
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). The level of cyclin D3 was relatively
higher in equivalent amounts of cellular proteins from the cell lines
compared with the adult testis (Fig. 1B).
To more precisely define the cellular and subcellular
localization of cyclin D3 in the testis, immunohistochemistry was
performed. In the testis, it is possible to recognize the particular
cell cycle stage of many of the cells based on morphological criteria
and their characteristic patterns of association within the
seminiferous tubule (30; reviewed in Ref. 31). In addition, there are
changes in the proportion of types of cells present and their mitotic
and meiotic stages during postnatal development. Cyclin D3-specific
localization was detected in nuclei of some spermatogonia in testis on
day 2.5 pn and day 17 pn development (Fig. 2, B and C, arrows) and at
even higher levels in the cytoplasm of elongating spermatids in adult
testis (Fig. 2D, curved arrow). Cyclin D3 was also detected
in pachytene spermatocytes in day 17 pn testis (Fig. 2C, asterisks). Cyclin D3 was strongly expressed in nuclei of
Leydig cells in adult testis (Fig. 2D, white arrows) and
also in somatic cells in the embryonic testis (Fig. 2A, white
arrows). There was also some weak staining in the nuclei of
Sertoli cells in day 17 pn and adult testis (Fig. 2, C and D,
arrowheads). No cyclin D3 was found in germ cells of
embryonic day 18.5 testis (Fig. 2A).
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). As a control for the specificity of the
competition reaction, cyclin D1 protein was used to examine whether it
could interact nonspecifically with cyclin D3 antibody and inhibit
subsequent binding to cyclin D3. As noted in Fig. 2F, cyclin D1 did not
block cyclin D3 antiserum binding to its specific antigen.
Distinct patterns of cyclin D3 expression during oogenesis
Development of the female germ line has a very different
temporal progression from that of the male (reviewed in Ref. 31). In
most mammals, the entire population of oogonia multiplies mitotically
during embryonic periods and differentiates into oocytes, which are
arrested in meiotic prophase at birth. At puberty, under hormonal
regulation, the growth of a few oocytes and follicles is resumed. The
growth phase of follicle development in the mouse has been well
characterized and divided into stages based on the size of the oocyte
and the number of granulosa cells surrounding the oocyte (32).
To ask whether cyclin D3 could function during oogenesis, immunoblot
analysis was used to show that cyclin D3 was indeed expressed in the
ovary (Fig. 1B). To localize cyclin D3 to particular ovarian cells,
ovaries during embryonic and postnatal development were examined by
immunohistochemistry. During embryonic ovarian development,
localization of cyclin D3 was limited to the somatic cells (Fig. 3A, arrowheads). Cyclin D3 was
not found in the germ cells during embryonic development (Fig. 3A, white arrows), but was found in oocytes as early as day 2.5
pn (Fig. 3B, black arrows). In day 2.5 pn ovary, cyclin D3
localization was observed in nuclei of both granulosa cells and oocytes
in primordial follicles (Fig. 3B, black arrow). The level of
cyclin D3 expression in nuclei of granulosa cells decreased early
during follicular development, with little cyclin D3 detected in nuclei
of granulosa cells of small follicles on day 4.5 pn (Fig. 3C). In adult
ovary, cyclin D3 was detected in nuclei of some luteal cells (Fig. 3D, white arrowheads). The low level of cytoplasmic staining of
cyclin D3 in granulosa and luteal cells was probably nonspecific, since
staining was observed after the antibodies were blocked with
antigen (Fig. 3F). During the progression of oocyte and follicular
growth in adult ovaries, the level of cyclin D3 in the nuclei of
oocytes decreased (Fig. 3, C and D). The levels of cyclin D3 were
almost undetectable in the nuclei of oocytes of preovulatory follicles
(Fig. 3E, black arrow).
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To determine whether Cdk4 could serve as a catalytic partner for cyclin
D3 and whether p27 could be involved with regulating the activity of
such complexes in vivo, the distribution of Cdk4 and p27
proteins in the testes and ovary was examined. Immunoblot analysis
indicated that both Cdk4 and p27 proteins were more abundant in lysates
from immature testes (day 7) than in adult testes (Fig. 4, A and B). Cdk4 was also higher in germ
cell-deficient homozygous (W/Wv) testes than in
heterozygous (W/+) testis (Fig. 4A). Both Cdk4 and p27
proteins were also detected in the adult ovary (Fig. 4, A and B).
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, A and B,
black arrows) and Leydig cells (Fig. 5, A and B, white
arrowheads), which was similar to the expression pattern of cyclin
D3. However, Cdk4 staining was not observed in the cytoplasm of
elongating spermatids (Fig. 5B) as was cyclin D3 (Fig. 2D). The
distribution of Cdk4 protein in these cells was consistent with the
distribution of Cdk4 mRNAs demonstrated in previous studies (34). In
the ovary, Cdk4 was highly expressed in follicular cells, but no
expression of Cdk4 could be detected in either the cytoplasm or nuclei
of oocytes at any stage of follicular development (Fig. 5C).
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, white arrowheads) and at
relatively lower levels in the cytoplasm of elongating spermatids (Fig. 5E, curved arrow). In the ovary, p27 was also highly
expressed in nuclei of oocytes in primordial follicles and in early
stages of oocyte growth, but at decreased levels in larger oocytes
(Fig. 5F, white arrows). The distribution of expression of
cyclin D3, Cdk4, and p27 in oocytes during follicular development is
summarized in Table 1.
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). This interaction could be blocked by
preincubation with peptides of Cdk4 (Fig. 6A) and p27 (data not shown),
but not with Cdk2 or Cdk5 (data not shown). Evidence of association of
Cdk4 and p27 was also seen. As noted above, p27 localized in the same
types of testicular cells as did cyclin D3. However, Cdk4 colocalized
with cyclin D3 only in spermatogonia and Leydig cells, but not in
elongating spermatids. This suggested that in certain cell types, there
may be alternative kinase partners and modulating proteins for cyclin
D3. To further identify the interacting proteins of cyclin D3 in
somatic cells, particularly in Leydig cells, the TM3 cell line was
examined. As shown in Fig. 6B, cyclin D3 immunoprecipitated with both
Cdk4 and p27 in TM3 cells.
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). As a control, another
Cdk inhibitor, p21, which is related to p27 (17), was shown to not
coimmunoprecipitate with cyclin D3 in ovarian lysates (Fig. 6C). There
is likely to be a cellular specificity to the association of cyclin D3
and either Cdk4 or p27, because the immunohistochemical analyses
indicated colocalization of p27 and cyclin D3 in the oocytes of
developing follicles (Figs. 3A and 5F), but not Cdk4 (Table 1). Cyclin
D3 and Cdk4 were both seen in the cytoplasm of granulosa cells and some
nuclei of luteal cells (Fig. 3, C and D, and Fig. 5C).
In vitro kinase activity of cyclin D3 in the testis
As cyclin D3 was detected in some testicular cell types that are
not mitotically active, it was of particular interest to determine its
kinase activity in these cells. As Rb has been reported as a preferred
substrate for cyclin D3 in fibroblast cells, whereas histone H1 was
shown to be the preferred substrate in sf9 insect cells (27), we
assayed for cyclin D3 kinase activity using both substrates. The kinase
activity of cyclin D3 in testicular lysates using Rb as a substrate was
too weak to be interpreted (data not shown). Using histone H1 as a
substrate, cyclin D3 kinase activity was detected in testes from day 7
mice (Fig. 7A). This decreased during
development, dropping to undetectable levels in the adult testis (Fig. 7A). The kinase activity of cyclin D3 was also detected in the TM3 cell
line. No signal clearly above background was observed in either TM4
cells or adult ovary (Fig. 7A), although cyclin D3 protein was detected
in both (Fig. 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of cyclin D3 in both spermatogonia and nonproliferating haploid, postmeiotic spermatids raised the possibility that it may have dual functions during spermatogenesis, involving both cell proliferation and cell differentiation. In the early stages of spermatogenesis, cyclin D3 protein was highly expressed in spermatogonia, indicating a possible role as a regulator to promote cell proliferation in the stem cell stages of the spermatogenic cycle. As the germ cells entered into meiosis and progressed to the leptotene stage, cyclin D3 was reduced to undetectable levels. However, at later stages of spermatogenesis, cyclin D3 mRNA was expressed in haploid, postmeiotic, round spermatids (6) and translated in terminally differentiating elongating spermatids. We were somewhat surprised that cyclin D3 was expressed in these cells, in that the D-type cyclins are presumed to be the cyclins associated with proliferation in response to growth factors (reviewed in Ref. 1). However, there is a report demonstrating that cyclin D3 was induced in terminally differentiated muscle cells (15). In addition, our data showed that cyclin D3 was detected in both proliferating Leydig cells during embryonic and early postnatal testicular development as well as in the differentiated Leydig cells of adult testis. Although adult Leydig cells do undergo cell division at a slow rate (35), they are not considered to be an actively proliferating class of cells (36, 37). The data from the present studies thus suggest that cyclin D3 function is not limited to cell cycle progression, at least in testicular cells. That is, cyclin D3 may be involved in other non-cell cycle functions, such as the specialized morphogenetic differentiation and chromatin remodeling that occur during spermiogenesis.
While this manuscript was in preparation, a report appeared that cyclin D3 protein was detected primarily in the cytoplasm of early stage primary spermatocytes in the rat testis (38). The discrepancy between their results and ours may be due to species differences in the expression of cyclin D3 or to the detection system and reagents used. The antibody we used was generated against protein corresponding to the full-length cyclin D3 complementary DNA (292 amino acids). The antibody used by Kang et al. (38) was produced against a cyclin D3 complementary DNA fragment encoding the C-terminal 236 amino acids. In addition, we treated our paraffin-embedded sections with microwaving to unmask antigenic determinants (24). This treatment might be critical for detection in cells undergoing nuclear condensation and compaction, such as spermatids. It should be noted that Kang et al. (38) reported that cyclin D3 was not present in spermatogonia, whereas we found cyclin D3 to be abundant in these cells. However, they also mentioned that they detected cyclin D3 mRNA and protein in day 7 testis, in which the major cell types are mitotically dividing spermatogonia and somatic cells, including Sertoli cells and Leydig cells. Therefore, our observation of the highest levels of cyclin D3 mRNA and protein in day 7 testis was consistent with the immunoblot and Northern blot results of Kang et al. (38).
In most mammals, female germ cells continue mitotic divisions after
entering the embryonic gonad and then enter meiosis and arrest at
diplotene of meiosis I. At birth, these resting oocytes are found in
primordial follicles. At puberty, any given follicle begins to grow.
The oocyte increases in size and completes its growth before antrum
formation (39). During this growth period, the oocyte increases in size
but remains arrested in the prophase of meiosis I. In response to
hormone regulation, oocytes complete maturation and are arrested in the
second meiotic metaphase. In this study, distinct expression of cyclin
D3 was found in the nuclei of oocytes, with the highest level in early
growing oocytes, particularly in primordial follicles. The level of
signal decreased after the oocytes completed growth and was virtually
undetectable immediately before the germinal vesicle breaks down, as
summarized in Table 1. In addition, cyclin D3 was found in nuclei of
follicular cells in primordial follicles, and the level decreased early
during follicular development. This raised the possibility that cyclin
D3 may play different roles in somatic and germ cells during follicular
development. During embryonic ovary development and early postnatal
follicular development, the potential role for cyclin D3 may be limited
to the regulation of granulosa cell proliferation. After birth, the
potential role for cyclin D3 may be switched to regulate oocyte
maturation. Targeted mutagenesis analysis revealed that cyclin
D2-deficient females were sterile (10), implicating cyclin D2 in oocyte
maturation through the influence of granulosa cell proliferation. It is
interesting to note that cyclin D3 levels drop in proliferating
granulosa cells, where cyclin D2 appears to be functioning.
Several proteins have been found to complex with cyclin D3 in vitro, including Cdk4 and p27 (11, 40). The parallel expression pattern of p27 and cyclin D3 in the growing oocyte suggested that p27 may be complexed with cyclin D3 during oocyte development, potentially regulating its function and playing a role in the maintenance of meiotic arrest and subsequent follicular development. Predepletion of p27 in the lysate increased the kinase activity of cyclin D3, suggesting an inhibitory effect of p27 on the kinase activity in cyclin D3/Cdk4 complexes. It is of interest to note that deletion of p27 has been reported to cause female-specific sterility in mice (41, 42). In these mice, secondary ovarian follicles developed, but did not progress to form corpora lutea.
By coimmunoprecipitation, we have found that cyclin D3 could complex with both Cdk4 and p27 in the testis and ovary, but not with Cdk2 or Cdk5. In the testis, Cdk4 colocalized with cyclin D3 only in spermatogonia and nondividing somatic cells, but not in elongating spermatids. These data suggest that the role of cyclin D3 during spermatogenesis may be mediated by different partners in a cell type-specific pattern. An alternative possibility is that cyclins and Cdks expressed in differentiated cells may form unusual complexes that help to stabilize the differentiated state (reviewed in Ref. 43).
Recent studies have shown that cyclin D1, without interacting with Cdk4, can stimulate estrogen receptor ligand-independent transcriptional activity (44, 45). Interestingly, estrogen receptor ß mRNA has been found in the early round spermatids of human testis (46), the same stage at which cyclin D3 mRNA was shown to be expressed in the mouse testis (6). Whether estrogen receptor ß protein is found in spermatids is not known. However, the predominantly cytoplasmic localization of cyclin D3 in elongating spermatids would seem to rule out a role in transcriptional activation. In the ovary, estrogen receptor ß is the predominant estrogen receptor type (47, 48). It is present in multiple cell types, including granulosa cells in small, medium, and large follicles, thecal cells, and corpora lutea (47, 48, 49). Whether cyclin D3 interacts with estrogen receptor ß during ovarian follicle development and luteinization remains to be determined.
Finally, previous studies have shown that cyclin D3/Cdk4 complexes phosphorylated the Rb protein better than histone H1, although cyclin D3/Cdk2 complexes preferred histone H1 in sf9 insect cells (27). In this study, we found cyclin D3 preferred histone H1 as a substrate in the mouse testis and immature ovary. Whether other isoforms of histone H1, notably the testis-specific variants (50), might be substrates for these complexes remains to be investigated.
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Acknowledgments |
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Footnotes |
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Received August 17, 1998.
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