(Received for publication, July 17, 1995; and in revised form, August 28, 1995)
From the
P-Labeled histone H1 was isolated from synchronized
Chinese hamster (line CHO) cells, subjected to tryptic digestion, and
fractionated into 15 phosphopeptides by high performance liquid
chromatography. These phosphopeptides were grouped into five classes
having different cell cycle phosphorylation kinetics: 1) peptides
reaching a maximum phosphorylation rate in S and then declining in
G
and M, 2) peptides reaching a maximum phosphorylation
rate in G
and then remaining constant or declining in M, 3)
peptides with increasing phosphorylation throughout S and G
and reaching a maximum in M, 4) one peptide that was
phosphorylated only in M, and 5) peptides that had low levels of
phosphorylation that remained constant throughout the cell cycle. Amino
acid analysis and sequencing demonstrated that the mitotic specific H1
phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide
Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is
phosphorylated on both the Ser and Thr, does not contain the consensus
sequence (S/T)PXZ (where X is any amino acid and Z is
a basic amino acid). This sequence is thought to be required by the
p34
/cyclin B kinase that has maximum
phosphorylating activity in mitosis. These data indicate that this
kinase either does not have an obligatory requirement for the consensus
sequence in vivo as generally believed or that it is not the
enzyme responsible for the mitotic specific H1 phosphorylation.
For over 27 years, the phosphorylation of histone H1 has been
thought to play a role in controlling the cell cycle (Ord and Stocken,
1968). To examine this possibility our laboratory used synchronized CHO ()cells to determine the cell cycle kinetics of histone
phosphorylation during cell proliferation (see review by Gurley et
al. (1978a)). In those studies it was found that histone H2A and
H4 phosphorylations were cell cycle-independent and probably not
involved in cell cycle control, while histone H1 and H3
phosphorylations were cell cycle-dependent and, therefore, more likely
to have a role in cell cycle control.
The phosphorylation of H3 was
highly restricted in the cell cycle, occurring only during mitosis. In
contrast, the phosphorylation of H1 was very complex, there being 1
phosphate/molecule in late G, 3 phosphates/molecule in S
and G
, and up to 6 phosphates/molecule in M (Hohmann et
al., 1976). All of the phosphates in H1 and H3 are lost during
telophase, thus resetting these histones to a phosphate-free state at
the beginning of the next cell cycle (Gurley et al., 1978b).
In our laboratory, Hohmann et al.(1976) demonstrated that the ``superphosphorylation'' of H1 in mitosis involved specific serine and threonine sites in the H1 molecule that were not phosphorylated during interphase. These sites were located in the short N-terminal tail of the H1 molecule. Because of this mitotic specificity we proposed that the phosphorylation of these sites may be necessary for the condensation of interphase chromatin into condensed chromosome structure (Gurley et al., 1974, 1978b) or for the orderly separation of chromosomes during anaphase (Gurley et al., 1974). This specificity has also led to the proposal that H1 superphosphorylation may be the ``trigger'' for mitosis (Bradbury et al., 1973). If this were true, the enzymes that phosphorylate the mitotic specific H1 sites would be the controlling molecules of the cell cycle. Thus, it has become important to understand the details of this molecular mechanism since the control of cell proliferation is one of the most important unanswered questions in cell growth and biological differentiation and since the disfunction of this mechanism lies at the heart of the cancer problem, i.e. uncontrolled cell proliferation.
Work on protein kinases has
revealed that the kinases p33and
p34
, when complexed with various cyclins, are
H1 kinases that have different cell cycle periods of maximum
phosphorylating activity (Evans et al.(1983); Pagano et
al.(1992); reviewed by Murray(1992)). Among these, the
p34
/cyclin B kinase has been found to have
maximum activity at mitosis (Pines and Hunter, 1989). Thus, one might
expect this kinase to be the enzyme responsible for the phosphorylation
of the mitotic specific serine and threonine sites in the N-terminal
portion of H1 (Langan et al., 1989). This enzyme is thought to
require the consensus sequence (S/T)PXZ, where X is
any amino acid and Z is a basic amino acid (reviewed by Moreno and
Nurse(1990)). In order to phosphorylate the serine or threonine
residues in H1 during mitosis, one would expect to find two such
sequences in the N-terminal portion of H1. However, this is not
strictly the case. For example, in calf thymus H1-1 there is no
SPXZ sequence in this part of the molecule, and in rabbit
thymus H1-3 there is no TPXZ sequence in this part of
the molecule (Liao and Cole, 1981). This raised a question of whether
our understanding of the mechanism of action of
p34
/cyclin B and its mitotic specific role is
complete or correct.
To examine this question we have fragmented CHO
H1 with trypsin and fractionated its phosphopeptides. Using
incorporation of [P]phosphate during various
phases of the cell cycle we have identified the mitotic specific
phosphopeptide and determined its sequence. This work has shown that it
contains neither an SPXZ nor a TPXZ consensus
sequence. These results indicate that further research will be
necessary to understand the mechanism of mitotic specific H1
phosphorylation and its role in the control of cell proliferation.
To obtain cells synchronized in M, Colcemid was added to cultures 5
h after release from hydroxyurea blockade. These cells traversed S and
G phases and were arrested and resynchronized in mitosis by
metaphase arrest. Three hours after adding Colcemid, the cells entering
M were used for histone measurements during mitosis.
In some
experiments the cells that were released from hydroxyurea were
resynchronized in G. This was accomplished by treating the
released cells with 7.5 µg of Hoechst 33342/ml of culture. This
treatment permits cells to traverse S and then reversibly arrests them
in late G
(Tobey et al., 1990). After 6 h in
Hoechst this culture was used for histone phosphorylation measurements
in G
phase arrest.
Cultures were also arrested in
G by treatment with the histone kinase inhibitor
staurosporine. This was accomplished by dissolving 1 mg of
staurosporine lyophilized powder (Kamiya Biomedical Co.) in 2 ml of
pure Me
SO and adding 100 µl of this stock solution to
1000 ml of exponentially growing CHO cells. This 50 ng/ml treatment
permits CHO cells to traverse the cell cycle and then arrests them in
G
(Crissman et al., 1991). After 10.5 h in
staurosporine this culture was used for histone phosphorylation
measurements in G
phase arrest.
The fraction of cells in
mitosis in the G and M synchronized cultures was determined
by staining a 1-ml aliquot of culture with acridine orange and counting
the percentage of mitotic cells using a fluorescence microscope (Gurley et al., 1973).
Following the extraction of H1, the core histones (H2A, H2B, H3, and
H4) were extracted from the chromatin using 0.4 N
HSO
. These histones were recovered by acetone
precipitation and dissolved in 200 µl of aqueous 0.2%
trifluoroacetic acid for fractionation and purification by HPLC.
The 50-µl sample was injected into a
Waters µBondapak CN column (10-µm irregular particle size,
125-Å pore size, 3.9-mm inner diameter 15-cm length)
attached to a Waters model 6000A HPLC solvent delivery system. H1 was
eluted from the column with a linear gradient of acetonitrile in water
containing 0.2% trifluoroacetic acid running from 5 to 35% acetonitrile
in 3 h at a flow rate of 1.0 ml/min.
The H1 eluting from the column
was monitored by UV absorption using a Waters model 481 flow variable
wavelength spectrophotometer set at 215 nm. The radioactivity of the P incorporated into the H1 was measured by flow liquid
scintillation counting using a Berthold LB504 HPLC radioactivity
monitor and Flo-Scint A liquid scintillation mixture (Packard). The UV
absorption and radioactivity data were collected in a Berthold LB510
Chromatography Data System computer, which calculated the specific
activity of each HPLC peak as counts/volt from the radioactivity counts
in each peak and the area of the UV peak measured in volts.
The remaining 150-µl H1 sample was then used to prepare purified H1 for tryptic peptide analysis. This was accomplished by subjecting the 150-µl H1 sample to the same HPLC procedure as the 50-µl sample except that the effluent from the column was not mixed with liquid scintillation fluid for radioactivity counting. Instead, the UV absorption of the column effluent was monitored, and the H1 peak was collected in a fraction collector. The purified H1, contained in about 10 ml of column effluent, was frozen and lyophilized to a dry powder in preparation for tryptic digestion and phosphopeptide analysis.
The core histones were fractionated by HPLC into H2B, H2A, H4, lhpH3, and mhpH3 (where lhp and mhp refer to the less hydrophobic and more hydrophobic variants of H3, respectively). This was accomplished by injecting the 200-µl sample of core histones into the same µBondapak CN column and eluting the histones with a linear gradient of acetonitrile in water containing 0.2% trifluoroacetic acid running from 0 to 20% acetonitrile in 10 min, followed by a linear gradient running from 20 to 50% acetonitrile in 180 min at a flow rate of 1.0 ml/min. The histones eluting from the column were monitored by UV absorption, and radioactivity was measured as described above.
Amino acid analysis was performed on the P1 peptide by hydrolyzing the sample to amino acids, derivatizing the amino acids using phenyl isothiocyanate, and separating the phenyl isothiocyanate derivatives by the Pico-Tag Method of Cohen et al.(1984). The details of this procedure using a Waters ``application-specified'' reversed-phase PICO-TAG C18 column have been described by Cohen et al.(1989), and the specific instrumentation and application in our laboratory has been described previously (Gurley et al., 1991). This system has been demonstrated to give excellent linear response with very high reproducibility and a 1-pmol detection limit (Bidlingmeyer et al., 1984; Cohen et al., 1984).
The P1 peptide was submitted to the Protein Structure Laboratory at the University of California, Davis, for amino acid sequence analysis. This analysis was performed by automated Edman degradation on an ABI model 477A protein sequencer. The peptide was blocked on its N terminus and could not be sequenced as submitted. Therefore, it was digested for 2 days with V8 protease, which cleaves peptides after glutamic acids (Drapeau, 1977). This produced a 14-amino acid peptide, which was sequenced, and a dipeptide, which was blocked on its N terminus and could not be sequenced. The dipeptide amino acid composition was determined to be Ser and Glu by amino acid analysis, and the sequence was deduced to be SE because V8 protease cleaves on the carboxyl side of Glu residues (Drapeau, 1977).
Figure 1:
Histone phosphorylation
during the cell cycle. A, UV absorption profile of histones H1
and H1 extracted from exponential cells and fractionated by
HPLC. B-I, 2-h [
P]phosphate
incorporation into the H1 of cells in various phases of the cell cycle. J, UV absorption profile of nucleosomal core histones (H2B,
H2A, H4, and two variants of H3) extracted from exponential cells and
fractionated by HPLC. K-R, 2-h
[
P]phosphate incorporation into the nucleosomal
cone histones of cells in various phases of the cell cycle. H1
is residual H1 that does not extract from chromatin with
perchloric acid.
As we have previously shown (reviewed in Gurley et al. (1978a)), the large amount of H2A phosphorylation and the small
amount of H4 phosphorylation in the nucleosomal core does not change
much throughout the cell cycle (Fig. 1, K-R), but
H3 is highly phosphorylated in mitosis (Fig. 1P), and
this phosphorylation is absent in all other phases of the cell cycle. (Fig. 1, L, M, N, Q, and R). The H3 phosphorylation observed in the
G-traversing culture is due to 12.4% mitotic cells that
contaminate this culture. When synchronized cultures are arrested in
G
with Hoechst 33342, there are only 1.6% mitotic cells
contaminating the culture, and there is no phosphorylation of H3 (Fig. 1Q), confirming that this phosphorylation is
restricted to M. This absence of H3 phosphorylation was also observed
when exponential cells were blocked in G
with staurosporine (Fig. 1R).
The absence of H1 phosphorylation in
early G is illustrated in Fig. 1C. However,
beginning in mid-G
we have previously shown that H1
phosphorylation is initiated (Gurley et al., 1975), so that
cultures arrested at the beginning of S have one site being
phosphorylated (Fig. 1D). When cells traverse through S
and G
their H1 phosphorylation rate increases (Fig. 1, E and F) and involves up to three
phosphorylation sites (Hohmann et al., 1976). In M it reaches
its maximum phosphorylation rate (Fig. 1G) and involves
up to six phosphorylation sites (Hohmann et al., 1976). While
arrest of cells in M did not inhibit the phosphorylation of H1 (Fig. 1G), the arrest of cells in G
significantly inhibited interphase H1 phosphorylation (Fig. 1, H and I).
Figure 2:
Phosphorylation of histone H1 tryptic
peptides during the cell cycle. A, HPLC UV absorption profile
of H1 tryptic peptides from exponential cells. H1 was purified by HPLC
as shown in Fig. 1A, digested with trypsin, and
subjected to fractionation by HPLC. The three large peaks eluting in the first 10 min and the large peak eluting at 20 min
are reagents from the tryptic digestion reaction. B-I,
2-h [P]phosphate incorporation into the tryptic
phosphopeptides of H1 of cells in various phases of the cell
cycle.
The three phosphopeptides of interphase cells all eluted
in the first 30 min (Fig. 2B), indicating that they
were small and highly charged. None of these peptides were
phosphorylated in every G (Fig. 2C).
However, all three interphase phosphopeptides incorporated
P in cells arrested at the G
/S boundary (Fig. 2D). Since we have previously shown by long
acid-urea gel electrophoresis that the H1 of these cells contains only
1 phosphate/molecule at G
/S (Hohmann et al.,
1976), these data indicate that any one of three sites is
phosphorylated in late G
.
The data in Fig. 2, E and F, indicate that the same three sites that are
phosphorylated singularly in late G are also phosphorylated
in S and G
. We have previously shown that cells traversing
S and G
contain H1 with up to 3 phosphates/molecule
(Hohmann et al., 1976). Thus, we conclude that there are three
different major sites of interphase H1 phosphorylation, but there is no
qualitative cell cycle specificity as to which site is phosphorylated
first.
In addition to the three major phosphorylation sites there
are some minor sites whose phosphorylation began in S phase and
increased in G. These minor phosphopeptides eluted between
the three major phosphopeptides at 11 and 21 min and at 65 and 67 min (Fig. 2, E and F). The minor phosphopeptide
eluting at 58 min in G
-traversing cells in Fig. 2F is really the major mitotic specific
phosphopeptide. Its detection in the G
-traversing culture
is due to the 12.4% mitotic cells contaminating the synchronized
culture as mentioned above. When the cells of this G
culture were prevented from traversing into M by Hoechst 33342
blockade, the phosphorylation of the major sites was reduced, and that
of the minor sites was inhibited below detection limits (Fig. 2H). The inhibition of interphase phosphorylation
sites by the kinase inhibitor, staurosporine, was even grater (Fig. 2I). Since staurosporine blocks CHO cell cycle
progression in G
(Crissman et al., 1991), these
data demonstrate a strong correlation between interphase
phosphorylation of H1 and the traverse of cells from G
into
M.
During M there was one phosphopeptide that was not observed in
any other phase of the cell cycle. This phosphopeptide eluted at 58 min
and contained the most incorporated P in the M
phosphopeptide chromatogram (Fig. 2G). Eluting late,
this peptide was completely resolved from all other peptides and from
the tryptic digestion reagents, making it possible to isolate and
purify this peptide for further analysis (Fig. 2A).
This peptide (P1) had the strongest UV absorption of all the peptides (Fig. 2A), suggesting that it was the largest.
Examination of the sequence of rabbit H1 suggested that the N-terminal
tryptic peptide was the largest peptide in mammalian H1 (reviewed in
van Holde(1989)). This proved to be the case when we sequenced this
peptide (see below). We had previously demonstrated that CHO cells
contain two mitotic specific phosphorylation sites in the N-terminal
tail of the H1 molecule, a serine site and a threonine site (Hohmann et al., 1976). Since we found only one tryptic phosphopeptide
that was mitotic specific, it was obvious that P1 contained both of
those mitotic specific phosphorylation sites.
Figure 3:
Quantification of H1 tryptic
phosphopeptides. The purified H1 from P-labeled mitotic
cells in Fig. 1G was subjected to trypsin digestion and
reversed-phase HPLC as shown in Fig. 2G. The HPLC
elution profile was expanded into three continuous panels (A, B, and C) to facilitate examination of the details of
fractionation. The top trace in each panel is the UV
absorption profile, and the bottom trace is the
P
incorporation profile. Fifteen phosphopeptides (numbered and shaded) could be identified above background. The
P counts in each phosphopeptide (indicated by the bar
under each peak or shoulder) was quantified.
Tryptic peptide P1 (panel B, upper trace) is the
mitotic specific phosphopeptide pp13 (panel B, lower
trace) eluting at 58 min.
The phosphorylation of these 15 phosphopeptides was normalized to column load, which was quantified from the UV absorption of P1 (Fig. 3B). These relative phosphorylations in various phases of the cell cycle are presented in the histograms in Fig. 4A. Examination of these histograms indicated that the major phosphorylation sites were phosphorylated to greater than twice the extent of the minor phosphorylation sites.
Figure 4:
Relative phosphorylation rate of each
phosphopeptide during the cell cycle. The P incorporation
of each phosphopeptide in each phase of the cell cycle (Fig. 2, B-I) was quantified as illustrated in Fig. 3.
These data were then normalized for preparation and HPLC yield by
dividing the
P counts in each phosphopeptide by the HPLC
column load measured by the UV absorption of peptide P1. This procedure
permits comparison of the phosphorylation rate of each phosphopeptide
with each other (pp1-pp15) and with the phosphopeptides of H1 in
other phases of the cell cycle. A, phosphorylation rate of
phosphopeptide in each phase of the cell cycle. B, comparison
of H1 phosphopeptide phosphorylation rates in cells traversing G
with that of cells arrested in G
by Hoechst 33342 and
staurosporine and with that of cells in exponential growth and
Colcemid-arrested mitosis.
The difference in H1
site phosphorylation in cells traversing G and in cells
arrested in G
with Hoechst 33342 or staurosporine (Stsp) is shown in Fig. 4B. It is seen that
when cell cycle traverse is arrested in G
with Hoechst the
phosphorylations of pp1, pp5, and pp9 are inhibited to 31.3, 31.3, and
33.4% of G
-traversing cells, respectively. When the cells
were treated with the H1 kinase inhibitor, staurosporine, the
phosphorylation of these three phosphopeptides was inhibited to 12.7,
19.4, and 17.0% of G
-traversing cells, respectively, and
their cell cycle traverse was arrested in G
. These data
suggest that the progression of cells from G
to M and the
phosphorylation of these three H1 sites are directly coupled.
Examination of the data in Fig. 4A revealed that
different phosphopeptides reached their maximum phosphorylation rate at
different phases of the cell cycle. When the phosphorylation of each
phosphopeptide was plotted versus cell cycle position it was
found that the phosphopeptides could be placed in five different
classes (Fig. 5), pp9 and pp15 having maximum phosphorylation in
S; pp1, pp5, pp6, pp10, and pp14 having a maximum rate in
G; pp3 and pp7 having a maximum rate in M but also having
some in interphase; pp13 having only phosphorylation in M; and pp2,
pp4, pp8, pp11, and pp12 having low constant levels of phosphorylation
in S, G
, and M. Thus, with the exception of pp13, there
appears to be a cell cycle preference for different phosphorylation
sites, but not an absolute dependence on cell cycle position. The
phosphorylation of pp13 on the other hand occurs exclusively in M and
must account for the M-specific phosphoserine and phosphothreonine
sites observed previously in the N-terminal one-third portion of H1
(Hohmann et al., 1976). The pp7, which is phosphorylated
predominately in M, is expected to contain the phosphothreonine site
observed previously in the C-terminal two-thirds portion of H1 (Hohmann et al., 1976).
Figure 5:
Classification of different
phosphopeptides by cell cycle phosphorylation kinetics. The
phosphorylation rate of each individual phosphopeptide in Fig. 4was plotted as a function of cell cycle position. The
various phosphopeptides (pp1-pp15) were then grouped into cohorts
having maximum phosphorylation rates in S phase (A), G phase (B), or M phase (C). D, pp13 was
the only phosphopeptide that was phosphorylated only in M. E,
five phosphopeptides had phosphorylation rates too low to accurately
classify.
Figure 6: Analysis of the N-terminal tryptic peptide (P1) of histone H1. A, amino acid analysis of CHO P1. B, amino acid sequence of CHO P1 compared with the P1 of other H1 molecules. C, demonstration that CHO H1 P1 belongs to the H1-1 subfraction class by comparison of the amino acid sequence of CHO H1 P1 with that of the P1 peptides of human placenta H1 subtypes. Sequences of the N-terminal tryptic peptide of H1 from calf thymus (Liao and Cole, 1981), rat liver (Cole et al., 1990), human spleen (Ohe et al., 1986, 1989), mouse (Yang et al., 1987), rabbit thymus (Rall and Cole 1971; Jones, et al., 1974), and human placenta (Parseghian et al., 1994) were compared with that from CHO (first line in panels B and C). In these comparisons the asterisk indicates that the amino acid in that position is the same as that in CHO H1. The dash indicates that the amino acid in that position is missing. The (Ac) indicates that the acetyl group on the N-terminal serine is assumed because the sequence was deduced from the H1's RNA sequence.
Attempts to sequence this peptide were initially unsuccessful, indicating that it was blocked on its N terminus. This indicated that P1 is the N-terminal peptide of H1, which is known to be blocked by an acetyl group (Phillips, 1963; Rall and Cole, 1971). To get a partial sequence on P1, the peptide was digested with V8 protease, which produced a dipeptide and a 14-amino acid fragment. The 14-amino acid fragment was sequenced and found to be TAPAAPAAAPPAEK. The dipeptide could not be sequenced, indicating that it was blocked on its N terminus, but it was determined to contain Ser and Glu by difference from the amino acid analysis (Fig. 6A). Since the V8 protease cleaves on the carboxyl side of glutamic acid (E) residues (Drapeau, 1977), it was concluded that the dipeptide sequence must be SE with an acetyl group attached to the N terminus of the serine. The V8 protease did not cleave the 14-amino acid fragment between Glu and Lys due to steric hindrance by the lysine (K). Thus it was concluded that the sequence of P1 is Ac-SETAPAAPAAAPPAEK (Fig. 6B, line 1), which is consistent with the amino acid analysis in Fig. 6A.
The sequence of this N-terminal fragment of CHO H1 was compared with those of eight other mammalian H1 N-terminal fragments (Fig. 6B). Differences between CHO H1 and the others ranged from none with calf thymus H1-1 and human spleen H1-d to six with human spleen H1-a. The two phosphorylatable sites in this fragment of CHO H1 occur as a serine and a threonine at the N-terminal end of the molecule in positions 1 and 3. These two sites must be the two mitotic specific phosphorylated sites we have previously detected (Hohmann et al., 1976). The N-terminal serine has been conserved in the eight other H1 molecules shown in Fig. 6B, but the position 3 threonine has not. The threonine is present in calf thymus H1-1, rat liver H1-d, and human spleen H1-a, H1-b, H1-c, and H1-d, but not in mouse H1 and rabbit thymus H1-3.
The most important observation is that the CHO H1
mitotic specific phosphopeptide does not contain the consensus sequence
(S/T)PXZ for phosphorylation by the p34/cyclin
B kinase (Fig. 6B). Thus it is concluded that either
this kinase is not the enzyme responsible for mitotic specific H1
phosphorylation, or it does not have an obligatory requirement for the
consensus sequence as generally believed.
Recently Parseghian et al.(1994) have proposed a classification of H1 subfractions based on the amino acid sequence of human placenta H1 subtypes. When the sequence of CHO H1 P1 was compared with that of human placenta H1 P1, the CHO N-terminal peptide was found to be identical to H1-1 of human placenta (Fig. 6C). The Ser and Thr residues are both conserved at the N-terminal end of all the human H1 subtypes in both spleen (Fig. 6B) and placenta (Fig. 6C), and like CHO there is no consensus sequence in any of these peptides.
Histone H1 is divided into three structural domains consisting of an apolar core, which interacts with the nucleosome and (like an elephant) has a long highly charged nonstructured polar ``trunk'' on the C-terminal end and a shorter nonstructured polar ``tail'' on the N-terminal end (Hartman et al., 1977). Some think the trunk and tail may interact with the linker DNA as it enters and exits the nucleosome (reviewed by Moreno and Nurse(1990)).
Recent work on protein kinases has suggested that
the p34 kinase, which is thought to be responsible for
some cell cycle-dependent H1 phosphorylations, requires a consensus
sequence (S/T)PXZ (Langan et al. 1989; Moreno and
Nurse, 1990). Sequence data on rabbit H1 show that the trunk of H1
contains four such consensus sites and that the tail contains one
(reviewed by van Holde(1989)). The apolar body of H1, which contains
many serines and threonines, contains no such consensus sites. This
suggests that cell cycle-dependent H1 phosphorylation occurs near the
DNA entry and exit sites on the nucleosome and promotes structural
changes there during S and G
, which leads to the ultimate
condensation of chromatin into chromosomes during mitosis (Gurley et al., 1978a). To test this model we initiated a study of the
cell cycle kinetics of H1 phosphorylation sites. In this work we hoped
to determine if the consensus sequences are the correct in vivo phosphorylation sites and also to determine the cell cycle order
in which the sites of phosphorylation occurred.
To do this we used
HPLC to quantify tryptic phosphopeptides in H1 isolated from
synchronized CHO cultures. Two surprising things were observed in these
studies. First, three major phosphopeptides were observed at the
G/S boundary, although only 1 phosphate/molecule has been
observed at this cell cycle stage using acid/urea gel electrophoresis
(Hohmann et al., 1976). This indicates that, while only one
phosphate is added to H1 during late G
, it is added to any
one of the three interphase phosphorylation sites on the C-terminal
trunk of H1. Thus, there does not appear to be an absolute order for
which site is phosphorylated first, second, or third.
The other
surprise was that the mitotic specific phosphopeptide eluted late
during HPLC like a large peptide rather than among the smaller peptides
predicted for the consensus site in the N-terminal tail of H1 (SPAK in
rabbit thymus H1-3 or TPVK in calf thymus H1-1 (Liao and
Cole, 1981)). Sequence analysis demonstrated that the mitotic specific
phosphorylation site of CHO H1 is on the end of the N-terminal tail and
does not contain the consensus sequence for the p34
kinase. Thus, the p34
/cyclin B kinase found exclusively
in mitotic cells (Pines and Hunter, 1989) is not responsible for
mitotic specific H1 phosphorylation, or this enzyme does not have an
exclusive requirement for the consensus sequence as previously thought.
In this work, 15 peaks and shoulders of P radioactivity
were quantified. Five major phosphorylated peaks were observed rising
and falling during the cell cycle: pp1, pp5, pp7, pp9, and pp13. Five
lesser phosphorylated peaks were also observed undergoing similar
fluctuations (pp3, pp6, pp10, pp14, and pp15), and five very small
peaks were detected just above the base line of detection (pp2, pp4,
pp8, pp11, and pp12). We have previously shown that CHO H1 consists
primarily of one variant of H1 (74%), but it does contain small amounts
of a second variant (17%) and trace amounts of two other variants
(about 8% combined) (Gurley et al., 1975). Thus, we suspect
that the five major phosphorylated peaks are from the major variant,
the five minor phosphorylated peaks are from the minor variant, and the
trace phosphorylated peaks are from the trace H1 variants.
Analysis
of each phosphopeptide with respect to the cell cycle showed that four
different types of cell cycle kinetics existed: peptides whose
phosphorylation peaked in S, those that peaked in G, those
that rose during interphase and peaked in M, and the one peptide that
was phosphorylated only in M. These preferences for different
phosphorylation sites during the cell cycle probably reflect
fluctuations in the various kinases and cyclins that are known to occur
during the cell cycle (Evans et al., 1983; Pagano, et al. 1992; Murray, 1992). However, there did not appear to be any cell
cycle exclusiveness for any phosphorylation site except the mitotic
specific ones in pp13. This means that either there is not great site
specificity for the various kinase/cyclin combinations, or there is
significant overlap of the various kinase/cyclin types during different
phases of the cell cycle. We have recently demonstrated that the former
is certainly true. (
)
Previous analysis of CHO H1 phosphorylation using acid/urea gel electrophoresis has shown that H1 contained six phosphates during mitosis (Hohmann et al., 1976). The demonstration of both a serine and a threonine in the same N-terminal tryptic peptide of H1 explains why we see six phosphates in mitotic H1 but only detect five major phosphopeptides. Comparison of CHO H1 with other mammalian species shows that the threonine site is not conserved. Therefore, its phosphorylation at mitosis is not an exclusive necessity for chromosome condensation but may simply reinforce the negative charge function of the N-terminal serine phosphate, which is conserved in mammals.
The fact that only one mitotic specific phosphopeptide was detected suggests that all of the CHO H1 variants probably have identical N-terminal tryptic peptides. Comparison with human placenta H1 variants suggested that all of the CHO H1 variants are of the H1-1 subtype at the N-terminal end of the molecule (or the H1-d subtype from human spleen). However, since this H1 N-terminal peptide homogeneity has not been conserved among various mammalian species, it is difficult at this time to determine what the universally important sequence is for normal function in the H1 tail.
Examination of the amino acid sequence of rabbit H1 revealed that it contains five consensus sequences, one in the tail of H1, four in the trunk, and none in the nonpolar ``body'' (reviewed by van Holde (1989)). In our laboratory, we have been unable to detect any phosphorylation in the tail of H1 except at mitosis, and all of that phosphorylation was in the non-consensus sequence of pp13 (P1). If CHO H1 is found to have a similar consensus sequence in the H1 tail following the P1 tryptic peptide (like rabbit and calf H1 (Liao and Cole, 1981)), it will demonstrate that the consensus sequence is not a requirement for H1 phosphorylation.
Using acid/urea gel electrophoresis we have previously shown that interphase H1 can have up to 3 phosphates/molecule, and all three of these phosphates occur in the C-terminal trunk of H1 (Hohmann et al., 1976). In the present work we have fractionated three different major phosphopeptides from H1 of interphase cells, which must correspond to those three interphase phosphates (ppl, pp5, and pp9). In our earlier work we also demonstrated an additional phosphothreonine in the C-terminal trunk of CHO H1, which was only detectable in mitotic cells. The work in this report suggests that phosphothreonine probably is in pp7, which reaches a maximum phosphorylation during mitosis. Our present work indicates that the phosphorylation of this site actually begins in S phase but remains at a low level until mitosis, where its phosphorylation rate then approaches that of the other major phosphopeptides.
In
conclusion, this work clearly demonstrates that the consensus sequence
(S/T)PXZ is not necessary for the mitotic specific
phosphorylation of H1. The cell cycle kinetics of phosphorylation at
various H1 sites indicates that there is no absolute cell
cycle-specific sites of phosphorylation during interphase. This
suggests that the various cyclin-dependent kinases overlap
significantly in the cell cycle and do not have different cell cycle
site specificities. It appears that, during interphase, the number of
phosphates per H1 molecule is more important than which sites are
phosphorylated. In contrast, during mitosis there is clearly a site
specificity for the N-terminal serine and threonine sites. Recent
experiments in our laboratory with individual H1 cyclin/kinases have
demonstrated these kinases' lack of site specificity. Also, Jerzmanowski and Cole(1992) have demonstrated that H1 must
be partially displaced from its chromatin-binding site before it can
become a preferred substrate for mitotic-type phosphorylation. Thus, it
appears that the displacement of the N-terminal tail of H1 from the
chromatin may be sufficient for p34
/cyclin B to perform
mitotic specific H1 phosphorylation and that the consensus sequence is
irrelevant for this particular cell cycle activity.