(Received for publication, September 12, 1996)
From the Department of Biology, Syracuse University,
Syracuse, New York 13244, the § Department of Biochemistry,
University of Wisconsin-Madison, Madison, Wisconsin 53706, the
Department of Microbiology and Immunology, Baylor College of
Medicine, Houston, Texas 77030, and the ** Department of Biology,
University of Rochester, Rochester, New York 14627
Micronuclear linker histones of the ciliated
protozoan, Tetrahymena thermophila, are extensively
phosphorylated in vivo. Each of these polypeptides, ,
,
, and
, contains sites for phosphorylation by cyclic-AMP
dependent protein kinase (PKA) but not Cdc2 kinase, and some data have
been presented implicating PKA kinase in their phosphorylation in
vitro and in vivo (Sweet, M. T., and Allis, C. D. (1993) Chromosoma 102, 637-647; Sweet, M. T., Jones, K., and Allis, C. D. (1996) J. Cell Biol., in press). In this
report we have extended these analyses by showing that Cdc2 and PKA
kinase are not evenly distributed between micro- and macronuclei.
Macronuclei, but not micronuclei, contain a 36-kDa polypeptide that is
immunoreactive with p34Cdc2 antibodies. In
contrast, a 40-kDa polypeptide is detected with PKA antibodies in
micronuclei, that is not detected in macronuclei. In support, extracts
from micronuclei, but not macronuclei, contain a kinase activity that
resembles some, but not all, characteristics of PKA from other sources.
Immunodepletion experiments using anti-PKA antibodies show that a
40-kDa polypeptide can be specifically removed from these extracts with
a concomitant loss in kinase activity. Microsequence analyses of
demonstrate that this linker histone is phosphorylated in
vivo on two PKA consensus sequences located in its
carboxyl-terminal domain, an optimum PKA consensus sequence,
Arg-Lys-Asn-
, and a less optimal PKA sequence,
Lys-Ser-
-Val. Collectively, these results suggest that
PKA or a PKA-like kinase is responsible for the phosphorylation of
linker histone in mitotically dividing micronuclei. In contrast,
macronuclei, which divide amitotically, phosphorylate linker histone H1
using a distinct, Cdc2-like kinase.
A variety of kinases and phosphatases have been shown to play an implicit role in the regulation of fundamental cell cycle processes. For example, a family of closely related cyclin-dependent protein kinases is thought to catalyze a series of phosphorylation events associated with cell-cycle progression (for reviews see Refs. 3, 4, 5, 6). During mitosis, several proteins such as linker histones (7, 8), lamins (9), and cytoskeletal proteins (10, 11) undergo stage-specific, reversible phosphorylation. The biological consequences of these phosphorylation events are largely unclear.
It has long been suggested that hyperphosphorylation of the linker histone H1 during mitosis is causally linked to mitotic chromosome condensation (reviewed in Ref. 8), although this relationship remains unproven and controversial (discussed in Refs. 12 and 13). It has also been proposed that phosphorylation of linker histone may act as a first-step mechanism to promote transient decondensation of the chromatin fiber, allowing access of specific factors (such as the SMC family of nonhistone proteins; reviewed in Refs. 13 and 14) in a variety of cell cycle-regulated processes including chromosome condensation (15). Mounting evidence has shown that chromosome condensation can occur in the absence of H1 or H1 phosphorylation in vitro (16, 17, 18) and in vivo (12, 19).
The ciliated protozoan, Tetrahymena thermophila, provides an
ideal model for unraveling complex relationships between H1
phosphorylation, gene expression, and mitotic chromosome condensation.
Each vegetative cell contains two types of nuclei, a somatic
macronucleus that divides amitotically and a germ-line micronucleus
that divides mitotically. Both nuclei contain linker-associated
polypeptides that differ dramatically. Macronuclei, for example,
contain a H1 that resembles vertebrate H1s in several properties
including growth or division-associated phosphorylation by a Cdc2-like
kinase (20). In contrast, micronuclei contains four distinct
polypeptides (,
,
, and
) that are also phosphorylated in
growing or dividing cells (21). If Cdc2 kinase is solely responsible
for linker histone phosphorylation and mitotic chromosome condensation
in Tetrahymena,
,
,
, and
would be expected to
be phosphorylated by this enzyme. However,
,
,
, and
do
not contain any obvious recognition sequence for Cdc2 kinase (22), and
none of these polypeptides are phosphorylated by this kinase in
vitro under conditions where macronuclear H1 is extensively
phosphorylated (1). Interestingly, all four of these polypeptides
contain at least one canonical phosphorylation site for
cAMP-dependent protein kinase
(PKA)1 (22), although none of the in
vivo sites of phosphorylation have been identified.
In this study, we have continued to explore the relationship between
PKA and linker histone phosphorylation in mitotic micronuclei in
Tetrahymena with emphasis on . Immunoblotting data
support the notion that PKA (or a PKA-like activity) exists in
micronuclei during most stages of the life of the cell, and antibodies
against PKA can immunodeplete
kinase activity from micronuclear
extracts. Microsequence analyses confirm that
is phosphorylated
in vivo on two serine residues embedded in the carboxyl
terminus; both sites conform to PKA recognition sites. Collectively,
these results suggest that a PKA or a PKA-like kinase is responsible
for phosphorylation of
in micronuclei and support the general
hypothesis that PKA kinase(s) play an important, and previously
unsuspected, role in mitosis.
Cell Culture and Labeling Conditions
T. thermophila strains CU428 (Chx/Chx-[cy-S]VII)
and CU427 (Mpr/Mpr [6 mp-s VI]) were grown in 1% enriched protease
peptone under standard conditions as described previously (23).
Previous data had shown that phosphorylation was maximal in early
mating cell cultures, 2-4 h after initiating mating (2). Therefore, 2.5-h mating cultures were utilized in this study. Conjugation was
induced according to Bruns and Brussard (24) with modifications described by Allis and Dennison (25). Mating cultures were
phosphorylated in vivo by starving cells of each mating type
separately at approximately 1-2 × 105 cells/ml in 10 mM Tris, pH 7.4, in the presence of
[32P]orthophosphate (12.5 mCi/ml) for 2-3 h before
adding 2 × growth medium and growing cultures for at least one
generation (2-5 × 105 cells/ml). Labeled cells were
then starved and mated as described above.
Preparation of Nuclei and Nuclear Proteins
Macronuclei and micronuclei were isolated from cells as
described by Gorovsky et al. (23), except that the nucleus
isolation buffer contained 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 10 mM
sodium butyrate, but not spermidine. Where indicated, purified
preparations of micro- and macronuclei were isolated following
sedimentation at unit gravity according to Allis and Dennison (25). In
order to better preserve labile phosphorylation modifications,
chloromercuriphenylsulfonic acid (final concentration 0.1 mM) was added to the cell homogenization buffer. was
partially purified from sulfuric acid extracts of micronuclei by
reverse phase (RP)-HPLC using a C8 column (Brownlee) with a linear
gradient of 5-90% acetonitrile containing 0.1% trifluoroacetic acid
(changing at a rate of 0.45%/min) with a flow rate of 1 ml/min.
Preparation of Nuclear Extracts
Macro- and micronuclei from log-phase growing cells were used to
prepare active kinase extracts immediately following isolation. Nuclei
were washed once in isolation buffer and then resuspended (at 5 × 107 macronuclei/ml and 1 × 109
micronuclei/ml) in 10 × phosphorylation buffer (1 M
NaCl, 250 mM MOPS, pH 7.4, 100 mM
MgCl2, 10 mM dithiothreitol, and 0.1% Nonidet
P-40). Nuclei were lysed in this buffer for 10 min before a 9-fold
volume of cold water was added; the lysate was then vortexed well and
then microcentrifuged for 15 min. The clarified supernatant was removed
and used immediately or frozen at 20 °C. Extracts prepared in this
fashion remained active for kinase activity for up to 1 week at
20 °C.
In Vitro Phosphorylation
Enzymes, HeLa Cdc2 kinase, bovine or Paramecium PKA
kinase, or crude Tetrahymena micronuclear extracts (extract
from approximately 1 × 107 micronuclei/reaction) were
added to each reaction immediately prior to the addition of
[-32P]ATP (100-150 units; 1 unit transfers 1 pmol of
Pi/min), as described previously (1). Incubations proceeded
at 30 °C for 15 min, and duplicate samples from each reaction were
removed, applied to P81 filter paper (Whatman International, Maidstone,
United Kingdom), and processed for liquid scintillation counting
(26).
Immunoprecipitation
Ten microliters of micronuclear extract was incubated with 5 µl of either preimmune serum or immune serum for 2 h with gentle shaking at 4 °C. As a negative control, immune serum was added to
buffer without nuclear extract. Protein A-Sepharose (15 µl) was added
to each mixture and the incubation was continued at 4 °C for 1 h more. Following centrifugation, the unbound supernatant was removed
from each incubation mixture, and 5 µl was analyzed in in
vitro kinase reactions with and without as substrate. Immunoblotting analysis was done on an aliquot of the
immunoprecipitation supernatant. Protein bound to the Sepharose beads
was also analyzed in this fashion after solubilization with Laemmli
sample buffer.
Chemical and Enzymatic Cleavages
N-BromosuccinimideRP-HPLC-purified dissolved in 180 ml
of 5% acetic acid was cleaved by adding 20 ml of 20 mM
N-bromosuccinimide (NBS) freshly dissolved in 5% acetic
acid and incubating 2 h at room temperature in the dark (27). The
resultant peptides were purified by RP-HPLC using a C18 column with a
0-90% acetonitrile linear gradient as described above. The single
in vivo phosphorylated NBS fragment from
, which eluted
at approximately 8% acetonitrile, was identified by scintillation
counting and gel analysis on 50% acid-urea gels (see below).
In vivo phosphorylated NBS
peptide from , isolated as described above, or in vitro
phosphorylated
or a synthetic
peptide was cleaved by mixing
endoproteinase Lys-C (Boehringer Mannheim) with peptide at 1:100 (w/w)
in 0.1 M ammonium bicarbonate buffer, pH 9.0, and
incubating at 37 °C for 1-2 h. A second equal aliquot of enzyme was
then added to the reaction mixture and incubation continued overnight
(28). Where indicated, peptides were purified by cation-exchange HPLC
using a polyCAT A column (PolyLC Inc.) with a 0-0.3 M
NaClO4 linear gradient (in a 10 mM phosphate
buffer, pH 6.5) increasing at a rate of 0.03 M
NaClO4/min. Peptides were also purified by elution with
water from acid-urea 50% polyacrylamide gels (29) as described
previously (30). Due to the small size and positive charge of several
of the
peptides analyzed in this study, carboxymethylcellulose
membrane was chosen for best retention of peptides (31). Peptides were
eluted from carboxymethylcellulose membranes for sequencing, as
described previously (31). Small peptides were also analyzed by thin
layer chromatography (TLC) using a phospho-chromatography buffer (37%
n-butanol, 25% pyridine, and 7% glacial acetic acid) as
described by Boyle et al. (32).
Preparation of Synthetic Peptides and Sequencing
Peptides used in this study (see Fig. 2 for details) were
synthesized by the solid phase procedure on a peptide synthesizer (model 430A; Applied Biosystems Inc., Foster City, CA). Peptides were
cleaved from the resin and analyzed by RP-HPLC, amino acid analysis
(Pico Tag system; Waters Associates, Milford, MA). Amino acid sequence
analysis (model 477A protein sequencing system; Applied Biosystems
Inc.) was used to confirm the sequence of peptides and to identify
sites of serine phosphorylation on purified peptides.
Electrophoresis and Immunoblots
One-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis systems have been described previously (33, 34). Acid-urea acrylamide gel electrophoresis of small peptides has also been described previously (29). Immunoblot analyses of SDS-polyacrylamide gels were done as described previously (35). Balanced protein loads of samples were ensured by staining parallel gels of equivalently loaded samples and by staining immunoblots directly with Ponceau red stain. Immunoreactivity was detected by alkaline phosphatase-conjugated secondary antibodies or by chemiluminesence and autoradiography as indicated. Polyclonal antiserum against the catalytic subunit of PKA was prepared as described (36). Antibodies against yeast p34cdc2 were a generous gift from the Beach laboratory.
The unique partitioning of mitosis-related events to
micronuclei along with a specialized set of linker-associated
polypeptides, ,
,
, and
, raises the question which kinase
is responsible for their phosphorylation in vivo. If, as
suggested by our earlier studies (1), PKA or a PKA-like activity is
responsible for micronuclear linker histone phosphorylation, we
reasoned that the catalytic subunit of this kinase should be present in
micronuclei. When an antibody raised against the catalytic subunit of
PKA from Paramecium is used to probe a blot of total nuclear
protein from extensively purified micro- and macronuclei, a single,
strongly immunoreactive band with an apparent molecular mass of 40 kDa is observed in lanes containing micronuclear protein (Fig.
1A). Unexpectedly, at all stages of the life
cycle examined (growing, starved, and 2 h mating), macronuclei
contain little or no detectable PKA catalytic subunit.
Opposite results are obtained when identical samples are probed with antibodies raised against yeast recombinant p34cdc2 (Fig. 1B). In agreement with our previous studies (20), a 36-kDa polypeptide is detected in macronuclear samples when probed with an antibody-generated yeast p34cdc2. Surprisingly, this polypeptide is not detected in micronuclei isolated from the same cells. Thus, macronuclei and micronuclei are distinguished not only by distinct, non-overlapping linker histones, but also by the kinases that might be responsible for phosphorylating them.
Micronuclear Extracts Phosphorylate SyntheticPrevious experiments suggested that , the smallest of
the micronuclear linker polypeptides, was phosphorylated in
vivo on at least one serine located in the carboxyl-terminal third
of the protein (1). It was suggested that PKA or a PKA-like kinase was
responsible for this phosphorylation since the catalytic subunit of
bovine PKA also phosphorylated the same CNBr-generated peptide in
vitro. As shown in Fig. 2A, three
putative sites for PKA phosphorylation are contained in the carboxyl
terminus of
. Two serines matching the most stringent PKA consensus
sequence (Arg-Arg/Lys-Xxx-Ser, underlined) are shown as
black boxes; one serine contained within a less stringent
consensus sequence (Arg-Xxx-Xxx-Ser) is indicated as an open
box. These two classes of PKA consensus motifs are utilized in the
majority of the PKA target sequences (37).
In order to determine whether macro- or micronuclei contain kinase
activities capable of phosphorylating PKA or Cdc2 substrates, a salt
extract was prepared from each type of nuclei and tested with either of
two contrasting peptides. One peptide, containing the three putative
PKA sites described above, was synthesized for use as a model PKA
substrate ( peptide, Fig. 2A); a second peptide,
containing two consensus sequences for Cdc2 kinase from macronuclear H1
(H1 peptide, Fig. 2B), was synthesized for use as a model
Cdc2 substrate. In vitro phosphorylation with macro- and
micronuclear extracts and purified bovine catalytic PKA are shown in
Fig. 3. Significant incorporation of
[32P]phosphate is observed when the synthetic
peptide
is used as a substrate with either crude micronuclear extract or
purified bovine catalytic PKA subunit. In contrast, the synthetic H1
peptide is not a good substrate for either of these kinase reactions, although this peptide is an excellent substrate with purified preparations of human Cdc2 kinase (data not shown). This result is
consistent with our inability to detect p34cdc2 in micronuclei
(Fig. 1B).
To determine if the PKA activities assayed in Fig. 3 display properties characteristic of PKA in higher organisms, parallel studies were repeated in the presence of the well known, competitive inhibitor of mammalian PKAs (PKI; Ref. 38). Unlike bovine PKA, which is clearly inhibited by this peptide, neither purified Paramecium (39) nor crude Tetrahymena PKA activity is inhibited by 5 mM PKI (Fig. 3). These data suggest that ciliate PKAs as well as PKAs from other sources (40) differ significantly from PKA in higher organisms. Another hallmark of PKA is its dependence on cAMP. While cAMP reproducibly increases the kinase activity of the crude micronuclear extract by about 20%, it does not show a significant requirement for cAMP. The significance of this result is not clear, but may be due to the high salt extraction conditions used to prepare the extract.
Immunodepletion of Kinase Activity from Micronuclear ExtractAntibodies against the Paramecium catalytic
subunit were used to precipitate PKA from crude micronuclear extracts
(Fig. 4A). A 40-kDa polypeptide is
precipitated with the anti-PKA (lane 2), but this peptide is
not precipitated with preimmune serum (lane 1) or when
extract is not added to the immune reaction (lane 3). Supernatants from these precipitation were then assayed for kinase activity as described above. Preimmune sera has little effect on the
kinase activity of the supernatant, while the supernatant from the
immune reaction shows an 80% decrease in kinase activity (Fig.
4B). These immunodepletion data are consistent with the suggestion that PKA or a PKA-like kinase is responsible for phosphorylation in micronuclei.
Mapping of Phosphorylation Sites in
The amino acid
sequence of the carboxyl terminus of suggests that there are three
potential phosphorylation sites that could be utilized by PKA. Shown in
Fig. 5 (top) are the expected peptides resulting from a limit digestion of synthetic
peptide with
lysine-specific (Lys-C) endoproteinase. If
is phosphorylated by PKA
using optimal PKA recognition motifs, we predicted that two
32P-labeled peptides would be produced following Lys-C
endoproteinase digestion, one with a single labeled serine,
Asn-
-Thr-Ser-Lys, and a second with two potential
phosphoserines, Arg-Arg-Ser-
-Lys (see the serines in
black and white boxes in Fig. 5).
To test this prediction, synthetic peptide was phosphorylated
in vitro by bovine PKA catalytic subunit or the crude
Tetrahymena micronuclear extract and subjected to a limit
digestion with Lys-C endoproteinase. In addition, RP-HPLC-purified
was phosphorylated in vitro and digested under identical
conditions. Phosphopeptides resulting from these digestion were then
resolved on a long (30 cm) 50% acid-urea acrylamide gel and identified
by autoradiography (Fig. 5). Regardless of the source of PKA,
micronuclear extracts or purified PKA from Paramecium (data
not shown) or bovine heart, the phosphopeptide maps produced are
essentially identical when assayed in this fashion. In addition, these
kinases produce identical phosphopeptide maps when either synthetic
peptide (Fig. 5, lanes 2 and 3) or intact
(data not shown) are used as in vitro substrates.
A comparison of in vivo versus in vitro phosphorylated
peptides was then performed to determine whether in vitro
phosphorylation of peptide produces a similar map as
phosphorylated in vivo.
, labeled in vivo with
[32P]orthophosphate, was recovered by RP-HPLC and cleaved
with Lys-C endoproteinase. Digestion products were then analyzed as
above. These results demonstrate that the in vivo
phosphopeptide map of
(Fig. 5, lane 1) is comparable
with, but not identical to, that of
phosphorylated in
vitro. Interestingly, not all of the peptides are phosphorylated
to the same extent. In all cases tested, in vitro and
in vivo, peptide c is labeled to the greatest extent. Peptide a (variable in different digests, see below) is phosphorylated to a lesser extent, while peptide(s) b, which resolve to varying extents, is typically poorly phosphorylated. To confirm that each of
the above peptides with similar acid-urea polyacrylamide gel mobilities
are indeed identical peptides, the bands were excised from the gel and
analyzed by thin layer chromatography. In all cases phosphopeptides
with the same electrophoretic mobility on the acid-urea gel had the
same mobility on the thin layer chromatography plates, and those with
different electrophoretic mobilities also differed in chromatographic
mobility (data not shown).
Phosphorylated
isoforms of linker histones migrate with a reduced mobility on SDS gels
(for example, see Ref. 41). Previous experiments suggested that, like
macronuclear H1, the mobility of on an SDS gel is reduced by
phosphorylation and that
is maximally phosphorylated during early
stages of the sexual pathway, conjugation (2). As shown in Fig.
6A, three distinct bands of
are well
resolved when HPLC-purified
from 2.5-h mating cells is analyzed in
long SDS gels by staining and autoradiography. Densitometric analysis
of the stained bands and corresponding autoradiogram indicate that the
relative specific activity of the slowest migrating band (labeled
S) was approximately twice that of the intermediate mobility
band (M) (data not shown), suggesting that
is
phosphorylated at two distinct sites during this stage of the life
cycle (see below). In contrast, the fastest band (F) is not
phosphorylated and most likely represents the dephosphorylated isoform
of
.
In order to determine if phosphorylation of occurs in a random or
ordered fashion, the labeled bands resolved in Fig. 6A were
excised and the individual isoforms extracted. Each band was then
separately cleaved with Lys-C endoproteinase, and the digestion
products were resolved in a high resolution acid-urea gel. The
resulting phosphopeptide map clearly documents a non-random pattern of
phosphorylation site utilization in the
isoforms resolved by
SDS-gel electrophoresis. Consistent with the idea that the band labeled
M is a monophosphorylated isoform of
, a single labeled
phosphopeptide is observed after Lys-C digestion of isoform M (Fig.
6B, peptide c). In contrast, the slowest
migrating species (band S), a putative diphosphorylated
isoform of
, produces two labeled peptides (b and c) with similar
relative mobility to the peptides labeled b and c
in Fig. 5.
To
rigorously determine the phosphorylation sites utilized in ,
,
labeled in vivo with [32P]orthophosphate, was
cleaved with NBS (see Fig. 7A) and the
resultant single phosphopeptide was purified by RP-HPLC. Direct
microsequence analysis verified that this peptide was indeed the
indicated carboxyl-terminal portion of
(the length of the
arrow in Fig. 7A corresponds to the amino acid
sequence obtained). During each cycle of microsequencing, an aliquot of
each cleaved phenylthiohydantoin-derivative was collected and the
presence of 32P was directly determined by scintillation
counting. As anticipated, the first indicated serine, contained within
the optimal PKA consensus sequence Arg-Lys-Asn-Ser, is phosphorylated,
yielding counts twice that of background (see Fig. 7B).
Although sequencing progressed through the second indicated PKA
consensus sequence, no additional site of phosphorylation could be
detected in this approach.
A second approach utilized peptides generated from Lys-C endoproteinase
digestion of in vivo phosphorylated , which were resolved
by electrophoresis on acid-urea gels shown previously. Resulting
phosphopeptides (labeled a, b, and c
in Figs. 5 and 6) were excised from the gel using the autoradiogram as
a template, eluted with water, and bound directly to
carboxymethylcellulose membrane for sequencing (31). Phosphopeptide
a was identified as Asn-Ser-Thr-Ser-Lys by microsequencing
(see arrow labeled a in Fig. 7A) and,
as expected, the first Ser residue in this peptide is well labeled with
32P (Fig. 7B). Thus, in agreement with the
results obtained with the NBS peptide, serine 201, contained within an
optimum PKA consensus site, is confirmed as an in vivo
phosphorylation site by this independent method.
Unfortunately, the other in vivo labeled peptides indicated
with bracket b and arrow c in Fig. 5, were not be
sequenced despite several attempts. However, because in vivo
and in vitro phosphorylated samples generate identical
phosphopeptides (Fig. 5) another approach was utilized to determine the
identity of the b and c peptides. In vitro phosphorylated
peptide was used as a model peptide substrate to confirm the
sequences of the remaining in vivo phosphorylated peptides.
The catalytic subunit of bovine PKA was chosen for these analyses
because of its purity and availability and because the peptide map
obtained with this kinase was identical to that obtained with the other
PKAs and, more importantly, to in vivo phosphorylated
.
In vitro labeled
peptide was cleaved with Lys-C
endoproteinase and the peptides so generated were isolated by acid-urea
gel electrophoresis and processed as described above. Peptide c
sequenced as a partial digest product containing the sequence
Arg-Lys-Asn-Ser-Thr-Ser-Lys (see Fig. 7A, arrow
c). The fact that the intensity of 32P labeling of
peptides a and c varied from preparation to preparation could be
explained as variability in the extent of Lys-C endoproteinase digestion (see Fig. 7A). Moreover, the first serine of this
in vitro labeled peptide was determined to be the only
phosphorylated residue in this peptide. This result demonstrates
agreement between in vitro phosphorylation of
peptide by
bovine PKA and in vivo phosphorylation of
.
Interestingly, phosphopeptide c, phosphorylated at serine 201, is the
same peptide as the single peptide observed in the digest of bands
M and S in Fig. 6B. Collectively,
these results strengthen our overall conclusion that serine 201 is a major site of in vivo phosphorylation in
.
Unfortunately the b peptide(s) was either not retained or eluted well
from the carboxymethylcellulose membrane used to immobilize peptides
for microsequencing. For that reason, the b peptide was recovered by
cation-exchange HPLC. An early eluting radiolabeled peptide was
recovered from this column and analyzed by acid-urea gel
electrophoresis to confirm its identity as a b peptide (data not
shown). Unexpectedly, upon microsequencing, this peptide was identified
as Gly-Lys-Ser-Ser-Val-Ser-Lys, the extreme carboxyl-terminal Lys-C
peptide (arrow b, Fig. 7A), with the second
serine in this peptide being phosphorylated (Fig. 7B). Since
this peptide was phosphorylated in vivo and in
vitro with a wide range of PKAs, including bovine,
Paramecium (data not shown), and Tetrahymena (Fig. 5), our data suggest that serine 215, two amino acids removed from a single lysine, can be phosphorylated by a wide range of authentic PKAs. Interestingly, peptide b, the slowest migrating of
these peptides, is detected only in band S (Fig.
6B). Collectively, these results suggest that PKA first
phosphorylates at serine 201 contained in the optimum consensus
sequence Arg-Lys-Asn-Ser and then becomes a maximally phosphorylated,
diphosphorylated isoform utilizing serine 215 contained in the less
optimal PKA consensus sequence Lys-Ser-Ser-Val.
Interestingly, the second "expected" optimum consensus sequence
Arg-Arg-Ser-Ser-Ser is not utilized for phosphorylation in vivo or in vitro with any of the substrates that we
have tested. However, we found it is possible to phosphorylate this
optimal PKA consensus sequence when the neighboring sequence of the peptide is altered. Omission of a single serine (serine 205, see Fig.
7A; a mistake made during the original synthesis of the
peptide) that lies between the two optimum PKA consensus sites changes
the Lys-C phosphopeptide map of
significantly. Using this
"mutated"
peptide, phosphopeptides a and c are not observed and
a new, faster migrating phosphopeptide with the sequence
Ser-Arg-Arg-Ser-Ser-Ser-Lys is obtained (data not shown). As expected
for a PKA optimal consensus motif, microsequence analysis shows that
the second serine following the second arginine is phosphorylated (data
not shown). The above peptide is phosphorylated when micronuclear
extract, Paramecium PKA, or bovine PKA is used as a source
of kinase with the altered peptide substrate, and thus as expected, the
second "expected" optimal PKA consensus motif is a good in
vitro substrate for most PKAs. However, unexpectedly, the
Arg-Arg-Ser-
-Ser (Ser-209 is underlined) motif is not
utilized when Ser-205 is present in the
model peptide substrate.
These results suggest that conformation of
plays an important role
in determining which sites of phosphorylation are utilized in
in vitro and in vivo.
One of the more unexpected results to emerge from this study is
the clear and non-overlapping partitioning of PKA and Cdc2 kinase
catalytic subunits between micro- and macronuclei of
Tetrahymena, respectively. Several lines of evidence suggest
that PKA or a PKA-like kinase, but not Cdc2, is responsible for
phosphorylation of (and likely other linker histones) in mitotic
micronuclei. First, micronuclei, but not macronuclei, contain a 40-kDa
polypeptide that is immunoreactive with antibodies raised against the
catalytic subunit of Paramecium PKA. Given the clear
evidence documenting the regulation of transcription by phosphorylation
(42) and, in particular, the involvement of PKA in a subset of these
phosphorylation events (43, 44), the complete absence of PKA catalytic
subunit in transcriptionally active macronuclei is striking and
unexpected. Equally remarkable is the absence of p34cdc2 in
micronuclei, given the suspected role of this kinase in a variety of
mitosis-associated events (3).
Second, micronuclear extracts and more purified PKAs from
Paramecium and bovine heart produced essentially identical
in vitro phosphopeptide maps with two in vitro
substrates, or a synthetic
peptide spanning all known potential
PKA phosphorylation sites in
(1). Importantly, phosphopeptide maps
obtained with all three sources of PKA are comparable with that
obtained with in vivo phosphorylated
. Third, antibodies
against the PKA catalytic subunit immunoprecipitated a 40-kDa protein
from micronuclear extracts, and this immune serum, but not preimmune
serum, depleted kinase activity from crude micronuclear extracts.
Finally, two in vivo sites of phosphorylation in
have
been identified. Both sites conform with recognition sites for PKA
(37), although other kinases with similar sequence requirements have
been reported (45). Collectively, these results lend strong support to
the idea that PKA or a PKA-like kinase is present in micronuclei and that this activity is at least partially responsible for
phosphorylation of
in vivo.
Our data suggest, however, that several differences exist between ciliate and mammalian PKAs. Unlike bovine PKA, neither Paramecium nor crude Tetrahymena PKA activity is inhibited by PKI. These results are in agreement with the findings that Paramecium and Dictyostelium PKAs, respectively, are poorly inhibited by this peptide (39, 40). Although, the catalytic core of the PKA is highly conserved between most species (Ref. 46; reviewed in Ref. 47), sequence differences in the recognition site between isomers of the PKA subunit in yeast and mammalian cGMP-dependent protein kinase reduce these enzymes' affinities for the Kemptide substrate and PKI (48, 49).
Our data strongly suggest that phosphorylation of by PKA is
influenced by both protein sequence and conformation. Studies using
model substrates with mammalian PKAs have suggested that substrates
containing a pair of basic residues (usually arginines) one to three
amino acids removed from the site of phosphorylation are generally
favored over substrates with a single basic residue (37). Based upon
this general rule, two putative sites of phosphorylation by PKA were
predicted from the sequence of the carboxyl terminus of
(1).
Our data demonstrate, however, that the above prediction is upheld for
only one of these sites. Serine 201, contained within the optimal
consensus sequence Arg-Lys-Asn-, is the exclusive site
of monophosphorylation within
in vivo and is a highly
preferred site in vitro. Unexpectedly, a second serine 209 contained within an optimal PKA recognition motif, Arg-Arg-Ser-Ser-Ser,
is not utilized in vivo or in vitro. However,
PKA, regardless of source, did utilize serine 209 when a synthetic
peptide spanning this region, but missing serine 205, was used a
substrate. Since this sequence is not utilized in vivo or
in vitro when the correct
peptide is used as substrate,
we favor the interpretation that some aspect of
's conformation,
possibly altered by the absence of serine 205, lowers the efficiency
that this site is utilized. Surprisingly, a second serine 215, embedded
within the sequence Lys-Ser-
-Val is phosphorylated when
is phosphorylated in vitro or in vivo
(site b, Fig. 6A). This less optimal PKA
phosphorylation site contains a hydrophobic valine one amino acid after
the phosphorylation site, a requirement shown to contribute to
efficient substrate binding with mammalian PKA (50). These data support
our contention that the overall structure of
plays an important
role in site utilization by PKA in vivo.
Previous studies have indicated that micronuclear linker histones are extensively phosphorylated in growing and mating cells, but not in starved cells (21). Our results (Fig. 1) demonstrate that PKA catalytic subunit is detected in micronuclei from growing, starved, and young mating cells, and at first glance, it appears that the amount of the 40-kDa subunit does not change appreciably with the physiological state of the cell. However, the crystal structure of PKA (50) demonstrated the importance of post-translational phosphorylation (at Thr-197) and suggested possible roles of phosphorylation in promoting PKA activation (reviewed in Ref. 47). Whether Tetrahymena PKA undergoes physiologically regulated modification during its life cycle is unknown.
Recent data suggest that an increasingly important mechanism in the role of phosphorylation events is the subcellular location of kinase or phosphatase subunits (reviewed in Ref. 51). For example, activation of PKA also requires a well known, cyclic AMP-dependent dissociation of catalytic subunit from an inhibitory regulatory subunit. Studies of fluorescence-tagged PKA injected into mammalian cells found that upon dissociation the free catalytic subunit moved from the cytoplasm to the nucleus while the regulatory subunit remained in the cytoplasm (52, 53). These data suggest the possibility that the activity of the catalytic subunit in the micronucleus may be modulated by a regulatory subunit that is selectively targeted to micronuclei during select stages of the life cycle. Along this line, the R subunit of PKA has been detected in Dictyostelium nuclei, and, interestingly, its amount in nuclei increases upon differentiation (54).
Although the biological function of linker histone phosphorylation is
presently unknown, it has been proposed that it may facilitate
chromatin decondensation facilitating factor access to DNA (15).
Condensed chromatin, in contrast, is thought to be stabilized by
dephosphorylated linker histone isoforms. Consistent with this model is
the recent finding that phosphorylation of in micronuclei is linked
(temporally and spatially) to a transient period of chromatin
decondensation and transcriptional activation during meiotic prophase
(2). We hypothesize that phosphorylation of the two carboxyl-terminal
PKA sites identified in
in this study acts to destabilize or
decondense micronuclear chromatin, thereby facilitating the binding of
factors required for activation including unique histone variants such
as hv1 (55). Moreover, Lamb and co-workers (56) have demonstrated that
inhibition of PKA in living mammalian cells results in rapid chromatin
condensation at all phases of the cell cycle. Based on these results,
we speculate that phosphorylation of PKA sites in mammalian H1s may
play a previously unsuspected role in chromatin decondensation.
We are grateful to Dr. Ned Lamb who helped us obtain carboxymethylcellulose membrane and to Dr. Elisabeth Alimi for sending us some of this membrane for preliminary experiments. Mammalian PKA and much invaluable information on this kinase were generously provided by Dr. David Brautigan.