From the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104-1079
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ABSTRACT |
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Heterochromatin-associated protein 1 (HP1)
is a nonhistone chromosomal protein with a dose-dependent
effect on heterochromatin mediated position-effect silencing. It is
multiply phosphorylated in vivo. Hyperphosphorylation of
HP1 is correlated with heterochromatin assembly. We report here that
HP1 is phosphorylated by casein kinase II in vivo at three
serine residues located at the N and C termini of the protein. Alanine
substitution mutations in the casein kinase II target phosphorylation
sites dramatically reduce the heterochromatin binding activity of HP1,
whereas glutamate substitution mutations, which mimic the charge
contributions of phosphorylated serine, have apparently wild-type
binding activity. We propose that phosphorylation of HP1 promotes
protein-protein interaction between HP1 and target binding proteins in heterochromatin.
In higher eukaryotes, chromosomes are composed of euchromatin and
heterochromatin. Heterochromatin is distinguished from euchromatin in
that it stays condensed throughout the cell cycle (1). In addition, it
replicates late in S phase, is enriched in repetitive DNA, and is
relatively poor in classical genes (2-4).
In Drosophila, when a euchromatic gene is placed next to or
within heterochromatin by chromosomal rearrangement or transposition, the euchromatic gene usually undergoes cell-specific silencing, called
"position-effect variegation" (5). This process indicates that
heterochromatin interferes with euchromatic gene expression, which
biochemical data suggest is caused by the acquisition of a distinct
chromatin structure with reduced accessibility to DNA-binding proteins
(6, 7). More than 50 different position-effect variegation regulators
have been identified, including genes encoding chromatin proteins and
protein modifiers (8). One such regulator encodes
heterochromatin-associated protein 1 (HP1),1 a nonhistone
chromosomal protein enriched in the pericentric heterochromatin of
interphase nuclei (9, 10). HP1 exerts dosage-dependent
effects on position-effect variegation (11, 12). HP1 localization
during the cell cycle is complex, perhaps reflecting the changes in
chromosome structures that accompany the cell cycle (13).
Current models suggest that HP1 functions as part of a chromosomal
protein complex (14). Proteins thought to bind to HP1-like proteins
include transcriptional intermediary factors (15), lamin B receptors
(16), and origin recognition complex proteins (17, 18).
HP1 is multiply phosphorylated in vivo (19). Using
two-dimensional gel electrophoresis, HP1 can be resolved into as many as eight charged isoforms. HP1 phosphorylation occurs predominantly at
serines and threonines, and increased phosphorylation of HP1 is
correlated with heterochromatin assembly during development. Phosphorylation may be correlated with the oligomeric state of HP1
in vivo (18). Phosphorylation of chromosomal proteins is implicated in the regulation of several nuclear functions. For example,
histones are multiply phosphorylated during mitotic and developmentally
programmed chromatin condensation (20), specific forms of
phosphorylated histone H1 are correlated with specific heterochromatic
satellite DNA sequences (21), and several transcription factors are
regulated by phosphorylation (22).
As a first step toward defining the role of HP1 phosphorylation in
heterochromatin assembly and position-effect variegation silencing, we
mapped three of the phosphorylation sites at the N- and C-terminal
domains of HP1. We present biochemical evidence for casein kinase II
(CKII) phosphorylation of HP1 in vitro and in
vivo. Alanine substitution mutation in the N-terminal CKII target
site dramatically reduces heterochromatin binding activity of HP1,
whereas glutamate substitutions mutations at either or both of the N-
and C-terminal sites have apparently wild-type binding activity,
suggesting that CKII phosphorylation is required for heterochromatin
binding. We propose that phosphorylation of HP1 promotes
protein-protein interaction between HP1 and target binding proteins
within heterochromatin.
Fly Culture and Tissue Preparation--
Fly stocks were
maintained at room temperature on standard cornmeal-yeast-sucrose-agar
medium containing methylparaben as a mold inhibitor. Third-instar
larvae were collected immediately before dissection and kept on ice
until dissection.
Expression and Purification of Recombinant HP1--
A
XbaI-BamHI fragment containing
Drosophila HP1 cDNA was cloned into expression vector
pET11a, and the recombinant plasmid was transformed into
Escherichia coli BL21(DE3) cells. A single colony of
transformed cells was grown in LB medium until
A600 = 0.6-1.0. Expression of HP1 was induced
by 1 mM
isopropyl-1-thio- Preparation of Drosophila Nuclear Extract--
Five g of frozen
0-6-h-old embryos (gift of Dr. S. C. R. Elgin) were used to make the
nuclear extract. Drosophila embryo nuclei were prepared
according to Wu et al. (23). The nuclear extract was made as
described (9), with modifications. Briefly, nuclei were suspended in 4 ml of extraction buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM In Vitro Phosphorylation Assay--
In a 20-µl reaction
mixture, purified recombinant HP1 (1 µg/µl) was mixed with 1 µl
Drosophila embryo nuclear extract and 2 µCi of
[ Two-dimensional Gel Analysis and Western
Blotting--
[32P]Phosphate metabolic labeling of
larval tissue and separation of in vivo labeled HP1 by
two-dimensional gel electrophoresis was done as described (19). For
analysis of in vitro phosphorylated HP1, protein was
precipitated with 10% trichloroacetic acid, washed successively with
100% ethanol and chloroform:methanol (1:1), dried, and resuspended in
80 ml of IEF sample buffer (9.5 M urea, 2% Triton X-100,
5% Trypsin Digestion and Radio-peptide Mapping--
In
situ trypsin digestion of protein immobilized on nitrocellulose
membrane was done according to Fernandez et al. (25). Briefly, the radiolabeled proteins were transferred electrophoretically to nitrocellulose membrane after SDS-PAGE. Radiolabeled HP1 was visualized by Western blotting and autoradiography. The region of
membrane containing immunolocalized HP1 was excised with a surgical
blade and blocked in 1.0 ml of 0.5% polyvinylpyrrolidone, Mr 40,000 (PVP-40, Sigma), 100 mM
acetic acid (w/v) in a 1.5-ml Eppendorf tube at 37 °C for 30 min.
The sample was washed six times with 1 ml of Milli-Q water, cut into
approximately 1 x 1-mm squares, and returned to the same tube. A
solution of 0.1 M NH4HCO3 and 2 M urea was added to just immerse the membrane strips.
Trypsin (0.5 µg/µg of substrate) was added, and the digestion was
incubated overnight at 37 °C. Following the digestion, the sample
was centrifuged at top speed in an Eppendorf centrifuge for 5 min. The
supernatant was stored at Sequencing of Radiolabeled Tryptic Peptides and Identification of
Radioactive Amino Acids--
Trypic phosphopeptide bands on the dried
PAGE gel were excised with a surgical blade. Peptide was eluted from
the gel slices with Milli-Q water. Peptides in the extract were
purified and desalted with a C-18 reverse phase HPLC column (130A
Separation System, Applied Biosystems) and subjected to protein
sequencing (477A Protein Sequencer, Applied Biosystems). Cleaved
phenylthiohydantoin derivatives from each cycle were collected, and
radioactive amino acid residues were determined by: 1) counting in a
scintillation counter (1500 Tri-Carb liquid scintillation analyzer,
Packard Instrument Co.) for the radioactivity and 2) concentrating to <10 µl and spotting onto a 0.1-mm cellulose thin layer
electrophoresis plate (EM Separations) for autoradiography.
Anti-CKII Immunodepletion of Extracts--
Antibody depletion of
CKII was done according to Birnbaum et al. (28) with
modifications. 5 µl of nuclear extract was incubated with 4 µl of
rabbit antiserum against native Drosophila CKII, 16 µl of
NET buffer (140 mM NaCl, 50 mM Tris-HCl, pH
8.0, 5 mM EDTA, and 0.05% Nonidet P-40), and 25 µg of
bovine serum albumin at 4 °C for 1 h. 25 µl of protein
A-acrylic beads suspension (Sigma) was added, and the mixture was
incubated at 4 °C for an additional 20 min. Following a brief
centrifugation, the supernatant was used in the in vitro
phosphorylation assay. As a control, 5 µl of the extract was treated
with rabbit nonimmune serum (no preimmune serum
remains),2 and the sample was
also used in the in vitro phosphorylation assay.
Site-directed Mutagenesis and Construction of the Transgene
Expression Vector--
Site-directed mutagenesis was performed using
the TransformerTM site-directed mutagenesis kit
(CLONTECH) according to manufacturer's instructions. Mutagenesis was done in a pBluescript SK(+) plasmid (Stratagene) with Drosophila HP1 cDNA inserted between
the XbaI and BamHI sites. Mutant cDNA was
then excised with SacI and BglII and cloned into
vector SP1 (29). A KpnI fragment from the SP1 vector was
cloned into transformation vector pV Expression and Localization of Drosophila Embryo Nuclear Extract Phosphorylates Recombinant HP1 in
Vitro--
HP1 is multiply phosphorylated in vivo, and
hyperphosphorylation is correlated with heterochromatin assembly (19).
As a first step toward understanding the functional significance of HP1
phosphorylation, we mapped phosphorylation sites in HP1. Our strategy
was to use recombinant HP1 (rHP1) as a substrate for phosphorylation by
Drosophila nuclear extract in vitro and then map
the sites of in vitro phosphorylation corresponding to sites used in vivo. Drosophila HP1 cDNA was
expressed in E. coli and purified to >95% homogeneity
(Fig. 1). To determine whether rHP1 is phosphorylated in bacteria, we
compared its mobility in two-dimensional gel electrophoresis to that of
HP1 from Drosophila (dHP1). Multiple dHP1 isoforms are
detected in Western blots of total Drosophila protein after
two-dimensional gel electrophoresis (Ref. 19; Fig.
2A, top). Similar Western
blots of rHP1 revealed a single major HP1 isoform (Fig. 2A,
middle). When rHP1 is added to total Drosophila protein
and co-electrophoresed, the rHP1 migrates at the extreme basic end of
the dHP1 pattern (Fig. 2A, bottom), consistent with
unmodified HP1.
Hyperphosphorylated isoforms of HP1 begin to appear by 2 h of
embryonic development (19). Therefore, we used a nuclear extract from
0-6-h Drosophila embryos to phosphorylate rHP1 in
vitro. When rHP1 is added to nuclear extract in the presence of
[32P]ATP, rHP1 is efficiently radiolabeled.
Co-electrophoresis with total Drosophila protein
demonstrates that radiolabeled rHP1 co-migrates with the second and
third most basic isoforms of dHP1 (Fig. 2B), consistent with
mono- and diphosphorylated protein. To verify that sites of rHP1
phosphorylation by embryo extract correspond to sites of dHP1
phosphorylation in vivo, the major tryptic phosphopeptides of in vitro labeled rHP1 were compared with those of dHP1
metabolically labeled with [32P]orthophosphate. In
vitro phosphorylated rHP1 was fractionated by SDS-PAGE and treated
with trypsin. Separately, total protein from larval tissue
metabolically labeled with [32P]orthophosphate was
fractionated by two-dimensional gel electrophoresis, and dHP1 protein
was digested with trypsin. Tryptic peptides from both preparations were
resolved electrophoretically on a 40% alkaline polyacrylamide gel.
Three major radioactive bands were common to both preparations (Fig.
2C). Thus, these major tryptic phosphopeptides contain
targets of phosphorylation used in vivo.
HP1 Is Phosphorylated by CKII--
The fact that embryo extract
can efficiently phosphorylate rHP1 at sites used in vivo
allowed us to map the residues phosphorylated by the extract. The major
tryptic peptides from in vitro phosphorylated rHP1 (peptides
1, 2, and 3 in Fig. 2C) were size-fractionated by 40%
alkaline PAGE, and each phosphopeptide was separately eluted from the
gel, desalted and purified by reverse phase HPLC, and subjected to
amino acid sequencing. As each amino acid was determined, the eluted
amino acid derivative was assayed for radioactivity. Results are
summarized in Fig. 2D. Peptides 1 and 2 are fragments of the
HP1 N terminus and contain the same phosphorylated serine residue, with
peptide 2 having one more undigested lysine at the N terminus. Peptide
3 is a C-terminal fragment containing a major phosphoserine and a minor
one three residues away on its N-terminal side. The two most terminal
serines occur within CKII recognition motifs, which is
S(T)XXE(D). These are the only consensus CKII site motifs in
HP1. In peptide 3, the N-terminal serine resembles a CKII target only
after its C-terminal partner is phosphorylated and becomes acidic.
To confirm the identity of the HP1 kinase activity in embryo extract,
we tested the sensitivity of the kinase to inhibitors and stimulators
of CKII. rHP1 phosphorylation by embryo extract is stimulated by
spermine and inhibited by heparin (Fig.
3). Both of these effects are also seen
with CKII (36, 37). As further confirmation, we tested whether antibody
to Drosophila CKII (a gift of Dr. C. V. C. Glover
III) inhibited in vitro phosphorylation. As shown in Fig. 3,
the CKII antisera almost completely eliminates HP1 phosphorylation by
embryo extract. Taken together, the results strongly implicate CKII in
HP1 phosphorylation in vitro and in vivo.
Mutations in CKII Sites Affect Heterochromatin Binding--
To
test the functional significance of HP1 phosphorylation, the serines in
the CKII target sites were replaced by either alanine (to prevent
phosphorylation) or glutamate (to mimic the spatial and charge
contributions of phosphorylated serine; Refs. 38 and 39). Five
mutations were assayed (Fig. 4):
replacement of the N-terminal serine by alanine (S15A) or by glutamate
(S15E), replacement of the very C-terminal serine by alanine (S202A) or by glutamate (S202E), and a combination of both Ser
For the S15A, S202A, and S15A,S202A mutations, comparable efforts to
obtain germline transformants resulted in no transgenic lines
(occasionally, transformed phaerate adults were observed, but these
failed to hatch or died shortly after hatching). To assay fusion
protein targeting for these mutations, we resorted to somatic
transformation. Plasmid DNA injected into early embryos stably endures
in somatic tissues through the third-instar larval stage, displaying
correct tissue-specific expression (33, 34). We had employed somatic
transformation in previous studies to assay HP1· Drosophila HP1 Is Phosphorylated by CKII in Vivo--
The methods
we used to identify HP1 phosphorylation sites involved direct
comparison of the in vivo and in vitro tryptic
peptide map by high concentration PAGE, rHP1 phosphopeptide sequencing, and radioactivity detection of each amino acid derivative. This strategy has been used to identify phosphorylation sites on other proteins (26, 27, 41, 42). For all three sites common to phosphorylated
rHP1 and dHP1, the targets are good fits to CKII consensus motifs,
which, together with the sensitivity of rHP1 phosphorylation to
spermine, heparin, and anti-Drosophila CKII serum, strongly
suggests that HP1 is a substrate for CKII. CKII is an ubiquitous cyclic
nucleotide-independent protein kinase that appears not to directly
mediate known signaling pathways (43). CKII activity has been found to
increase in response to some mitogens, and its substrates include a
number of transcription factors involved in growth control (44).
Because CKII is found both in the nucleus and the cytoplasm (36), and
because we found that alanine substitution had no effect on nuclear
targeting, HP1 phosphorylation by CKII could occur in either compartment.
CKII consensus target sites are found at the N and/or C terminus of HP1
homologs from Drosophila virilis, Schizosaccaromyces pombe, mealybug, mouse, and human. Not all HP1 homologs have CKII targets at both ends (some have neither), but in several such cases the
homologous position is occupied by glutamate. Little or nothing is
known about the functional homology between Drosophila melanogaster HP1 and its structural homologs in other species, but
such apparent structural conservation suggests functional conservation.
Nevertheless, the data presented here showing that CKII phosphorylation
is required for efficient heterochromatin targeting by the unique
D. melanogaster HP1 suggest that such structural
conservation is likely to be functionally significant.
The only detailed structural information for any HP1 homolog is a
solution NMR peptide structure based on the N-terminal chromo domain of
a single mouse HP1 homolog (45), and the sequences corresponding to the
targets of CKII lie outside of the solved structure. So far, we have
found no effect of CKII phosphorylation on HP1 multimerization in
solution.3 Although two of
the CKII sites occur within a previously identified nuclear targeting
domain (40), we observed no impairment in nuclear targeting for any of
the CKII site mutant fusions. CKII phosphorylation could contribute to
heterochromatin binding by HP1 by promoting a conformational shift that
permits 1) additional kinases to phosphorylate internal targets in, for
example, the HP1 linker region between the chromo domains; or 2) the
exposure of sites for protein-protein interactions. Either of these
results could facilitate heterochromatin assembly. As previously noted (19), this interval is serine/threonine-rich and includes two consensus
targets for protein kinase A and one for protein kinase C.
Phosphorylation on Both the N- and the C-terminal CKII Sites Is
Required for Heterochromatin Binding--
Although the Ser
Although transformants were recovered with Ser CKII Phosphorylation and HP1 Function--
The most basic HP1
isoforms in vivo are phosphorylated at CKII sites. Thus,
CKII phosphorylation does not directly account for the
hyperphosphorylation that accompanies the appearance of heterochromatin
in the early embryonic development. Indeed, it probably accounts for
the maternally loaded HP1 isoforms seen in unfertilized oocytes (19).
Nevertheless, the mutational analysis shows that CKII phosphorylation
is essential for heterochromatin binding.
CKII is an ubiquitous eukaryotic serine/threonine protein kinase that
phosphorylates more than 100 substrates, many of which control cell
division or signal transduction. These substrates include a striking
number of nuclear proteins involved in DNA replication and
transcription (47). CKII modifies protein-DNA binding (48-51) and
protein-protein interaction (52, 53). In Drosophila, CKII is
present in both the cytoplasmic and nuclear compartments. CKII
phosphorylation enhances the DNA binding activity of the engrailed
protein (51) and modulates Antennapedia activity (39) and
dorsoventral patterning (54). Drosophila DNA topoisomerase II is stimulated by CKII phosphorylation (55). We showed previously that significant HP1 phosphorylation still occurs in vivo in
tissues treated with sufficient cycloheximide to block all detectable nascent protein synthesis (19). This turnover of phosphate uncoupled from a new synthesis suggested that HP1 phosphorylation could regulate
its chromatin association, an example being the dynamic dissociation
and reassociation of HP1 that reportedly takes place during mitosis
(13). Alternatively, phosphorylation-dephosphorylation may be regulated
during decondensation of heterochromatin to permit DNA replication in
late S phase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside at 30 °C for
2-3 h. Cells were spun down at 5,000 × g for 5 min in
4 °C, and the cell pellet was frozen and stored at
70 °C. For
protein purification, the pellet was thawed on ice and suspended in 40 ml of lysis buffer (100 mM Tris-HCl, pH 7.3, 4 mM EDTA, 0.4 mM EGTA, 4 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride).
All subsequent steps were done on ice or at 4 °C unless indicated
otherwise. 24 mg of lysozyme was added to the sample and incubated on
ice for 20 min. Cells were lysed either by freezing and thawing or by
sonication. NaCl was added to a final concentration of 0.3 M to get maximum HP1 solubilization. After centrifugation at 18,000 rpm for 30 min in a Sorvall SS-34 rotor, the supernatant was
diluted 3-fold and applied to a DEAE-Sepharose column. The DEAE column
was eluted with 0.4 M NaCl. Eluted fractions containing HP1
were pooled, diluted 4-fold, and applied to a 6-ml Resource Q FPLC
(Amersham Pharmacia Biotech FPLC system) column. The Resource Q column
was developed with a 0.1-0.4 M NaCl gradient, and HP1 was
eluted in the 0.2-0.25 M fractions. These fractions were
pooled and loaded on an S-200 Sephacryl FPLC gel filtration column. HP1 was collected in an elution volume corresponding to a molecular mass of
~40 kDa. HP1-containing fractions were pooled again and applied to a
1-ml Mono Q FPLC column. The Mono Q column was developed with a
0.1-0.4 M NaCl gradient, and HP1 was purified as a single sharp, symmetrical peak at ~0.25 M NaCl. At each
chromatographic step, fractions containing HP1 were determined by 12%
SDS-PAGE and Coomassie Brilliant Blue staining. Relative purification
at each step is shown in Fig. 1.
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Fig. 1.
Purification of recombinant HP1 from E. coli. Drosophila HP1 cDNA was cloned
into a pET11a expression vector and expressed in transformed E. coli cells as described under "Experimental Procedures."
Pooled HP1-enriched fractions are shown; MWM, molecular
weight markers. Sizes in kDa are given to the left of the
gel. Lane 1, DEAE-Sepharose fraction; lane 2,
Resource Q FPLC fraction; lane 3, S-200 Sephacryl FPLC
fraction; lane 4, Mono Q FPLC fraction. Fractions enriched
for recombinant HP1 were resolved by 12% SDS-PAGE and stained with
Coomassie Brilliant Blue R.
-mecaptoethanol, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride,
0.4% Triton X-100) and homogenized at 4 °C with a glass-Teflon homogenizer. Homogenate was centrifuged in an Eppendorf 5415 C centrifuge at top speed for 10 min in 4 °C. The supernatant was aliquotted and stored at
70 °C as nuclear extract. Protein
concentration of the extract was found to be about 1 mg/ml with a BCA
protein assay (Pierce).
-32P]ATP at 100 µCi/µmol in 20 mM
Tris-HCl, pH 7.4, 10 mM MgCl2, and 100 mM KCl or 100 mM NaCl (both salts work equally
well). The reaction proceeded at room temperature for 20 min and was stopped by adding 5 µl of 5× SDS-PAGE sample buffer and boiling for
3 min. Products were separated by a 12% SDS-PAGE, and radiolabeled proteins were visualized by exposing the gel to x-ray film or imaging
using a Molecular Dynamics PhosphorImager.
-mercaptoethanol, 1.6% Bio-Lyte 5-8 ampholytes (Bio-Rad),
0.4% Bio-Lyte 3-10 ampholytes (Bio-Rad)) before being applied to an
isoelectric focusing gel. After two-dimensional gel electrophoresis,
proteins were transferred to nitrocellulose (Millipore) membrane using
a Bio-Rad mini-Trans-Blot electrophoretic transfer cell according to
manufacturer's instructions. After transfer, the membrane was blocked
with 1% bovine serum albumin (fraction V, Sigma) in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween
20) at room temperature for 30 min. The anti-HP1 serum was a gift of
Drs. R. F. Clark and S. C. R. Elgin (Washington University, St.
Louis, MO) and was used at a 1:10,000 dilution in TBST buffer. The
secondary antibody was an anti-rabbit IgG-alkaline phosphatase
conjugate (Promega) and was used at a 1:7,500 dilution in TBST buffer.
Detection was with 5-bromo-4-chloro-3-indoyl phosphate and nitro blue
tetrazolium (Promega) as described (24). To detect radiolabeled
protein, Western blots were either subjected to autoradiography using
x-ray film (Kodak XAR-5) or imaged using a Molecular Dynamics PhosphorImager.
20 °C before peptide mapping. Tryptic
peptide mapping was done on a 40% PAGE under alkaline conditions as
described (26, 27). The gel was dried and subjected to autoradiography or PhosphorImager analysis to visualize the phosphopeptides.
206 in which HP1 cDNA is
fused downstream of and in frame with E. coli lacZ under the
Drosophila Hsp70 heat-shock promoter (29), inserted in the P-element vector, pYC1.8 (30).
-Galactosidase Fusion
Protein--
Germline transformation was performed by injecting
v36F;ry506 embryos with each of
the constructs together with the helper plasmid p
25.7wc (31),
essentially as described (32). Go survivors were mated to
v36F;ry506 flies. F1
adults were screened for rescue of the vermilion eye color. Somatic
transformation was performed essentially as described (33, 34). Host
strain in both cases was a
v36F;ry506 stock. Third-instar
larvae were collected from each of the germline and somatic
transformants carrying the pv
206 mutant fusion constructs, heat-shocked at 37 °C for 30 min, and recovered at room temperature for 1 h. Salivary glands were dissected and stained with X-gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside) as
described (35). Stained tissue was placed on a microscope slide and
mounted in 95% glycerol, 5% phosphate-buffered saline.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Mapping of phosphorylation sites in HP1 using
recombinant HP1 protein. A, Western blots of
two-dimensional gels, aligned to show multiple dHP1 isoforms in larval
tissue (top), the single major isoform of rHP1
(middle), and the co-electrophoresis of rHP1 with the most
basic dHP1 isoforms (bottom). B, two-dimensional
gel analysis of rHP1 after in vitro phosphorylation. Western
blot of a two-dimensional gel containing a mixture of dHP1 and
phosphorylated rHP1 (top) and an autoradiograph of the same
blot show the relative mobility of phosphorylated rHP1
(bottom). C, co-electrophoresis of the major
in vitro and in vivo tryptic phosphopeptides.
Phosphorylated rHP1 and dHP1 was labeled, purified, and analyzed as
described under "Experimental Procedures." Phosphopeptides were
imaged using a Molecular Dynamics PhosphorImager. The band between
peptides 1 and 2 was also observed in some digests of in
vitro phosphorylated samples and probably represents an incomplete
digestion product. D, positions of phosphorylated residues
in rHP1, mapped by direct sequencing. The N- and C-terminal amino acids
of HP1 are shown. The relative positions of peptides 1, 2, and 3 from
panel C are shown below. Asterisks
indicate the positions of phosphorylated residues detected by sequence
analysis.
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Fig. 3.
CKII phosphorylates HP1 in
vitro. 1 µg of rHP1 was incubated for 10 min with 1 µl of embryo nuclear extract (treated as indicated) in the presence
of [ -32P]ATP. Proteins were resolved by 12% SDS-PAGE,
and subjected to autoradiography. Phosphorylation of rHP1 by embryo
extract (lane 1) is stimulated by 2 mM spermine
(lane 2). Phosphorylation is inhibited by 1 µg/ml heparin
(lane 3). Phosphorylation of rHP1 by embryo extract
(lane 4) is inhibited by immunodepletion using
anti-Drosophila CKII (lane 6) but not by mock
immunodepletion using a nonimmune serum (lane 5). Higher
molecular weight phosphoproteins represent phosphorylation of embryo
extract proteins.
Ala mutations (S15A,S202A). The mutant HP1 cDNA were fused downstream from, and
in frame with, E. coli lacZ, and the fusion cDNA was
placed under the control of the Drosophila Hsp70 heat shock
promoter. P-element-mediated germline transformations were generated
for the two glutamate substituted mutants. Transgenic larvae were subjected to heat shock, and larval polytene tissue was stained with
X-gal to localize the fusion protein.
-Galactosidase alone is a
cytoplasmic protein when expressed in Drosophila cells (Fig. 4a). As reported previously (29, 40), full-length
HP1·
-galactosidase fusion protein targets
-galactosidase
activity to the nucleus and decorates the heterochromatin with
-galactosidase; this results in blue-staining nuclei with X-gal,
with one or two intensely staining dark blue spots corresponding to the
pericentric heterochromatin (Fig. 4c). For both the S15E and
S202E mutations, X-gal staining showed that these two mutant HP1 fusion
proteins resulted in nuclear localization and heterochromatin binding
indistinguishable from wild-type HP1 fusions (Compare Fig. 4,
e and g-c; see also Refs. 29 and
40).
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Fig. 4.
Mutational analysis of CKII phosphorylation
sites in HP1. Top, codons for Ser-15
(S15) and Ser-202 (S202) were
replaced with codons for either alanine or glutamate. Bottom
(a-h), nuclear and subnuclear localization of
-galactosidase fusion proteins in Drosophila polytene
tissues, detected by X-gal staining. Arrows indicate
heterochromatin staining. a, E. coli
-galactosidase in transgenic salivary gland cells, showing
predominantly cytoplasmic staining; b,
-galactosidase
fused to amino acids 151-206 of HP1, showing nuclear localization but
not heterochromatin binding; c, HP1·
-galactosidase
fusion protein from Platero et al. (29) with wild-type CKII
sites, showing heterochromatin binding; d,
S15A·
-galactosidase fusion protein, showing nuclei from highly
expressing cells in which reduced heterochromatin binding can sometimes
be seen; e, S15E·
-galactosidase fusion protein;
f, S202A·
-galactosidase fusion protein; g,
S202E·
-galactosidase fusion protein; h,
S15A,S202A·
-galactosidase fusion protein. In the nuclei of highly
expressing cells, a faint spot of staining is sometimes seen
(arrowhead in the right
nucleus).
-galactosidase
fusions (29, 40). Somatically transformed third-instar larvae were
heat-shocked, and polytene tissues were dissected out and stained with
X-gal. By this assay, the S202A mutant appeared similar to the wild
type (Fig. 4f), but the S15A mutant showed decreased
heterochromatin binding activity visible only in highly expressing
cells (Fig. 4d). Only in overstained nuclei did we observe
the darkly staining spots that represent heterochromatin binding. For
the double mutation (S15A,S202A), the loss of heterochromatin targeting
was even more dramatic. Although the fusion protein still appeared to
concentrate in the nuclei, it no longer bound to heterochromatin (Fig.
4h). These results demonstrate a requirement for CKII
phosphorylation for efficient heterochromatin binding.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala
mutation on the C-terminal site did not discernibly alter the
heterochromatin binding activity of the mutant fusion protein, the Ser
Ala mutation on the N-terminal site conspicuously reduces
heterochromatin binding. The double Ser
Ala mutation (S15A,S202A)
almost completely eliminated heterochromatin binding, although the
protein could still get into the nucleus. The double mutant appeared to
have a generally more severe effect. However, care should be taken in
interpreting quantitative differences, because levels of fusion protein
expression vary from cell to cell in these assays. Although the effect
of the single C-terminal substitution was not detectable by the X-gal
staining method, it is possible that each mutation exerts some effect
on HP1 heterochromatin binding activity because the combined mutations
had the most dramatic effect on heterochromatin binding. Although there
are two CKII sites at the C terminus of HP1, we chose to mutate the
more downstream site. The upstream site is dependent on the
phosphorylation of the downstream serine, so when we mutated the first
serine to alanine, we also disabled the second one as a CKII target.
Thus, in our assay, any effect attributable to the downstream serine could also reflect a requirement for phosphorylation of the upstream serine. Experiments are in progress to identify additional sites of HP1
phosphorylation and to test their role in HP1 localization and
silencing activity.
Glu mutations in the
N or C terminus, we were unable to recover germline transformants with
Ser
Ala mutations. The significance of this finding is unclear, but
it may represent a kind of "dominant negative" phenotype (46). In
the absence of heat shock, basal levels of wild-type
HP1·
-galactosidase fusion protein are not toxic (29, 40), although
such transgenic lines are not as healthy as wild-type flies.3 Mutant HP1 fusion protein, on the other hand, may
be toxic at low basal levels. A reasonable speculation is that the
nonphosphorylated HP1 participates in only some
HP1-dependent activities or sequesters heterochromatin
factors in an inactive form.
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ACKNOWLEDGEMENTS |
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We thank R. Bell for constructing the pET11a-based HP1 expression vector and for preliminary expression and purification studies, Y.-H. Chang and J. S. Huang for advice and help in rHP1 purification and phosphopeptide analysis, R. Islam and S. Tomatsu for help with the mutagenesis, J. Ma for help with molecular biology and fly stock-keeping, S. C. R. Elgin for anti-HP1 serum and frozen embryos, and C. V. C. Glover for anti-CKII serum. We also thank Y.-H. Chang, A. Shilatifard, and A. Waheed for critical reading of the manuscript.
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FOOTNOTES |
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* This project was supported by National Science Foundation Grant IBN 9506103.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Edward A. Doisy Dept.
of Biochemistry and Molecular Biology, St. Louis University Health
Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104-1079. Tel.: 314-577-8154; Fax: 314-577-8156; E-mail:
eissenjc{at}wpogate.slu.edu.
2 C. V. C. Glover, personal communication.
3 T. Zhao and J. C. Eissenberg, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
HP1, heterochromatin-associated protein 1;
dHP1, HP1 from
Drosophila;
rHP1, recombinant HP1;
CKII, casein kinase II;
FPLC, fast protein liquid chromatography;
PAGE, polyacrylamide gel
electrophoresis;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
HPLC, high pressure liquid
chromatography.
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REFERENCES |
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