From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
Received for publication, November 6, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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HP1 is an essential heterochromatin-associated
protein in Drosophila. HP1 has dosage-dependent
effects on the silencing of euchromatic genes that are mislocalized to
heterochromatin and is required for the normal expression of at least
two heterochromatic genes. HP1 is multiply phosphorylated in
vivo, and HP1 hyperphosphorylation is correlated with
heterochromatin assembly during development. The purpose of this study
was to test whether HP1 phosphorylation modifies biological activity
and biochemical properties of HP1. To determine sites of HP1
phosphorylation in vivo and whether phosphorylation affects
any biochemical properties of HP1, we expressed Drosophila
HP1 in lepidopteran cultured cells using a recombinant baculovirus
vector. Phosphopeptides were identified by matrix-assisted laser
desorption ionization/time of flight mass spectroscopy; these peptides
contain target sites for casein kinase II, protein tyrosine kinase, and
PIM-1 kinase. Purified HP1 from bacterial (unphosphorylated) and
lepidopteran (phosphorylated) cells has similar secondary structure.
Phosphorylation has no effect on HP1 self-association but alters the
DNA binding properties of HP1, suggesting that phosphorylation could
differentially regulate HP1-dependent interactions.
Serine-to-alanine and serine-to-glutamate substitutions at consensus
protein kinase motifs resulted in reduction or loss of silencing
activity of mutant HP1 in transgenic flies. These results suggest that
dynamic phosphorylation/dephosphorylation regulates HP1 activity in
heterochromatic silencing.
Heterochromatin protein 1 (HP1)1 is a
heterochromatin-associated protein in Drosophila
melanogaster (1, 2) and a context-dependent regulator
of transcription. HP1 has dosage-dependent effects on the
silencing of euchromatic genes (3-5), and it is required for the
proper expression of certain heterochromatic genes (6). HP1 is
essential in Drosophila (5); mutations cause recessive late
larval lethality, associated with misregulation of essential heterochromatic genes and mitotic defects (6-8). HP1-like proteins have been identified in yeast, nematodes, insects, amphibians, and
mammals (reviewed in Ref. 9). HP1 family proteins in
Schizosaccharomyces pombe and mouse have also been shown to
participate in position-dependent silencing (10, 11).
HP1 contains two copies of a structural motif called the chromo domain,
connected by a short "hinge domain." The chromo domain structure
consists of three HP1 is multiply phosphorylated by serine-threonine kinases in
Drosophila (27). Hyperphosphorylation of HP1 correlates with heterochromatin assembly during development. In Tetrahymena,
the HP1 family protein Hhp1p becomes hyperphosphorylated in response to
starvation; this is associated with reduction in macronuclear volume
and increased chromatin condensation (28). Human HP1 family proteins
are phosphorylated in vivo (29) and have been shown to be
substrates in vitro for multiple kinases (21, 30).
Huang et al. (31) recovered differentially phosphorylated
HP1 in distinct protein complexes from Drosophila embryo
extracts, suggesting that phosphorylation could regulate the assembly
of HP1 into higher order chromatin structures. This study also found a
correlation between the extent of HP1 phosphorylation and salt extractability from embryo nuclei. These findings suggest that HP1
phosphorylation regulates distinct macromolecular interactions in
vivo.
Consensus kinase target sites are found near the N- and C-terminal ends
of Drosophila HP1 and in the hinge domain between the chromo
domain motifs. We recently reported that casein kinase II (CKII) from
embryo nuclear extract phosphorylates HP1 at three target sites
in vitro and that alanine substitutions for CKII target
serines interfere with efficient heterochromatin targeting in a
transient expression assay (32). Our study did not test the
transcriptional regulatory activity of mutant HP1 proteins.
In this report, we compare the biochemical properties of phosphorylated
and unphosphorylated HP1. We map sites of phosphorylation in HP1 by
expressing Drosophila HP1 in lepidopteran cells using a
recombinant baculovirus. MALDI-TOF mass analysis of proteolytic cleavage products identified products consistent with phosphorylation at consensus casein kinase II sites and a protein tyrosine kinase consensus, as well as a phosphopeptide containing a putative PIM-1 kinase target site. We find that phosphorylation leads to no
significant change in HP1 secondary structure or self-association but
inhibits HP1 binding to DNA in vitro. Amino acid
substitutions in consensus phosphorylation sites reduced or eliminated
silencing activity in transgenic flies, implicating phosphorylation in
the regulation of HP1 in vivo.
Expression and Purification of Recombinant HP1--
HP1 was
expressed in Escherichia coli and purified as described
previously (32). The BacPAKTM Baculovirus Expression system
(CLONTECH) was used to express HP1 in Sf21
cells. An XbaI-KpnI fragment with HP1 cDNA
was inserted into the pBacPAK8 vector, and infections were performed
according to manufacturer's instructions. Cells were grown at room
temperature. After 5 days of infection, the cells were harvested, and
HP1 expression was verified by Western blot analysis. For both the
bacterially expressed and baculovirus expressed proteins, the glycine
residue that is normally Gly2 in HP1 is preceded by the
amino acid sequence MARVDL in both recombinant proteins.
To purify HP1 from the infected cells, 10 75-cm2 flasks of
cells or 500 ml of suspension cell culture was infected with passage two virus. After 5 days, the cells were collected by centrifugation at
1000 × g for 5 min. The cell pellet was lysed in a
buffer of 20 mM HEPES, pH 7.4, 0.1 M NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1 mM NaF, 0.1 M
Na3VO4, 0.1% Triton X-100 by sonication on
ice. The lysate was centrifuged at 18,000 rpm in an SS34 rotor
(Sorvall) for 30 min, and the supernatant liquid was applied to a 1-ml
DEAE column. Proteins were eluted with 0.5 M NaCl in lysis
buffer from the column and diluted to 0.1 M NaCl in the
same buffer. The sample was then loaded onto a 1-ml Mono Q column on a
Smart System high pressure liquid chromatography (Amersham Pharmacia
Biotech). The column was developed with a 0.1-0.5 M NaCl
gradient, with HP1 eluting in a single peak at about 0.4 M
NaCl. Two-dimensional gel electrophoresis was done as described
(27).
Phosphatase treatment of baculovirus-expressed HP1 was performed as
described (27). Briefly, 2 µg of potato acid phosphatase (0.8 units/mg; Sigma) was added to a 20-µl solution of 50 mM
Tris-HCl, pH 6.5, containing 2 µg baculovirus-expressed HP1, and the
mixture was incubated at room temperature for 15 min. 4 µl of 10×
shrimp alkaline phosphatase buffer (200 mM Tris-HCl, pH
8.0, 10 mM MgCl) and 1 µl of shrimp alkaline phosphatase
(Amersham Pharmacia Biotech; 1 units/µl) was added to the reaction,
and the volume was brought to 40 µl with distilled water. After 15 min of incubation at room temperature, the sample was stored at
Circular Dichroism--
CD spectra were recorded using AVIV 60DS
CD spectrophotometer. Five spectra were collected every 0.5 nm at room
temperature for each sample. The spectra were then averaged and
corrected for the buffer.
DNA Binding and Cross-linking Assays--
Purified HP1 (10 µM) was mixed with 1 mM disuccinimidyl
suberate (Pierce) in the presence of 0.2 M NaCl and 0.05%
bovine serum albumin and was incubated on ice for 15 min. The reaction
was stopped by adding SDS-PAGE sample buffer and boiling for 5 min. After electrophoresis, the proteins were transferred to nitrocellulose membrane (Millipore) using a Mini-TransBlot electrophoretic transfer cell (Bio-Rad) according to the manufacturers' 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 rabbit anti-HP1 serum was a gift of Dr. S. C. R. Elgin (Washington University) 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 1:7500 dilution in
TBST buffer. Detection was with 5-bromo-4-chloro-3-indolyl phosphate
and nitro blue tetrazolium (Promega) as described (33). DNA binding
assay was performed as described (17), using a 146-bp sea urchin 5 S
rDNA end-labeled with the fluorescent dye Cy5.
Proteolytic Digestions of HP1 for Mass Spectroscopy--
Two
methods were used to prepare peptides for MALDI-TOF mass spectroscopy.
One method involved solution digestion by one of three enzymes. (i) For
trypsin of Glu-C, 5 µg of HP1 was mixed with 0.25 µg of trypsin
(Sigma) or Glu-C (Sigma) in a 20-µl buffer of 25 mM
NH4HCO3, pH 8.0. The mixture was incubated at
37 °C overnight. (ii) For Lys-C, 5 µg of HP1 was mixed with 0.25 of Lys-C (Sigma) in 20 µl of 25 mM Tris-HCl, pH 7.7, 1 mM EDTA. The mixture was incubated at 37 °C for 24 h. After digestion, the peptides were purified and desalted by C18
reverse phase chromatography (ZipTip; Millipore), dried in a vacuum,
and subject to mass spectroscopy. The second method for peptide
preparation was to subject intact HP1 to SDS-PAGE, transfer the protein
to polyvinylidene difluoride membrane electrophoretically, localize
transferred protein by transient staining with 0.5% Ponceau S for
30 s, wash briefly with water, and excise the region of
membrane-containing protein. The membrane fragment was further sliced
into small pieces, washed in water until free of stain, and incubated
in 200 µl of 0.1% polyvinylpyrrolidone 40 (Sigma) in methanol for 30 min. The blocked membrane was then incubated in 200 µl of water at
37 °C for 30 min, rinsed three more times in water, and submerged in
~20 µl of protease in appropriate buffer for overnight digestion at
37 °C overnight to achieve extensive digestion, or for 1-2 h at
room temperature to yield a higher fraction of partial digestion
products. Following digestion, the sample was vortexed briefly, and the supernatant liquid was recovered. The membrane fragments were washed
once in 20 µl of water or 10% acetonitrile, and this supernatant was
combined with the first for mass spectroscopy.
MALDI-TOF Analysis--
All MALDI spectra were obtained using a
Voyager Biospectrometry Work station (Perspective Biosystems Inc.,
Framingham, MA) in positive ion linear mode. Three matrices were used
as follows: sinapinic acid was used for intact HP1 analysis, and
Site-directed Mutagenesis and Construction of the Transgene
Expression Vectors--
Site-directed mutagenesis was performed using
the TransformerTM site-directed mutagenesis kit
(CLONTECH) according to the manufacturer's instructions. Site-directed mutagenesis was performed directly on a
pYC1.8-containing HP1 cDNA under the Hsp70 heat shock
promoter (34).
Genetic Assays of Phosphorylation Site Mutations--
To measure
the ability of the different kinase target mutations to affect
heterochromatic position-effect silencing, the following crosses were
performed: In (1)wm4/In(1)wm4;
Su(var)2-101/InCyRoi × v/Y; transgene/Sco;
ry506 (when the transgene is on the second
chromosome) or In 1)wm4/In(1)wm4;
Su(var)2-101/InCyRoi × v/Y; transgene
ry506/Sb ry506 (when the
transgene is on the third chromosome), where "transgene" refers to
either wild-type or mutant HP1 cDNA transgene constructs. Progeny
from each cross were heat-shocked twice daily at 37 °C for 45 min
throughout development. Heat shocks were spaced at least 8 h
apart. Sons were collected, aged for 3 or more days, and red eye
pigment was extracted and measured spectrophotometrically according to
the method of Ephrussi and Herold (35). Pigment values are expressed as
percentage of wild-type (Canton S) red eye pigment, and each
measurement was made using a minimum of 30 fly heads.
HP1 Is Multiply Phosphorylated When Expressed from Recombinant
Baculovirus--
Most of HP1 phosphorylation occurs at serines and
threonines (27). Inspection of the amino acid sequence of HP1 reveals consensus target sites for several different protein kinases (Fig. 1). We previously showed that the N- and
C-terminal CKII consensus sites are phosphorylated in vitro
by an embryo nuclear extract CKII activity, suggesting that these sites
are used in vivo. However, the CKII sites cannot explain the
7-8 distinct HP1 isoforms previously observed in whole tissue extracts
(27). To identify sites of HP1 phosphorylation, we expressed HP1 in
Sf21 lepidopteran (Spodoptera frugiperda; the fall
armyworm) cells using recombinant baculovirus. We reasoned that since
HP1 is highly conserved, and since lepidoptera are insects, these cells
would likely phosphorylate HP1 at the same sites as for HP1 expressed
in Drosophila. Fig. 2 shows
two-dimensional gel analysis of total cellular protein from Sf21
cells infected with recombinant baculovirus expressing HP1. At least
seven HP1 isoforms are visible, the most basic of which represents
unphosphorylated HP1. We refer to the baculovirus-expressed recombinant
HP1 as "SfHP1" and to the bacterially expressed recombinant HP1 as
"rHP1." Previous work (32) suggested that rHP1 is unphosphorylated, so rHP1 was used as a reference polypeptide with which SfHP1 was compared.
After purifying Sf HP1 from infected cells (see "Experimental
Procedures"), we used matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy to examine the
posttranslational modifications in SfHP1. A single major mass is
present in the rHP1 preparation (Fig.
3A) corresponding to the
expected mass of HP1 minus the N-terminal methionine, confirming that
rHP1 is not posttranslationally modified beyond N-terminal processing. In contrast, the SfHP1 is distributed over multiple masses (Fig. 3B), many separated by the mass of one phosphate (80) and
all major masses larger than the mass of HP1. After phosphatase
treatment, the SfHP1 mass profile collapses into a single major peak
whose mass is equivalent to that of HP1 minus the N-terminal methionine (Fig. 3C). Thus, SfHP1, like the endogenous HP1 in
Drosophila, is multiply phosphorylated. This analysis also
shows that there are no significant posttranslational modifications in
SfHP1 besides phosphorylation and N-terminal processing.
Mapping Sites of Phosphorylation in HP1 Using MALDI-TOF--
To
determine whether consensus kinase sites in HP1 are phosphorylated
in vivo, we looked for evidence that these motifs are phosphorylated in SfHP1. We digested purified SfHP1 with different endopeptidases and subjected the peptides to MALDI-TOF mass analysis to
identify phosphopeptides (36).
Phosphopeptides were identified that contained each of the consensus
CKII sites (Fig. 4, A and
B). We previously showed that these sites are phosphorylated
in bHP1 by an embryo nuclear extract in vitro; these results
strongly suggest that the CKII consensus sites are significantly
phosphorylated in vivo. Interestingly, a LysC peptide
containing the N-terminal CKII site was found to be doubly
phosphorylated (Fig. 4A). This peptide also contains a
consensus protein tyrosine kinase (PTK) motif. Platero et
al. (34) showed that the conservative substitution of
phenylalanine for tyrosine in this PTK motif resulted in loss of
silencing activity of the mutant protein. The detection of a
doubly-phosphorylated peptide containing both a CKII and PTK motif
suggests that the consensus PTK site is used in vivo.
Unexpectedly, we found a LysC peptide whose mass corresponds to that of
a singly phosphorylated internal peptide of HP1 without a consensus
kinase motif. While this study was in progress, Koike et al.
(30) reported that the human HP1 family protein HP1Hs
We found no consistent evidence for phosphorylation at either of the
two PKA/CaCKII consensus sequences or at the PKC consensus sequence motif.
Phosphorylation Does Not Affect HP1 Secondary Structure or
Dimerization in Vitro--
To test whether phosphorylation causes
significant changes in HP1 structure in solution, circular dichroism
spectra were obtained using purified rHP1 (unphosphorylated) and SfHP1
(phosphorylated). No significant differences were observed (Fig.
5A), indicating that
phosphorylation does not significantly affect HP1 secondary structure.
The HP1 C-terminal chromo domain can dimerize in vitro (16),
which could account for self-association of rHP1 in solution (17). To
test whether phosphorylation can affect HP1 self-association, we
exposed solutions of purified rHP1 and SfHP1 to the bifunctional cross-linking agent dissuccinimidyl suberate and then separated proteins by SDS-PAGE and immunolocalized HP1 by Western blot. As shown
in Fig. 5B (compare lanes 2 and 4),
both SfHP1 and rHP1 preparations are cross-linkable as dimers to
similar extents, indicating that phosphorylation has no significant
effect on the efficiency of HP1 self-association.
Phosphorylation Inhibits HP1 Binding to DNA--
We showed
previously that rHP1 binds to DNA in vitro (17). To test
whether phosphorylation affects DNA binding activity of HP1, we
compared rHP1 and SfHP1 binding to a 146-bp DNA fragment in
vitro. As shown in Fig.
6A, only rHP1 detectably binds
DNA. To test whether the SfHP1 protein preparation contains an
inhibitor of DNA binding, equal amounts of each protein were mixed and
challenged to bind DNA; the mixture retards DNA mobility somewhat more
than rHP1 alone, suggesting that SfHP1 contributes to a band shift, but
only in the presence of unphosphorylated HP1. A likely interpretation of this is that SfHP1 can form heterotypic complexes with rHP1 but does
not contribute directly to DNA binding.
Surprisingly, phosphatase treatment of SfHP1 fails to restore DNA
binding activity (data not shown). Judging from the MALDI mass
analysis, phosphatase treatment of SfHP1 yields a spectrum nearly
identical to that of unphosphorylated rHP1, with no evidence of
residual modifications. This suggests that phosphorylation causes
tertiary conformational changes in HP1 structure that inhibit DNA
binding and that this structural modification remains after phosphatase
treatment. Consistent with this possibility, we find that
dephosphorylated SfHP1 becomes insoluble.
To test the contribution of CKII phosphorylation to HP1 DNA binding
activity, mutant HP1 protein in which both target serines were
substituted with either alanine or glutamate (to mimic phosphorylation) were expressed in E. coli and purified. As shown in Fig.
6B, the double alanine mutation band-shifts labeled DNA with
similar efficiency to wild-type HP1, but the double glutamate mutation
band-shifts DNA very poorly and with a distinct mobility from the
wild-type protein. This suggests that CKII phosphorylation of HP1
contributes significantly to the loss of DNA binding in phosphorylated
HP1 protein.
Mutations in HP1 Phosphorylation Sites Abolish or Antagonize
Silencing Activity--
To test whether HP1 phosphorylation is
required for heterochromatin-mediated gene silencing in
vivo, we replaced serines with either alanines (to block
phosphorylation) or glutamate (to mimic constitutive phosphorylation)
at sites containing serine/threonine kinase consensus motifs.
Ser15 and Ser202 fall within consensus motifs
for CKII. Furthermore, these sites were shown previously to be
phosphorylated in vitro by Drosophila embryo
nuclear extract (32). To test the role of CKII consensus motifs in
silencing, serine codons 15 and 202 were replaced with alanine or
glutamate codons. Similarly, serines 89-91 and 102-104 are found
within consensus motifs for PKA and CaCKII. In these cases, three
serines were simultaneously replaced with three alanines or three
glutamates. Transgenic lines were established for these mutant HP1
proteins using P-element mediated germ line transformation.
Mutant proteins were tested by expression under an Hsp70
heat shock promoter in transgenic flies carrying the
white-variegating chromosome inversion
In(1)wm4. The dominant suppressor of position effect
variegation Su(var)2-1 was included in the background to
give a strong background of pigmentation on which enhancement can
readily be detected. If the mutant protein retains wild-type silencing
function, transgene expression will enhance the silencing of the
white gene in the In(1)wm4 inversion,
giving flies with reduced levels of red eye pigment compared with sibs
that lack the transgene. Heat shock resulted in similarly elevated
levels of HP1 antigen by Western blot in all transgenic lines,
indicating that mutant proteins were expressed at comparable levels
(data not shown).
Alanine and glutamate substitutions at either the N-terminal or
C-terminal CKII sites resulted in proteins that had little or no
ability to enhance position effect silencing (Fig.
7). Surprisingly, simultaneous
substitution of alanines or glutamates at both target sites results in
a protein that retains some silencing activity. However, the magnitude
of the silencing conferred by CKII mutants (1.5-2-fold) was less than
that seen for wild-type HP1 overexpression from the same promoter
(~3.5-fold), suggesting that the CKII mutant proteins are not fully
functional in this assay. Similarly, an S15E/S202A double mutation also
has reduced silencing activity.
A triple alanine substitution at the upstream PKA/CaCKII consensus
motif (S89A/S90A/S91A) results in loss of silencing activity, suggesting that these serines are used in vivo (Fig. 7).
Strikingly, a triple alanine substitution at the downstream PKA/CaCKII
consensus motif (S102A/S103A/S104A) results in protein that
suppresses silencing in two of three transgenic lines (Fig.
7). Since this is the opposite of the normal activity of HP1, this
mutation results in an antimorphic or "dominant negative" phenotype
suggesting that the mutant proteins antagonize heterochromatin in
vivo (37). Finally, a triple glutamate substitution at the
downstream consensus also results in a protein defective in silencing.
Thus, both PKA/CaCKII motifs are functionally important in HP1
silencing activity.
Our mutational studies (summarized in Table
I) strongly implicate multiple kinases in
the regulation of HP1 silencing activity in vivo. The fact
that silencing activity is reduced or lost with mutations that block
phosphorylation as well as with mutations that mimic constitutive
phosphorylation suggests that both phosphorylated and unphosphorylated
isoforms are functionally important and that HP1 phosphorylation is
dynamic. This is consistent with a previous observation that HP1
phosphorylation continues to occur in the absence of nascent protein
synthesis (27). We cannot exclude, however, that kinase target site
mutations may exert their effects by affecting HP1 structure or HP1
protein-protein interactions required for silencing.
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-strands packed against an
-helix (12, 13), a
motif found in an archeal DNA-binding protein (14, 15). The C-terminal
chromo domain self-associates in vitro (16). HP1 binds DNA
and nucleosomes in vitro (17). Heterotypic interactions
between HP1 family proteins and silencing factors (18-22), nuclear
membrane proteins (23, 24), and replication factors (25, 26) have also
been reported, suggesting that HP1 family proteins may participate in
multiple distinct nucleoprotein complexes in vivo.
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70 °C until use.
-cyano-4-hydroxycinnaminic acid and 2.5-dihydroxybenzoic acid were
used for peptides. 10 mg/ml sinapinic acid or
-cyano-4-hydroxycinnaminic acid was dissolved in 50% acetonitrile,
0.3% trifluoroacetic acid. 25 mg/ml 2.5-dihydroxybenzoic acid was
dissolved in 0.6% trifluoroacetic acid, 33% acetonitrile. Typically,
0.1-5 pmol of protein or peptide was dried and resuspended in 2.5-10
µl of matrix and then 1-2 µl was immediately spotted on the sample
plate. Spectra were obtained from different regions of the dried
sample, and representative spectra were selected for further analysis.
Time-to-mass conversion was achieved by internal calibration using
angiotensin II (Sigma; m/z = 1046.54), Peptide
Sequencing Standard (Sigma, catalog number P2046; m/z = 1657.84), and specific HP1 peptides.
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Fig. 1.
Consensus kinase target sites in HP1.
The consensus target sites for serine/threonine kinases and protein
tyrosine kinase are highlighted over a bar representing
Drosophila HP1. Relative positions of the chromo domain
(striped bar) and chromo shadow domain
(stippled bar) motifs are indicated.
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Fig. 2.
Two-dimensional gel analysis of recombinant
baculovirus-expressed HP1. Whole cell proteins were extracted,
fractionated by two-dimensional gel electrophoresis, and stained with
Coomassie Blue. Asterisks are placed over each HP1 isoform.
Top, total cell lysate. Bottom, unphosphorylated
bacterially expressed HP1 (~1 µg) was added to the lysate as a
mobility standard (arrow), demonstrating that
baculovirus-expressed HP1 isoforms are more negatively charged.
Identities of the HP1 spots were independently confirmed by Western
blot using anti-HP1 serum.
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Fig. 3.
MALDI-TOF mass analysis of recombinant
HP1. A, rHP1 is unphosphorylated as judged by
two-dimensional electrophoresis (32). The mass of 23,593.5 coincides
with the mass of HP1 minus the N-terminal methionine (23,594.8). The
asterisk denotes a cluster of peaks centered at 23,818, corresponding to the expected mass of HP1 minus the N-terminal
methionine plus a single molecule of matrix adduct. B, SfHP1
is extensively modified. Arrow labeled "0" indicates the
mass position for unphosphorylated HP1 minus the N-terminal methionine.
Numbered arrows indicate the expected positions for mono-,
di-, tri-, tetra-, penta-, and hexaphosphorylated HP1 masses.
C, SfHP1 after treatment with phosphatase. Again, the major
peak at 23,595.0 corresponds to unphosphorylated HP1 minus the
N-terminal methionine, and the asterisk denotes the position
of the sinapinic acid adduct of this protein.
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Fig. 4.
MALDI identification of phosphopeptides in
baculovirus expressed HP1. A, an N-terminal LysC
peptide containing CKII and PTK consensus sites is phosphorylated. Peak
labeled a has a mass of 2781 and corresponds to the peptide
R[126-151]K. B, C-terminal peptides containing a CKII
site are phosphorylated. Left, tryptic peptide
peak a has a mass of 1223 and corresponds to the
peptide M[194-202]R. Right, GluC peptide peak
b has a mass of 1346 and corresponds to the peptide Y[41-50]E.
C, linker domain peptide containing PIM-1 kinase target
sites is a phosphorylated LysC peptide. Peak a has a
mass of 1497 and corresponds to the peptide E[101-115]K; peak
b has a mass of 1391; its identity is unknown.
is
a target for the pim-1 kinase proto-oncogene.
Multiple sites of Pim-1 phosphorylation occur in HP1Hs
,
at and downstream from the sequence motif KRKS in the hinge region.
Fig. 4C shows that a peptide containing the single KRKS motif, which also occurs in the hinge domain of
Drosophila HP1, is singly phosphorylated.
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Fig. 5.
Phosphorylation does not affect HP1 secondary
structure or self-association. A, circular dichroism
was determined for rHP1 (dashed line) and SfHP1 (solid
line). Each line represents the average of five
separate measurements. B, purified recombinant HP1 expressed
in bacteria (lanes 1and 2) or in Sf21
cells (lanes 3 and 4) was cross-linked in
solution. Cross-linked samples are shown in lanes
2 and 4. Proteins were then resolved by 10%
SDS-PAGE and subjected to Western blot analysis using an anti-HP1
serum. Lane M shows the relative mobilities of protein
standards whose masses are given on the left of the
panel.
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Fig. 6.
Phosphorylation inhibits HP1 binding to
DNA. A, 10 µM bacterially expressed
(1st and 4th lanes) or baculovirus
expressed protein (3rd and 4th
lanes) was mixed with 10 nM Cy5-labeled 146-bp
DNA and then electrophoresed in a 8% native polyacrylamide gel. Note
that the baculovirus expressed protein alone does not shift the DNA but
enhances the shift caused by bacterially expressed HP1 (compare
2nd and 4th lanes). B, 10 µM wild-type HP1, HP1 (S15A/S202A), or HP1 (S15E/S202E)
was mixed with 10 nM Cy5-labeled 146-bp DNA and
electrophoresed in an 8% native polyacrylamide gel.
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Fig. 7.
Mutations in HP1 phosphorylation sites reduce
or abolish silencing activity. Males hemizygous for a transgene
expressing wild-type or mutant HP1 were crossed to
In(1)wm4/In(1)wm4;
Su(var)2-101/InCyRoi females. Vials
were heat-shocked twice daily throughout development to express the
transgenic HP1. Red eye pigment was extracted from sons with and
without the transgene. A minimum of 30 flies was used for each
genotype, and typical standard deviations were 10% (never more than
20%). For each cross, a ratio of transgene-bearing to control siblings
was calculated. This ratio is log-transformed for graphical
presentation.
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Summary of consensus kinase target site data for HP1
Functional Significance of HP1 Phosphorylation-- A recent study found a correlation between the extent of HP1 phosphorylation in embryos and salt extractability (31). Extensively phosphorylated HP1 was extractable at relatively low salt, while high salt concentration was required to solubilize hypophosphorylated HP1. Hypophosphorylated HP1 was also found associated with a high molecular weight (~1 MDa) complex, while extensively phosphorylated HP1 was found in small (<100 kDa) complexes. The functional significance of differential salt extractability and molecular complex formation is unknown, but these observations suggest that phosphorylation may regulate HP1 trafficking in the nucleus. We reported previously that mutations in CKII sites at either end of HP1 interfered with efficient heterochromatin targeting in vivo (32), consistent with this inference. The results reported here showing reduced HP1 silencing activity for CKII site alanine and glutamate substitutions further support the inference that CKII phosphorylation is important to the mechanism of HP1-mediated heterochromatin formation.
Pim-1 kinase was found to associate with the human HP1 family protein
HP1Hs in a yeast two-hybrid protein assay.
pim-1 is a proto-oncogene with multiple targets (38,
39). The functional significance of Pim-1 phosphorylation is unknown,
but Pim-1 expression is correlated with cell proliferation in several
systems. Since previous work indicated that several serines are
targeted downstream of the Pim-1 kinase consensus target motif in
HP1Hs
(30), we did not test mutations in the two serines
and single threonine within the Pim-1 consensus peptide. The D. melanogaster genome contains three Pim-1 kinase homologs (CG3105,
CG11870, and CG8201). No mutations are known for any of these genes, so it is not possible to test the role of these putative kinases in HP1
phosphorylation or heterochromatic silencing.
Human HP1 family proteins associate with the silencing transcriptional
intermediary factors TIF1 and TIF1
, and all three human HP1-like
proteins are substrates for the intrinsic kinase activity of these
factors (21). The physiological significance of these associations,
however, are unknown, as are the target sites for phosphorylation in
any of the human HP1s. The D. melanogaster genome contains
six putative genes with significant homology to TIF1 family proteins
(bonus, GH10646, CG 14306,
CG 1624, CG 3815, and CG 8419). Of
these, only bonus has known mutant alleles (40). bonus is a late larval recessive lethal required for
peripheral nervous system, midgut, fat body, and cuticle development
(41). It will be interesting to test whether HP1 phosphorylation is affected in bonus mutant embryos or larvae.
Unexpectedly, serine-to-alanine substitutions in the hinge region
connecting the chromo and chromo shadow domains result in proteins that
interfere with heterochromatic silencing. This dominant negative
activity suggests that mutant proteins are sequestering certain factors
in heterochromatin but are unable to function in a competent silencing
complex. Thus, phosphorylation in the hinge domain is also implicated
in HP1 regulation in vivo, although we were unable to detect
significant amounts of phosphopeptides containing either of the
PKA/CaCKII consensus motifs using MALDI analysis of
baculovirus-expressed HP1. DNA binding activity has been localized to
the hinge domain in the human HP1 family protein HP1Hs
(42), suggesting that phosphorylation in the hinge could act by
regulating HP1-DNA contacts. We were unable to delimit DNA binding
activity in rHP1 to a single domain (17), suggesting that multiple
contacts mediating DNA binding are dispersed in Drosophila
HP1. Further studies on HP1-DNA interaction, as well as interactions
between HP1 and histone and nonhistone proteins, will be required to
determine the mechanism of phosphorylation-mediated HP1 regulation.
Identification of Sites of HP1 Phosphorylation in HP1-- We have identified phosphopeptides consistent with four distinct sites of HP1 phosphorylation. Together with the unphosphorylated HP1 isoform, this accounts for five of the seven isoforms visible in the two-dimensional gel of baculovirus-expressed HP1. The mutational results suggest that both PKA/CaCKII consensus kinase sites may also be used, which could account for two more spots. There are several possible reasons why phosphopeptides containing these motifs were not detected in our MALDI-TOF analysis. 1) These sites may only be phosphorylated in a small fraction of the total purified protein. 2) Phosphopeptides containing these sites may not desorb and/or ionize efficiently during the MALDI procedure. 3) These sites may not be efficiently phosphorylated in S. frugiperda cells. Finally, our studies cannot rule out additional sites of phosphorylation in vivo. The two-dimensional electrophoresis pattern only reveals the number of modifications in a given isoform, not the sites of modification, so in principle there could be more than six potential phosphorylation sites.
The distributions of phosphorylated peptides and consensus motifs implicate at least four classes of protein kinases in the phosphorylation of HP1: CKII, PKA/CaCKII, protein tyrosine kinase, and Pim-1 kinase. All of these kinases are known to have multiple nuclear targets that include transcriptional regulators. Thus, HP1 joins a substantial fraction of nuclear factors as a transcriptional regulator subject to posttranslational regulation by phosphorylation.
Biochemical Consequences of HP1 Phosphorylation-- We found that phosphorylation of HP1 has no detectable effect on HP1 secondary structure or dimerization but strongly interferes with HP1 binding to DNA. Serine-to-glutamate substitutions at the CKII consensus target serines in HP1 have a severe effect on DNA binding of rHP1 in vitro, suggesting that CKII phosphorylation contributes significantly to the loss of SfHP1 DNA binding activity. A simple mechanism by which phosphorylation could alter DNA binding is by contributing to electrostatic repulsion between a phosphorylated domain and the DNA sugar-phosphate backbone. Such a mechanism would be expected to have little effect on the protein secondary structure, which is what we observe. Thus, phosphorylation can provide a mechanism to regulate selectively HP1-DNA interactions while preserving HP1-protein contacts.
Phosphorylation is known to control the activity of several
transcription factors (43, 44). In some cases, phosphorylation has been
shown to regulate DNA binding activity. For example, phosphorylation of
the POU transcription factor GHF-1 inhibits DNA binding activity (46),
and phosphorylation of the zinc finger transcription factor SP1 by
casein kinase II reduces its DNA binding activity (45). Phosphorylation
of the architectural transcription factor HMG-I reduces its binding to
DNA and nucleosomes in vitro (47). Hyperphosphorylation of
the yeast DNA damage-responsive transcription factor Crt1 inhibits its
ability to bind DNA (48). Calcium-dependent phosphorylation
inhibits DNA binding by the Ets-1 transcription factor 50-fold by
stabilizing an inhibitory secondary structure in the Ets-1 protein
(49). We found that phosphorylated HP1 lacks detectable DNA binding
activity in vitro, suggesting that phosphorylation could
regulate HP1 in vivo by modulating its ability to bind
chromatin. In addition, phosphorylation may regulate interactions
between HP1 and other heterochromatin-associated proteins. As
candidates for HP1-associated proteins are identified, the role of HP1
phosphorylation in modulating such associations should be tested.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. C. R. Elgin for HP1 antibody; Dr. J. C. Lee for providing the circular dichroism measurements; Dr. Y.-H. Chang for helpful insights into MALDI mass spectroscopy and its interpretation; Drs. A. Waheed and D. Dorsett for critical reading of the manuscript; and Dr. A. Waheed for much helpful discussion on methodology and interpretation and for encouragement.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R55 GM57005 (to J. C. E.).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 School of
Medicine, 1402 South Grand Blvd., St. Louis, MO 63104 Tel.:
314-577-8154; Fax: 314-577-8156; E-mail: eissenjc@slu.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010098200
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ABBREVIATIONS |
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The abbreviations used are: HP1, heterochromatin protein 1; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; CKII, casein kinase II; PAGE, polyacrylamide gel electrophoresis; bp, base pair; PTK, protein tyrosine kinase; PKA, cAMP-dependent protein kinase; CaCKII, calcium/calmodulin-dependent kinase II.
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