phosphorylation site mutations in heterochromatin protein 1 (hp1
TRANSCRIPT
(HP1) Reduce or Eliminate Silencing Activity
Tao Zhao, Tomasz Heyduk, and Joel C. Eissenberg*
Edward A. Doisy Department of Biochemistry
and Molecular Biology
1402 South Grand Blvd.
St. Louis, MO 63104
*corresponding author
Ph: (314)577-8154
FAX: (314)577-8156
Email: [email protected]
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on December 19, 2000 as Manuscript M010098200 by guest on A
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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 MALDI-TOF 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.
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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 S. 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 ß-strands packed against an
alpha 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.
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).
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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.
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EXPERIMENTAL PROCEDURES
Expression and purification of recombinant HP1—HP1 was expressed in E. coli and purified as
described previously (32). The BacPAK™ 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 manufacturers instructions. Cells
were grown at room temperature. After five days of infection, the cells were harvested, and HP1
expression 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, ten 75 cm2 flasks of cells or 500 ml suspension cell
culture was infected with passage two virus. After five days, the cells were collected by
centrifugation at 1000 x 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 DTT / 1 mM PMSF / 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 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 HPLC
(Pharmacia). 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. 2D gel electrophoresis was done as described (27).
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Phosphatase treatment of baculovirus-expressed HP1 was performed as described (27). Briefly, 2
µg potato acid phosphatase (0.8 U/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 X shrimp alkaline phosphatase buffer (200 mM Tris-HCl, pH
8.0 / 10 mM MgCl) and 1 µl shrimp alkaline phosphatase (Amersham; 1 U/µl) was added to the
reaction and the volume was brought to 40 µl with distilled water. After 15 min. incubation at
room temperature, the sample was stored at –70°C until use.
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% BSA, 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
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 U.), 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-indoyl phosphate and
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nitro blue tetrazolium (Promega) as described (33). DNA binding assay was performed as
described (17), using a 146 bp sea urchin 5S 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) Trypsin of Glu-C: 5 µg of HP1 was mixed with 0.25 µg or trypsin (Sigma) or
Glu-C (Sigma) in a 20µl buffer of 25 mM NH4HCO3, pH 8.0. The mixture was incubated at 37°C
overnight. (ii) 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 PVDF membrane
electrophoretically, localize transferred protein by transient staining with 0.5% Ponceau S for 30
sec., 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 water at 37°C for 30 min, rinsed three more
times in water, and submerged in ~20µl 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 water or 10% acetonitrile, and this supernatant was combined with the first for mass
spectroscopy.
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MALDI-TOF analysis—All MALDI spectra were obtained using a Voyager Biospectrometry
Workstation (PerSeptive Biosystems Inc., Framingham MA) in positive ion linear mode. Three
matricies were used: sinapinic acid was used for intact HP1 analysis, and α-cyano-4-
hydroxycinnaminic acid (CHCA) and 2.5-dihydroxybenzoic acid (DHB) were used for peptides.
10 mg/ml sinapinic acid or CHCA was dissolved in 50% acetonitrile / 0.3% trifluoroacetic acid.
25 mg/ml DHB was dissolved in 0.6% trifluoroacetic acid / 33% acetonitrile. Typically, 0.1-5
pmol protein or peptide was dried and resuspended in 2.5-10 µl of matrix, 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 Cat. # P2046; m/z = 1657.84), and specific HP1 peptides.
Site-directed mutagenesis and construction of the transgene expression vectors—Site-directed
mutagenesis was performed using the Transformer™ site-directed mutagenesis kit (Clontech)
according to the manufacturers 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 X 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 X v/Y;
transgene ry506 / Sb ry506(when the transgene is on the third chromosome), where “transgene”
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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 eight hours apart. Sons were collected, aged for three 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 percent of wild-type (Canton S) red
eye pigment each measurement was made using a minimum of 30 fly heads.
RESULTS
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 casein kinase II (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 2D 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)
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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 Methods), we used matrix-assisted laser-
desorbtion/ionization time-of-flight (MALDI-TOF) mass spectroscopy to examine the
postranslational 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 (35).
Phosphopeptides were identified that contained each of the consensus CKII sites (Figs. 4A and
4B). 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
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phosphorylated in vivo. Interestingly, a Lys C 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 HP1Hsγ is a target for
the Pim-1 kinase proto-oncogene. Multiple sites of Pim-1 phosphorylation occur in HP1Hsγ, at
and dowstream of 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.
We found no consistent evidence for phosphorylation at either of the two PKA/CaCK II
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
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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 crosslinking
agent dissuccinimidyl suberate, 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 crosslinkable 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
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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
cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent kinase II (CaCKII).
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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 a 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 to
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, a S15E,S202A double mutation also has reduced silencing activity.
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A triple alanine substitution at the upstream PKA/CaCK II consensus motif (SSS89,90,91AAA)
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/CaCK II consensus motif
(SSS102,013,014AAA) 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/CaCK II motifs are
functionally important in HP1 silencing activity.
DISCUSSION
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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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 (ca. 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 HP1Hsγ 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 HP1Hsγ (30), we did not test mutations
in the two serines and single threonine within the Pim-1 consensus peptide. The Drosophila
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.
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Human HP1-family proteins associate with the silencing transcriptional intermediary factors
TIF1α and TIF1β, 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 Drosophila
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/CaCK II 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 HP1Hsα
(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
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proteins--will be required in order 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 2D 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 phosphopepetides 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; or (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 2D
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 implicates 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.
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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-
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phosphorylation in modulating such associations should be tested.
Acknowledgements—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. This work was supported by NIH grant R55 GM57005 to JCE.
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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.
Fig. 2. 2-D gel analysis of recombinant baculovirus-expressed HP1. Whole cell proteins were
extracted, fractionated by 2-D gel electrophoresis, and stained with Coomassie blue. Asterisks
are placed over each HP1 isoform. (top) Total cell lysate. (bottom) Unphosphorylated bacterially
expressed HP1 (ca. 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.
Fig. 3. MALDI-TOF mass analysis of recombinant HP1. (left) rHP1 is unphosphorylated as
judged by 2-D electrophoresis (32). The mass of 23593.5 coincides with the mass of HP1 minus
the N-terminal methionine (23594.8). The asterisk denotes a cluster of peaks centered at 23818,
corresponding to the expected mass of HP1 minus the N-terminal methionine plus a single
molecule of matrix adduct. (center) 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. (right) SfHP1 after treatment with phosphatase. Again, the major peak at 23595.0
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corresponds to unphosphorylated HP1 minus the N-terminal methionine and the asterisk denotes
the position of the sinapinic acid adduct of this protein.
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 labeled “a” has a mass of
1223 and corresponds to the peptide M[194-202]R. (right) GluC peptide Peak labeled “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 phosphorylated. LysC peptide. Peak labeled “a” has a mass of 1497
and corresponds to the peptide E[101-115]K ; peak labeled “b” has a mass of 1391; its identity
is unknown.
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 crosslinked in solution. Crosslinked samples
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 (lanes 1
and 4) or baculovirus expressed (lanes 3 and 4) was mixed with 10 nM Cy5-labeled 146 bp
DNA, 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 lanes 2 and 4). (B) 10 µM wild-type HP1, HP1 (SS15,202 AA) or HP1
(SS 15,202 EE) was mixed with 10 nM Cy5-labeled 146 bp DNA, and electrophoresed in a 8%
native polyacrylamide gel.
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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Table I. Summary of consensus kinase target site data for HP1 Consensus site
HP1 isoform Silencing activity1 Corresponding phosphopeptide detected
Wild type +++ S15DAE S15A
yes
RPS89S90S91 SSS89,90,91AAA no enhancement no
RAS102S103S104 SSS102,103,104AAA SSS102,103,104EEE
1scoring system: +, enhanced silencing; 0, no effect on silencing; -, reduced silencing (dominant negative activity)
2from Ref. 34.
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kinase C proteinkinase II
Fig. 1
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23593.5 23595.0
Fig. 3
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Phosphorylation Site Mutations in Heterochromatin Protein 1 (HP1) Reduce or
published online December 19, 2000J. Biol. Chem.
10.1074/jbc.M010098200Access the most updated version of this article at doi:
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Tao Zhao, Tomasz Heyduk, and Joel C. Eissenberg*
Edward A. Doisy Department of Biochemistry
and Molecular Biology
1402 South Grand Blvd.
St. Louis, MO 63104
*corresponding author
Ph: (314)577-8154
FAX: (314)577-8156
Email: [email protected]
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on December 19, 2000 as Manuscript M010098200 by guest on A
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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 MALDI-TOF 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.
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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 S. 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 ß-strands packed against an
alpha 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.
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).
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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.
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EXPERIMENTAL PROCEDURES
Expression and purification of recombinant HP1—HP1 was expressed in E. coli and purified as
described previously (32). The BacPAK™ 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 manufacturers instructions. Cells
were grown at room temperature. After five days of infection, the cells were harvested, and HP1
expression 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, ten 75 cm2 flasks of cells or 500 ml suspension cell
culture was infected with passage two virus. After five days, the cells were collected by
centrifugation at 1000 x 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 DTT / 1 mM PMSF / 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 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 HPLC
(Pharmacia). 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. 2D gel electrophoresis was done as described (27).
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Phosphatase treatment of baculovirus-expressed HP1 was performed as described (27). Briefly, 2
µg potato acid phosphatase (0.8 U/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 X shrimp alkaline phosphatase buffer (200 mM Tris-HCl, pH
8.0 / 10 mM MgCl) and 1 µl shrimp alkaline phosphatase (Amersham; 1 U/µl) was added to the
reaction and the volume was brought to 40 µl with distilled water. After 15 min. incubation at
room temperature, the sample was stored at –70°C until use.
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% BSA, 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
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 U.), 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-indoyl phosphate and
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nitro blue tetrazolium (Promega) as described (33). DNA binding assay was performed as
described (17), using a 146 bp sea urchin 5S 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) Trypsin of Glu-C: 5 µg of HP1 was mixed with 0.25 µg or trypsin (Sigma) or
Glu-C (Sigma) in a 20µl buffer of 25 mM NH4HCO3, pH 8.0. The mixture was incubated at 37°C
overnight. (ii) 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 PVDF membrane
electrophoretically, localize transferred protein by transient staining with 0.5% Ponceau S for 30
sec., 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 water at 37°C for 30 min, rinsed three more
times in water, and submerged in ~20µl 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 water or 10% acetonitrile, and this supernatant was combined with the first for mass
spectroscopy.
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MALDI-TOF analysis—All MALDI spectra were obtained using a Voyager Biospectrometry
Workstation (PerSeptive Biosystems Inc., Framingham MA) in positive ion linear mode. Three
matricies were used: sinapinic acid was used for intact HP1 analysis, and α-cyano-4-
hydroxycinnaminic acid (CHCA) and 2.5-dihydroxybenzoic acid (DHB) were used for peptides.
10 mg/ml sinapinic acid or CHCA was dissolved in 50% acetonitrile / 0.3% trifluoroacetic acid.
25 mg/ml DHB was dissolved in 0.6% trifluoroacetic acid / 33% acetonitrile. Typically, 0.1-5
pmol protein or peptide was dried and resuspended in 2.5-10 µl of matrix, 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 Cat. # P2046; m/z = 1657.84), and specific HP1 peptides.
Site-directed mutagenesis and construction of the transgene expression vectors—Site-directed
mutagenesis was performed using the Transformer™ site-directed mutagenesis kit (Clontech)
according to the manufacturers 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 X 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 X v/Y;
transgene ry506 / Sb ry506(when the transgene is on the third chromosome), where “transgene”
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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 eight hours apart. Sons were collected, aged for three 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 percent of wild-type (Canton S) red
eye pigment each measurement was made using a minimum of 30 fly heads.
RESULTS
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 casein kinase II (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 2D 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)
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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 Methods), we used matrix-assisted laser-
desorbtion/ionization time-of-flight (MALDI-TOF) mass spectroscopy to examine the
postranslational 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 (35).
Phosphopeptides were identified that contained each of the consensus CKII sites (Figs. 4A and
4B). 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
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phosphorylated in vivo. Interestingly, a Lys C 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 HP1Hsγ is a target for
the Pim-1 kinase proto-oncogene. Multiple sites of Pim-1 phosphorylation occur in HP1Hsγ, at
and dowstream of 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.
We found no consistent evidence for phosphorylation at either of the two PKA/CaCK II
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
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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 crosslinking
agent dissuccinimidyl suberate, 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 crosslinkable 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
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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
cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent kinase II (CaCKII).
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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 a 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 to
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, a S15E,S202A double mutation also has reduced silencing activity.
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A triple alanine substitution at the upstream PKA/CaCK II consensus motif (SSS89,90,91AAA)
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/CaCK II consensus motif
(SSS102,013,014AAA) 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/CaCK II motifs are
functionally important in HP1 silencing activity.
DISCUSSION
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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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 (ca. 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 HP1Hsγ 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 HP1Hsγ (30), we did not test mutations
in the two serines and single threonine within the Pim-1 consensus peptide. The Drosophila
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.
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Human HP1-family proteins associate with the silencing transcriptional intermediary factors
TIF1α and TIF1β, 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 Drosophila
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/CaCK II 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 HP1Hsα
(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
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proteins--will be required in order 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 2D 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 phosphopepetides 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; or (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 2D
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 implicates 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.
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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-
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phosphorylation in modulating such associations should be tested.
Acknowledgements—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. This work was supported by NIH grant R55 GM57005 to JCE.
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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.
Fig. 2. 2-D gel analysis of recombinant baculovirus-expressed HP1. Whole cell proteins were
extracted, fractionated by 2-D gel electrophoresis, and stained with Coomassie blue. Asterisks
are placed over each HP1 isoform. (top) Total cell lysate. (bottom) Unphosphorylated bacterially
expressed HP1 (ca. 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.
Fig. 3. MALDI-TOF mass analysis of recombinant HP1. (left) rHP1 is unphosphorylated as
judged by 2-D electrophoresis (32). The mass of 23593.5 coincides with the mass of HP1 minus
the N-terminal methionine (23594.8). The asterisk denotes a cluster of peaks centered at 23818,
corresponding to the expected mass of HP1 minus the N-terminal methionine plus a single
molecule of matrix adduct. (center) 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. (right) SfHP1 after treatment with phosphatase. Again, the major peak at 23595.0
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corresponds to unphosphorylated HP1 minus the N-terminal methionine and the asterisk denotes
the position of the sinapinic acid adduct of this protein.
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 labeled “a” has a mass of
1223 and corresponds to the peptide M[194-202]R. (right) GluC peptide Peak labeled “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 phosphorylated. LysC peptide. Peak labeled “a” has a mass of 1497
and corresponds to the peptide E[101-115]K ; peak labeled “b” has a mass of 1391; its identity
is unknown.
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 crosslinked in solution. Crosslinked samples
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 (lanes 1
and 4) or baculovirus expressed (lanes 3 and 4) was mixed with 10 nM Cy5-labeled 146 bp
DNA, 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 lanes 2 and 4). (B) 10 µM wild-type HP1, HP1 (SS15,202 AA) or HP1
(SS 15,202 EE) was mixed with 10 nM Cy5-labeled 146 bp DNA, and electrophoresed in a 8%
native polyacrylamide gel.
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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Table I. Summary of consensus kinase target site data for HP1 Consensus site
HP1 isoform Silencing activity1 Corresponding phosphopeptide detected
Wild type +++ S15DAE S15A
yes
RPS89S90S91 SSS89,90,91AAA no enhancement no
RAS102S103S104 SSS102,103,104AAA SSS102,103,104EEE
1scoring system: +, enhanced silencing; 0, no effect on silencing; -, reduced silencing (dominant negative activity)
2from Ref. 34.
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kinase C proteinkinase II
Fig. 1
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23593.5 23595.0
Fig. 3
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Phosphorylation Site Mutations in Heterochromatin Protein 1 (HP1) Reduce or
published online December 19, 2000J. Biol. Chem.
10.1074/jbc.M010098200Access the most updated version of this article at doi:
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