From the III. Zoologisches
Institut-Entwicklungsbiologie, Universität Göttingen,
Göttingen D 37073, Germany, ¶ Wydzia
Max Planck Institute for Biophysical
Chemistry, Department of Biochemistry, Göttingen D 37077, Germany, and §§ MDS Proteomics A/S,
5230 Odense M, Denmark
Received for publication, December 8, 2000, and in revised form, March 23, 2001
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ABSTRACT |
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The high mobility group (HMG)
proteins of the AT-hook family (HMGA) lie downstream in regulatory
networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein
kinase 2 (CK2) as final effectors. In the cells of the midge
Chironomus, almost all of the HMGA protein (cHMGA) is
phosphorylated by CK2 at two adjacent sites. 40% of the protein
population is additionally modified by MAP kinase. Using spectroscopic
and protein footprinting techniques, we analyzed how individual and
consecutive steps of phosphorylation change the conformation of an HMGA
protein and affect its contacts with poly(dA-dT)·poly(dA-dT) and a
fragment of the interferon- High mobility group
(HMG)1 proteins are
nonhistone proteins that are thought to play various roles in the
assembly of chromatin and the regulation of transcription (for a review
see Refs. 1 and 2). This group comprises three families of structurally unrelated proteins: HMGB,2
HMGN, and HMGA (previously HMG-1/2, HMG-14/17 and HMGI/Y; for a review
see Refs. 3 and 4). The presence of 2-15 AT-hooks, the putative DNA
binding domains (DBD), is common for the HMGA family (for comparison of
multiple AT-hook-containing proteins see Ref. 5). The AT-hooks bind
within the minor groove of AT-rich DNA (6). Simultaneous binding of two
or more of these domains increases the strength of interaction of these
proteins with DNA (7-9). Mammalian HMGA1a, HMGA1b, HMGA2, and insect
cHMGA (formerly cHMGI) have three DBDs. Depending upon the type of DNA
examined, all or only two of these DBDs are directly involved in
binding (10). DBD2 plays a central role in the organization of the
protein-DNA complexes (9-14), whereas binding of the two other DBDs to
DNA depends on the DNA sequence and conformation (9, 10, 14). Comparative analyses of binding of the HMGA proteins to the
interferon- The proteins of the HMGA family are phosphorylated in vivo
by Cdc2 kinase (17, 18), MAP kinase (19), and CK2 kinase (20, 21). In
addition, efficient phosphorylation of these proteins by PKC has also
been demonstrated (19, 22, 23). These posttranslational modifications
attenuate the DNA binding affinities of mammalian HMGA1a (17, 18, 22,
24), HMGA1b (14), murine HMGA2 (12), and insect cHMGA (19). Structural studies revealed that phosphorylation of HMGA2 protein by Cdc2 kinase
impairs DNA binding of DBD2 (12), whereas phosphorylation of HMGA1b by
this kinase mainly affects the contacts of DBD1 with DNA (14).
The cHMGA protein from Chironomus tentans is the only
characterized HMGA protein originating from a non-mammalian species. Like its mammalian counterparts, it has three AT-hook domains and a
C-terminal stretch of mainly acidic amino acid residues (7). Moreover,
mammalian and insect proteins are phosphorylated by the same kinases.
In this work we studied the consequences of phosphorylation of cHMGA on
the conformation of the protein and its interaction with DNA. We show
that phosphorylation of cHMGA by CK2, Cdc2 kinases, and MAP kinase
affects the conformation of the protein. Phosphorylation of the protein
by CK2 and Cdc2 kinase impairs DNA binding of the AT-hook domains
located close to the N and C termini of the protein. In contrast,
phosphorylation of Ser-22 by MAP kinase does not
affect protein binding to DNA and appears to have another function,
e.g. modulation of protein-protein contacts.
Construction and Expression of cHMGA Mutants--
Three double
mutants of the cHMGA protein, in which two residues were mutated to Cys
and Trp, respectively, were constructed by means of a polymerase chain
reaction. The cysteine and tryptophan residues were introduced at
positions 3 and 94 ((Cys-3)cHMGA), 15 and 1 ((Cys-15)cHMGA), and 94 and
1 ((Cys-94)cHMGA; see Fig. 2). The constructs were cloned into the
expression vector pET3a, and the proteins were overexpressed in BL 21 (DE3) cells as described (25-27).
Protein Extraction and Purification--
The proteins were
isolated by extraction with 5% (v/v) HClO4 in three
freezing-thawing cycles from cultured Chironomus cells or
from bacteria expressing cHMGA or its mutants (7). The cell supernatants were acidified with HCl to 0.35 M, and
proteins were precipitated with 6 volumes of acetone and dried. Crude
extracts were separated on a cation-exchange column (Poros SP20,
4.6 × 100 mm, PerSeptive Biosystems) using a 0.3-1 M
NaCl gradient in 25 mM sodium borate, pH 9.4. To the
fraction containing the mutated proteins 1 M dithiothreitol
was added to the final concentration of 50 mM. After a
30-min incubation at 20 °C the samples were concentrated and
desalted on Sep-Pack Plus C18 cartridges (Waters) using
70% CH3CN, 0.1% CF3COOH in
H2O as the eluent. Finally, the proteins were
chromatographed on a reverse-phase C18 Zorbax SB-300 column
using a linear CH3CN gradient in 0.1% CF3COOH
in H2O as described previously (25). The Trp residues
introduced into the mutants allowed quantification of the proteins by
spectrophotometry using an absorption coefficient for tryptophan of
5,500 M Phosphorylation of the Proteins--
50 µg of the native or
recombinant cHMGA protein were phosphorylated at 30 °C with 10 units
of recombinant human Cdc2 kinase (New England Biolabs Inc.) for 5 h in the presence of 4 mM ATP in 8 µl of Cdc2 kinase
buffer (50 mM Tris/HCl, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM
ethylene glycol-bis( Modification of the Mutants of the cHMGA Protein with
Acrylodan--
50 µM protein in 25 mM
MOPS/NaOH, 150 mM NaCl, pH 7.0 was incubated with a
3-4-fold molar excess of acrylodan
(6-acryloyl-2-dimethylaminonaphthalene; Molecular Probes). The reaction
was carried out at 4 °C for 3-5 h, and the reaction products were
separated by reverse-phase HPLC as described above. To verify that the
proteins were phosphorylated and modified by acrylodan at the desired
sites, we digested the proteins with trypsin in 0.1 M
Tris/HCl, pH 7.5 at 37 °C for 3 h. Peptides were resolved and
identified by reverse-phase C18 chromatography as described
previously (19). The use of the diode array spectral detector enabled
easy identification of peptides containing tryptophan or
acrylodan-modified cysteine residues.
DNA and Oligonucleotides--
Synthetic linear
poly(dA-dT)·poly(dA-dT) DNA was obtained from Amersham Pharmacia
Biotech. The approximate average length of this DNA was 5000 base
pairs. The 34-base pair fragment of the promoter of the IFN Hydroxyl Radical Protein Footprinting--
The protein
footprinting reactions and quantitative analyses of the products
were performed as described previously (10, 12-14, 28, 29). Briefly,
10 pmol of the radioactively end-labeled protein (10-20 kcpm) were
digested at room temperature in the presence or absence of DNA in a
total volume of 10 µl containing 180 mM NaCl and 10 mM MOPS buffer, pH 7.2. The chemical digestions were
started by sequential addition of 1 µl each of the following freshly
prepared solutions: (i) 20 mM EDTA and 10 mM
(NH4)2Fe(II)(SO4)2, (ii) 0.2 M sodium ascorbate, and (iii) 0.375% (v/v)
H2O2. Reactions were stopped after 30 min by
the addition of 3.3 µl of 4× concentrated SDS sample buffer (4%
SDS, 16% glycerol, 25 mM Tris/HCl, pH 6.8, 6%
Limited Proteolytic Digestion--
A mixture of native
phosphoforms and the recombinant protein (molar ratio of 1:1:1), each
labeled at Ser-3 by Cdc2 modification, was digested with endoproteinase
Glu-C in 25 mM sodium phosphate, pH 7.8 and 180 mM NaCl at 20 °C. The protein to enzyme ratio was 50:1
(mol/mol). Reactions were terminated by addition of an equal volume of 10 M urea solution containing 5% (v/v) acetic
acid, 4% (v/v) Mass Spectra--
Mass spectra were recorded on a Finnigan MAT
TSQ 700 triple-stage quadrupole mass spectrometer equipped with an
electrospray ion source. Samples were typically dissolved in a
methanol/water/acetic acid (47:48:5, v/v/v) solution at a concentration
of 50 pmol/µl and introduced into the electrospray needle by
mechanical infusion through a microsyringe at a flow rate of 1 µl/min. A potential difference of 4.5 kV was applied between the
electrospray needle. Nitrogen gas was used to evaporate the solvent
from the charged droplets. At least 20 scans were averaged to obtain
each spectrum. The resulting spectra were transformed using a BioWorks
software package (Finnigan).
Fluorescence Measurements--
Emission spectra with or without
quenching were taken on an SLM-Aminco-Bowman series 2 luminescence spectrometer in 10 mM MOPS/NaOH, pH 7.2, 100 mM sodium acetate, 100 µg/ml bovine serum albumin.
Measurements were corrected for buffer background, dilution, and
instrumental factors. Excitation was at 391 nm. Emission spectra were
recorded using 4- and 2-nm slits for excitation and emission, respectively. The titrations with KI were performed using 2- and 16-nm
slits for excitation and emission (530 nm), respectively.
Circular Dichroism Measurements--
CD spectra of 20-50
µM (Cys-94)cHMGA protein in 10 mM sodium
phosphate, pH 7.5 were recorded at a scanning speed of 50 nm/min from
190-280 nm in cuvettes of 1-mm path length with a Jasco 720 spectropolarimeter using a 1-nm bandwidth. For each sample three CD
spectra were averaged to improve the signal-to-noise ratio. Prior to
each CD measurement the protein concentration of the sample in the
cuvette was determined in a Cary 100 spectrophotometer (Varian Inc.)
using an absorption coefficient of Lifetime Measurements--
Fluorescence lifetimes of
acrylodan-labeled proteins were determined by time-correlated single
photon counting. The excitation was at 405 nm with a pulsed diode laser
(IBH, Glasgow, Scotland) operating at 1 MHz, and emission was collected
using a 520-nm interference filter. The fluorescence was detected by an
integrated photon detection module (TBX-04, IBH) containing a
photomultiplier, pre-amplifier, and constant fraction discriminator,
and its output was processed by time-correlated single photon counting.
The instrument response function, measured with a LUDOX (DuPont)
suspension, had a full width at half-maximum of 245 ps. Decay
curves were analyzed with decay analysis software supplied by IBH.
Because the decay of all samples was double exponential,
number-averaged, and intensity-averaged (31), lifetimes were calculated
according to Equations 1 and 2.
The Most Prominent Fraction of the cHMGA in Chironomus Cells Is
Phosphorylated by CK2 at Two Sites within Its Acidic
Tail--
Previously we reported that cHMGA is phosphorylated at
distinct sites by Cdc2, MAP kinase, and PKC, respectively (19). These conclusions were drawn from peptide mapping studies of in vivo 32P-labeled protein. Because the major fraction of the
protein, having the highest electrophoretic mobility, was not
detectable on autoradiograms, and a phosphatase treatment of this form
did not change its mobility, we considered this form as
dephosphorylated cHMGA. Unexpectedly, mass spectroscopic analysis of
native cHMGA revealed that the major fraction of the protein had an
Mr of 10,544. Because the calculated relative
mass of the cHMGA protein is 10,340, this value suggested that this
protein fraction could be doubly phosphorylated and singly acetylated
(Fig. 1, A and B,
Table I). Minor fractions with
Mr of 10,383 and 10,463 could reflect protein forms that carry a single acetylation and both one acetylation and one
phosphorylation, respectively (Table I). The fraction of
Mr of 10,623 could reflect the protein with
three phosphorylations and a single acetylation (Table I, Fig.
1B). To map the sites of modifications we isolated the main
fraction of native cHMGA protein and digested the protein with trypsin.
Peptide separation followed by mass analysis showed that the main
subfraction of the C-terminal peptide 85-98 was doubly phosphorylated
and that the N-terminal peptide 1-7 was acetylated (Table I, Fig.
1D). A small portion (<20%) of the peptide 85-98 was
found in a singly phosphorylated form. Peptide 85-98 contains two
serine residues in a sequence matching the consensus sequence
phosphorylated by casein kinase 2; however, these two serine
residues do not represent a preferred recognition site (32). To
determine whether CK2 can modify Ser-96 and Ser-97 residues, we
incubated the cHMGA recombinant protein in the presence of the CK2 and
[
Taken together, our present and previous results (19) show that the
cHMGA protein has at least five distinct phosphorylation sites located
at serine residues at positions 3, 22, 72, 96, and 97 (Fig.
2). Phosphorylation of residues 96 and 97 appears to have a constitutive character, because more than 90% of the
protein extracted from Chironomus cells is phosphorylated at
both sites. The fully dephosphorylated form of the protein was rare,
suggesting that in cells only minute amounts of this form are present.
Up to 40% of the cHMGA population occurs in a form that is
additionally phosphorylated at Ser-22 by MAP kinase (19). The fraction
of protein phosphorylated at Ser-3 by Cdc2 kinase is below 5% and reflects the portion of cells that are in the late G2 and M
phases. Furthermore, a small fraction, usually below 2% of the cHMGA
protein, was found to be modified at Ser-72. This amino acid represents a putative target site of PKC (19).
The consequences of the HMGA phosphorylation on protein binding to DNA
have been analyzed (12, 14, 17-19, 22, 23). Those studies led to the
conclusion that this type of posttranslational modification attenuates
the strength of protein-DNA binding. However, it is unknown how changes
in the binding affinity are accomplished and how phosphorylation of
distinct sites affects the conformation of HMGA proteins. To obtain
insights into these processes we examined the changes in protein
structure and binding properties by means of hydroxyl radical protein
footprinting, limited proteolytic digestion, and fluorescence and CD spectroscopy.
Fluorescence and CD Spectroscopy Reveals CK2-induced Structure
Alteration--
Single cysteine mutants of cHMGA labeled covalently
with acrylodan were used to study the conformational changes of the
protein in response to CK2 phosphorylation (Fig.
3, A-C). The fluorescence intensity and/or the position of the fluorescence emission maximum of
this label depend on its microenvironment (33), and therefore its
fluorescence can be used as a sensitive indicator of conformational changes in proteins (34-36). For this purpose, we constructed three mutants of cHMGA in which Ser-3, Ala-15, or Val-94 was substituted with
a cysteine residue. After production in bacteria and purification, a
portion of each mutant was phosphorylated in vitro by CK2.
Both the phosphorylated and non-phosphorylated single cysteine mutants were then modified with acrylodan and purified.
The spectral properties of the acrylodan attached to cHMGA were
different, depending upon the location of the label within the
polypeptide chain, and were differentially affected by phosphorylation (Fig. 3). They are distinct in their quantum yields, their maxima of
fluorescence emission (Fig. 3; Table II),
and their lifetimes (Table III),
suggesting that the fluorophore at each position is in a distinct
environment. The phosphorylation of the protein by CK2 at Ser-96 and
Ser-97 results in strong red shifts of the emission spectra of
acrylodan at positions 15 and 94. In this case the phosphorylation
seems to create a more polar environment for the probes (Fig. 3,
B and C; Table II). For the protein labeled with
acrylodan at Cys-3 phosphorylation by CK2 led to a weak blue shift,
which seems to indicate a decrease in the polarity of the environment
around the probe (Fig. 3A; Table II). The
phosphorylation-dependent changes in the spectral
properties of the proteins labeled at positions 3 and 15 disappeared in
the presence of 6 M guanidine·HCl, suggesting structural
differences between phosphorylated and non-phosphorylated forms of
cHMGA (Fig. 3, D and E). In contrast, for the
protein labeled at position 94 the great differences in fluorescence
intensity between phospho and dephosphoforms persists even in the
presence of 6 M guanidine·HCl (Fig. 3, C and
F). This effect can be attributed to the vicinity of the
fluorophore and the site of phosphorylation on the primary structure
level. Because free acrylodan coupled to
Analysis of fluorescence decays of acrylodan-labeled samples revealed
that for all samples the decays were double exponential. The lifetimes
of the two decay components were quite similar for all samples and were
not affected greatly by phosphorylation (Table III). The observation
that the lifetimes of acrylodan were relatively insensitive to
phosphorylation and that the quantum yields were changed as a function
of phosphorylation suggests that the quantum yield changes were
entirely due to differences in static quenching. This can be
illustrated by calculating the ratio of
Iodide is a collisional quencher, whose efficiency depends on the
polarity of the environment of the fluorophore. For each of the
analyzed forms of the cHMGA protein a strong quenching was observed
(Fig. 3, G-I). Because a straightening of the plots in the
presence of denaturant was observed (Fig. 3I), the downward curvature of the Stern-Volmer plots must originate from multiple conformers of the protein rather than from multiple chemically distinct
forms of the probe (38). Because the intensity-averaged lifetimes
(Table III) did not change significantly in response to
phosphorylation, any changes in quenching efficiency in response to
phosphorylation could be interpreted as being due to changes in the
accessibility to the quencher. The phosphorylation of the protein
mutants resulted in a weakening of quenching. In the case of the Cys-94
mutant, the changes can be directly attributed to the vicinity of the
phosphate groups. In contrast, changes of the quenching efficiency at
positions 3 and 15 must reflect changes in protein conformation.
To confirm that the changes in fluorescence properties induced by CK2
phosphorylation indeed originate from perturbations in protein
conformation, we analyzed the CD spectral properties of the
phosphorylated and non-phosphorylated forms of cHMGA. The comparison of
the CD spectra at 20 °C revealed small but distinct differences
between the CK2-phosphorylated and unmodified proteins (Fig.
4, black lines).
Phosphorylation by CK2 led to a slightly reduced molar ellipticity in
the region around 200 nm and, perhaps more importantly, caused a local
maximum at 223 nm and a local minimum at 228 nm, accompanied by a
general reduction of the ellipticity in the region of 215-250 nm (Fig.
4, inset). Because the shapes of the CD spectra were
different, the observed changes cannot be a result of limited accuracy
in determination of protein concentration. To show that the alterations
induced by CK2 phosphorylation were due to structural changes, we also
measured the CD spectra of phosphorylated and non-phosphorylated forms
at 80 °C, assuming the denaturation of the proteins at this
temperature (Fig. 4, gray lines). At 80 °C the
ellipticity at 208 nm was reduced, whereas in the range of 208 to
~255 nm it was increased. No difference between CK2-phosphorylated
and non-phosphorylated cHMGA was observed upon denaturation.
Partial Digestion Confirms a Conformational Change of
the cHMGA Protein by Individual Phosphorylations--
Limited
proteolytic digestion is a sensitive tool for the detection of the
changes in the structure of proteins. The mixture of
32P-labeled cHMGA[PCdc2],
cHMGA[PCdc2,PCK2], and
cHMGA[PCdc2,PMAPK,PCK2] was
partially digested by proteinase Glu-C (Fig.
5). The experiment revealed differences
between the various phosphoforms in the susceptibility for degradation.
Quantification of the radioactive bands of the labeled proteins showed
that the resistance of the protein to degradation increased with the
progress of the phosphorylation. Fitting of the experimental points to
the single exponential decay function enabled calculation of the
half-life for the individual protein forms (Fig. 5B). The
half-life value of 53 min for
cHMGA[PCdc2,PMAPK,PCK2] was 2.7 and 6 times greater than the values obtained for
cHMGA[PCdc2,PCK2] and
cHMGA[PCdc2], respectively. Because the sites that can be
attacked by this protease are not in the proximity of the
phosphorylation sites, the observed differences in the susceptibility
to proteolysis suggest different conformations of the phosphoforms. A
similar experiment in which cHMGA[PCK2] and
cHMGA[PMAPK,PCK2] were compared revealed that
phosphorylation at Ser-22 by MAPK increases resistance of the protein
to digestion (data not shown). From these experiments the conclusion
can be drawn that an increase in the extent of cHMGA phosphorylation is
accompanied by an augmentation of structural compactness and metabolic
inertness of the protein. Interestingly, phosphorylation within the C
termini of structurally unrelated HMG1 box (HMGB) proteins has a
similar effect on protein conformation, as manifested in increased
protein stability (39). This may suggest that phosphorylation of HMG
proteins by CK2 and MAPK may play an important role in regulating the
turnover rates of these proteins.
Protein footprinting is a technique that can monitor structural changes
in proteins (40). Using phosphorylation at Ser-3 for end labeling of
the protein (10), we compared the native protein, modified in
vivo at Ser-22 by MAP kinase, with the unphosphorylated protein.
The resulting digestion patterns easily showed visible differences
between the proteins (Fig.
6A). Quantitative analysis according to Frank et al. (10) enabled localization of the
changes in accessibility induced by MAPK phosphorylation (Fig.
6B). For clearer documentation we transformed the data in
plots of normalized differences (Fig. 6C). The region of
residues 9-23 was exposed by modification of Ser-22, whereas small
regions around residues 25-27, 34-37, and 44-51 and the whole C
terminus from residue 76 were protected. Thus, among the three putative
DNA-binding sites of the protein, only the solvent accessibility of the
first AT-hook appears to be directly affected by MAPK
phosphorylation.
In another set of experiments we investigated the modification of
Ser-3, which is the target for Cdc2 kinase. To obtain more detailed
information on the effect of this modification to the conformation of
the protein and its DNA binding, we labeled the C terminus of cHMGA
with 32P using CK2 and performed hydroxyl radical
footprinting analysis. For the size assignment of the bands in the
hydroxyl radical patterns, a series of limited digestions with the
sequence-specific proteinases thermolysin, trypsin, proteinase Glu-C,
or proteinase Arg-C were performed to generate end-labeled peptides of
defined length (Fig. 7, A and
B). The comparison of the peptide patterns obtained by digestion of the CK2-labeled protein in its Cdc2-phosphorylated and
non-phosphorylated forms revealed only slight differences. There were
only a few regions for which the susceptibility was significantly
altered (Fig. 8), with the strongest
effect in the region comprising the second AT-hook (residues
52-58).
Phosphorylation of cHMGA by CK2 and Cdc2 Affects DNA Binding of the
AT-hooks in the Proximity of the Phosphorylation Site--
Because the
primary function of HMGA proteins is binding to DNA, we analyzed how
the individual phosphorylations that appear to occur in vivo
affect the organization of the cHMGA-DNA complexes. Poly(dA-dT)·poly(dA-dT) and a 34-base pair fragment of the IFN
Using the Ser-96/97 labeling we found that all three AT-hooks are
involved in binding to poly(dA-dT)·poly(dA-dT) (Fig.
9A). In contrast, binding to
the IFN
Simultaneous phosphorylation of the protein by Cdc2 and CK2 resulted in
significant changes in the protection patterns. In both complexes, with
poly(dA-dT)·poly(dA-dT) and IFN Extensive phosphorylation is a characteristic feature of the
mammalian (41, 42) and invertebrate (19, 43) HMGA proteins. Different
kinases have been shown to be involved in phosphorylation of this group
of proteins. In our previous study, we have reported that up to 40% of
the cHMGA protein in Chironomus cells is phosphorylated by
Cdc2 kinase, MAPK, and PKC (19). In these studies the phosphorylation sites were mapped in the protein that was in vivo
pulse-labeled with radioactive orthophosphate. This technique leads to
the detection mainly of those phosphorylation sites that are frequently
modified during labeling time. Because cHMGA protein has a long
turnover rate (44), we were unable to detect the sites of
phosphorylation at Ser-96 and 97 by radioactive labeling (19) because
phosphorylation at these sites seems to be coupled to the rate of
protein synthesis. The presented analysis, based on mass spectroscopic
measurements, revealed that nearly the entire population of cHMGA is
phosphorylated by CK2 at Ser-96 and Ser-97. Our previous and
present results suggest that phosphorylation by CK2 is coupled to
translation, whereas the other kinases confer specific properties to
the protein in a dynamic and reversible manner. Because the
modification by CK2 affects nearly the entire population of the protein
in the cell and appears to persist through longer periods, we called this constitutive phosphorylation (39). In contrast, the other phosphorylations within the cHMGA were termed facultative (39).
Studies performed in several laboratories have shown that the DNA
binding affinity of HMGA proteins is attenuated upon phosphorylation (12, 14, 17-19, 22, 23). Our previous analyses of the binding properties of the insect HMGA revealed that coincident phosphorylation of the protein at two positions is necessary to elicit a significant change in the affinity of the protein to AT-rich satellite DNA (19). In
this work we investigated the consequences of phosphorylation of cHMGA
with respect to protein conformation and the organization of the
cHMGA-DNA complex.
HMGA proteins are usually considered randomly coiled polypeptides.
Indeed, circular dichroism spectra of mammalian HMGA1a protein suggest
that about 70% of the polypeptide does not adopt any defined secondary
structure (45). When we fitted the CD spectra of the cHMGA with
secondary structure estimation software using the algorithm of Yang
et al. (46), the results indicated that as much as 23% of
the polypeptide may be in the The protein footprinting DNA binding studies show that phosphorylation
at two sites must occur to impair contacts between a single AT-hook and
DNA. The observed effects of protein phosphorylation are distinct with
respect to the type of DNA bound to the protein (10). Phosphorylation
of both termini of the protein leads to a reduction of the contacts of
the third AT-hook in the complex with poly(dA-dT)·poly(dA-dT) (Fig.
10, a and c). In
the case of IFN promoter. We demonstrate that
phosphorylation of cHMGA by CK2 alters its conformation and modulates
its DNA binding properties such that a subsequent phosphorylation by
Cdc2 kinase changes the organization of the protein-DNA complex. In
contrast, consecutive phosphorylation by MAP kinase, which results in a
dramatic change in cHMGA conformation, has no direct effect on the
complex. Because the phosphorylation of the HMGA proteins attenuates
binding affinity and reduces the extent of contacts between the DNA and
protein, it is likely that this process mirrors the dynamics and
diversity of regulatory processes in chromatin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter have shown that each of the four analyzed HMGA
proteins binds in a specific manner (14). This supports the idea
that each of the HMGA proteins has an individual function,
e.g. in the regulation of specific sets of genes (15,
16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
cm
1 at 280 nm. The wild type cHMGA was
quantified on Coomassie Blue R-stained polyacrylamide gels using one of
the cHMGA mutants as a standard.
-aminoethyl ether) tetraacetic acid, pH 7.5). 50 µg of purified cHMGA protein or Cdc2-phosphorylated cHMGA were
phosphorylated at 37 °C with 500 units of recombinant human CK2 (New
England Biolabs Inc.) for 2.5 h in the presence of 200 µM ATP in 50 µl of CK2 buffer (20 mM
Tris/HCl, 50 mM KCl, 10 mM MgCl2,
pH 7.5). For 32P end labeling 100-150 µCi of
[
-32P]ATP were added to the reaction mixture. The
reaction products were separated by reversed-phase HPLC. The
cHMGA[PMAPK,PCK2] form of cHMGA was isolated
from Chironomus cells as described previously (19).
gene
containing the positive regulatory domains III-1 and II and the
negative regulatory domain I was prepared from synthetic
oligonucleotides (10).
-mercaptoethanol, and 0.01% bromphenol blue). The reaction products
were separated on Tricine-SDS-polyacrylamide gels and analyzed by
phosphorimaging as described previously (10). Size markers were
generated by limited digestion of 10 pmol of end-labeled HMG protein by
trypsin, thermolysin, proteinase Arg-C, or proteinase Glu-C in a
10-µl reaction volume. Cleavage in the presence of 10 ng of trypsin
or thermolysin was carried out in 180 mM NaCl, 20 mM Tris/HCl, pH 7.5 at 0 or 20 °C, respectively. The
reactions with trypsin were stopped by addition of 1 µl of 0.14 mM N
-p-tosyl-L-lysine chloromethyl ketone. Cleavage with proteinase Glu-C was carried out in
the presence of 5 or 50 ng of enzyme in 25 mM sodium
phosphate, pH 7.8 and 180 mM NaCl at 20 °C. Digestion of
the protein in the presence of 20 ng or 0.5 µg of Arg-C was performed
in 90 mM Tris/HCl containing 8.5 mM
CaCl2, 5 mM dithiothreitol, and 0.5 mM EDTA, at 20 °C. Finally, the reactions were stopped
by addition of 4× concentrated SDS sample buffer containing 20 mM EDTA.
-mercaptoethanol, 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.2 mM
N
-tosyl-L-lysine chloromethane. Reaction products were separated on 15% polyacrylamide urea-acetic acid-Triton X-100 gels (30). Gels were scanned with a PhosphorImager (Molecular Dynamics), and the intensities of the bands were quantified.
206 = 268,550 M
1 cm
1.
Jasco Secondary Structure Estimation software (version 1.00.00, 1998)
was used to estimate the amount of secondary structure elements.
(Eq. 1)
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. Rapid incorporation of labeled phosphate
into the protein was observed (Fig. 1C). In contrast, under
the same conditions the native protein was labeled only marginally
(Fig. 1C, inset), indicating that nearly all CK2
target residues are already modified in vivo. To check
whether the phosphorylated sites in vitro are identical to
those modified in vivo, we digested cHMGA with trypsin and
analyzed the products by HPLC. Two radioactive peptides were found
(Fig. 1E, rec. cHMGA/CK2 modified).
Their retention times were identical to those of the singly and doubly
phosphorylated peptide 85-98 (Fig. 1D), indicating that
Ser-96 and Ser-97 are phosphorylated by CK2 in vivo.
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Fig. 1.
The phosphorylation at residues 96 and 97 is
the main posttranslational modification in the cHMGA protein and is
caused by CK2. A, electrospray ionization mass
spectrum obtained for a mixture of native cHMGA. B, a
deconvoluted spectrum of A shows the presence of four
components with Mr of 10,383, 10,463, 10,544, and 10,623, differing by ~80 units (indicated by bold
numbers). The value of 10,383.0 is in agreement with the calculated
Mr of acetylated cHMGA. The identity of two
other signals is unknown (Mr values are given in
parentheses). C, time course of the in
vitro phosphorylation of recombinant cHMGA. The proteins were
phosphorylated using recombinant human CK2 in the presence of
[ -32P]ATP. At the indicated times, the
reactions were terminated by precipitation with 33%
CCl3COOH, and the incorporated radioactivity was measured.
Inset, a 2.5-h reaction resulted in an incorporation of 1.51 and 0.112 mol of phosphate per 1 mol of recombinant and native protein,
respectively. D, mapping of the phosphorylation sites in the
main fraction of the native cHMGA. ~50 µg of the native protein
were digested with 5 µg of trypsin for 3 h, and the digestion
products were separated on a reverse-phase C18 column. The
identity of the individual peptides was determined by mass spectrometry
(Table I). E, comparative mapping of the in vitro
phosphorylation sites of CK2. Either radioactively phosphorylated or
nonphosphorylated recombinant cHMGA was digested with trypsin
and chromatographed as described above. Fractions carrying the
radioactive label are indicated by shaded areas.
Mass spectrometric analysis of the native cHMGA protein and its
peptides obtained by tryptic digestion
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Fig. 2.
The primary structures of the cHMGA protein
and its mutants. The sites phosphorylated by Cdc2 kinase
(Cdc2), MAPK, PKC, and CK2 within cHMGA are indicated by
arrows. The putative DBDs (or AT-hooks) are
boxed.
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Fig. 3.
Fluorescence spectroscopy reveals differences
in conformation between nonphosphorylated and CK2-phosphorylated
protein. A-F, fluorescence emission spectra of
nonphosphorylated (black lines) and phosphorylated
(gray lines) single cysteine mutants modified with
acrylodan in the absence (A-C) and presence
(D-F) of 6 M guanidine·HCl. The excitation
wavelength was 391 nm. G-I, Stern-Volmer plots for
fluorescence emission quenching by KI measured at 530 nm. Closed
symbols, non-phosphorylated proteins; open symbols,
CK2-phosphorylated proteins. The squares in I represent
quenching in the presence of 6 M guanidine·HCl.
-mercaptoethanol has an
emission maximum of 538 nm (Table II), the emission maximum of 523 nm
of the label at position 94 in the non-phosphorylated protein suggests
that, even in the presence of 6 M guanidine·HCl, this
region of the protein is not fully unfolded.
Spectral properties of cHMGA mutants and -mercaptoethanol modified
with acrylodan
Summary of the lifetime measurements
num to quantum yield (Table III). In the absence of static quenching
num and quantum yield should be proportional (37). The
data in Table III show the existence of large differences in this ratio
between different proteins and between phosphorylated and
unphosphorylated forms of the same protein. Thus, it can be concluded
that acrylodan fluorescence detected conformational changes of the
protein in response to phosphorylation.
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Fig. 4.
CD spectra confirm conformational changes of
cHMGA induced upon CK2 phosphorylation. The spectra of
(Cys-94)cHMGA (dashed lines) and
(Cys-94)cHMGA[PCK2] (solid lines) were
recorded in 10 mM sodium phosphate, pH 7.5 at 20 °C
(black lines) or 80 °C (gray lines) in
the range of 190-280 nm. Inset, enlarged representation of
CK2 modification-induced alteration in the range of 215-250 nm.
deg, degree.
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Fig. 5.
Time course of digestion of phosphoforms of
the cHMGA protein. The recombinant, native, and native
Ser-22-phosphorylated proteins were phosphorylated by Cdc2 kinase in
the presence of [ -32P]ATP at the residue
Ser-3, yielding cHMGA[32PCdc2],
cHMGA[32PCdc2,PCK2], and
cHMGA[32PCdc2,PMAPK,PCK2]
forms. The three phosphoforms were mixed in the molar ratio of 1:1:1
and digested by proteinase Glu-C. At the indicated times, the reaction
was terminated by addition of CCl3COOH to a final
concentration of 33%. The precipitated mixture of proteins and
peptides was separated on 15% acrylamide gels in the discontinuous
acetic acid/urea/Triton system (A). The gel was dried, and
the radioactivity in the individual protein bands was quantified by
phosphorimaging. B, analysis of the data from A.
The ratio of [intact protein]t to [intact
protein]0 was plotted against time. The lines are
theoretical curves calculated from the relationship [intact
protein]t/[intact protein]0 = e
kt. The calculated half-life times
of cHMGA[32PCdc2],
cHMGA[32PCdc2,PCK2], and
cHMGA[32PCdc2,PMAPK,PCK2]
were 8.9, 19.9, and 53.2 min, respectively.
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Fig. 6.
Phosphorylation at Ser-22 affects the
conformation of the cHMGA protein. A, representative
electrophoretic patterns of hydroxyl radical digestions of the
cHMGA[32PCdc2] and
cHMGA[32PCdc2,PMAPK] proteins
from an individual experiment. B, plot of corrected
PhosphorImager intensities. C, difference plot
showing averaged data from eight independent experiments. Positive
values mean less cutting in
cHMGA[32PCdc2,PMAPK] compared
with cHMGA[32PCdc2] at this particular
position. Bold lines above the plot indicate regions where
the observed MAPK-induced protection or exposition was statistically
significant according to a Student's t test (significance
level of = 0.05). The schematic primary structure of the cHMGA
protein with AT-hooks (boxes) is shown in the lower
part of the panel.
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Fig. 7.
Molecular weight markers and assignment of
the bands for protein footprinting using C-terminal labeling at Ser-96
and Ser-97 residues. A, molecular weight markers were
generated by site-specific cleavage of 32P end-labeled
cHMGA [PCK2] with thermolysin, endoproteinases Glu-C (5 ng) and Arg-C (20 ng), and trypsin for the indicated time. Lanes marked
with an asterisk represent extended digestions with 50 ng of
Glu-C or 0.5 µg of Arg-C. OH·, hydroxyl radical
lanes show peptide patterns of the protein digested with the chemical
proteinase. B, plot of size of peptide markers
versus relative mobility. The relative mobility of intact
cHMGA was defined as 0, and the most rapidly migrating band of hydroxyl
radical cleavage was defined as 1.
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Fig. 8.
Changes in the conformation of
the cHMGA protein upon phosphorylation by Cdc2 at Ser-3.
A, representative electrophoretic patterns of hydroxyl
radical digestions of the cHMGA[32PCK2] and
cHMGA[32PCK2,PCdc2] proteins from
an individual experiment. B, plot of corrected
PhosphorImager intensities. C, difference plot showing
averaged data from 12 independent experiments. Positive values mean
less cutting in
cHMGA[32PCK2,PCdc2] compared with
cHMGA[32PCK2] at this particular position.
Bold lines above the plot indicate regions where the
observed Cdc2-induced protection or exposition was statistically
significant according to a Student's t test (significance
level of = 0.05). The schematic primary structure of the cHMGA
protein with AT-hooks (boxes) is shown in the lower
part of the panel.
promoter were footprinted on the end-labeled proteins. For the analysis
of the effects of the phosphorylation at Ser-96/97 and Ser-22 the
protein was labeled at Ser-3, whereas labeling at Ser-96/97 using CK2
was used to study changes in the complex organization upon
phosphorylation by Cdc2 kinase at Ser-3.
fragment involved mainly the regions around the second and
third AT-hooks (Fig. 9B). These results are in good
agreement with previous studies in which the Ser-3 labeling was used
(10).
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Fig. 9.
Protein footprints of
poly(dA-dT)·poly(dA-dT) (A and C)
and IFN fragment
(B and D) on
different phosphoforms of the N- (A and
B) and C-terminally (C
and D) labeled cHMGA. Difference
plots showing averaged data from 6-8 independent experiments. Positive
values mean less cutting in DNA-bound cHMGA compared with unbound
protein at this particular position. Bold lines above the
plots indicate regions where the observed protection or exposition
induced by the DNA binding was statistically significant according to a
Student's t test (significance level of
= 0.05).
The schematic primary structure of the cHMGA protein with AT-hooks
(boxes) is shown in each panel.
, an impairing of the contacts via
the third AT-hook was observed (Fig. 9, C and D).
In the complex with IFN
, an increase of the extent of protection in
the regions flanking the central AT-hook was concomitant with an
attenuation of protection in the region comprising the third AT-hook
(Fig. 9D). Consecutive modification of the protein at Ser-22
by MAP kinase did not significantly change the protection pattern in protein footprints obtained with either
poly(dA-dT)·poly(dA-dT) or IFN
(Fig. 9, C and
D, gray lines). Thus, it appears that this phosphorylation has no direct effect on cHMGA binding to the studied ligands.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet conformation and that about
21% may be in
-turns. Thermal denaturation of the protein changed
the CD spectra in such a way as to emphasize the presence of
conformational preferences of the native protein. However, the
estimation of the secondary structure of the denatured protein yields a
higher content of
-sheet (~48%
-sheet and ~6%
-turn), so
that such estimations should be interpreted cautiously, especially in
the case of HMGA proteins. The highly reproducible digestion patterns
obtained using either hydroxyl radicals or proteinases suggest that
HMGA1a, HMGA1b, HMGA2, and cHMGA have distinct conformations (10, 12,
14). The results presented in this study strongly indicate that the
polypeptide of the cHMGA is not fully randomly coiled either and that
phosphorylation by CK2, MAPK, and, to a lesser extent, Cdc-2 modulate
its spatial arrangement.
fragment binding this double phosphorylation causes
a loss of the interaction of the first and third AT-hooks (Fig. 10,
e and g). These findings resemble the results
obtained during the studies of phosphorylation of human HMGA1b (14) and
murine HMGA2 (12). For those proteins, simultaneous phosphorylation by
CK2 and Cdc2 leads to changes in the nature of the contacts between individual elements of IFN
and a single AT-hook. In HMGA1a, this phosphorylation affects binding of the N-terminal AT-hook to the positive regulatory domain III-1 element. In contrast,
posttranslational modification of the HMGA2 results in structural
perturbations of the binding of the central and C-terminal
AT-hook to positive regulatory domain II and negative regulatory
domain I elements, respectively.
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Fig. 10.
Summary of the footprinting
experiments. The upper box schematically represents the
cHMGA protein. The hatched and checkered areas
indicate the positions of the DBDs and the PKRP sequence motifs,
respectively. Regions of cHMGA that are significantly protected from,
and exposed to, the hydroxyl radical cleavage in the presence of DNA
are indicated by horizontal black and gray boxes,
respectively. P indicates a phosphate group;
asterisks indicate sites of end labeling with
32P. Data in a and e are from
(10).
Consecutive phosphorylation of the Ser-22 by MAPK has no additional
effect on the extent of contacts between the protein and either
poly(dA-dT)·poly(dA-dT) or the IFN fragment (Fig. 10, d and h). Modification of this residue may be important for
modulation of the cHMGA interaction with other types of DNAs or proteins.
The biological significance of multiple sites of phosphorylation in the HMGA proteins is currently unknown. In addition to modulating DNA binding, the phosphorylation of specific residues may affect the turnover rate and cellular distribution of the proteins. Dephosphorylation of the C-terminal serines in the insect HMGA protein leads to conformational changes that, under in vitro conditions, facilitate degradation of the protein. Because the phosphorylation of the C-terminal acidic "tails" appears to be common within the HMG families (39), it is possible that it represents a widespread mechanism to increase stability.
Cytological studies have revealed that HMGA proteins occur mainly in the G/Q and C bands of mitotic chromosomes (47), which represent constitutive heterochromatin. In interphase nuclei of mammalian cells (48, 49) and in the Drosophila polytene chromosomes, HMGA and the related D1 protein (50), respectively, are also abundant in heterochromatin. But the HMGA proteins have also been localized to decondensed chromatin, to chromosomal puffs, and to nucleolus organizer (44, 48), which are chromosomal sites active in transcriptional processes. Thus, the presence of subpopulations of HMGA proteins may suggest their functional variability, which could be achieved by distinct phosphorylations.
The HMG1 box domain proteins (HMGB) and the nucleosome-binding HMGN
proteins are structurally unrelated to HMGA but share with HMGA several
physicochemical properties, e.g. their small size,
amphipathic character, and unusual solubility in acids. Therefore, phosphorylation by particular kinases can have common consequences for proteins that are members of the HMG family. Microinjection experiments of phosphorylated and non-phosphorylated HMGB proteins (51), as well as mass spectroscopic studies on the
subcellular distribution of HMGN proteins (52), suggest that the
dephosphorylated and phosphorylated states are characteristic of
nuclear and cytoplasmic locations. Future studies could answer this
question by comparing the modification status within the HMGA
proteins in the nuclear and cytoplasmic fractions.
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ACKNOWLEDGEMENT |
---|
Thanks are due to Dr. Claudette Klein (St. Louis University) for rereading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant Wi-1210/2-1 from the Deutsche Forschungsgemeinschaft (to J. R. W.).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.
§ Present address: Max-Planck-Institut für Experimentelle Medizin, Göttingen D 37075, Germany.
Present address: Inst. of Biochemistry, Warsaw University,
Warsaw PL 02096, Poland.
¶¶ To whom correspondence should be addressed: MDS Proteomics A/S, Stærmosegårdsvej 6, 5230 Odense M, Denmark. Fax: 45 6557 2040; E-mail: j.wisniewski@protana.com.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M011053200
2 The nomenclature of the mammalian HMG proteins has been revised recently; see the HMG protein home page. Consequently, we also renamed the cHMGI protein of Chironomus "cHMGA." In this paper we use the new nomenclature of HMG proteins (the old nomenclature is given in parentheses). HMGA (HMGI/Y family); HMGA1a (HMGI), HMGA1b (HMGY); HMGA2 (HMGI-C); HMGB (HMG-1/2 family); HMGN (HMG-14/17 family); cHMGA (cHMGI).
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ABBREVIATIONS |
---|
The abbreviations used are:
HMG, high
mobility group;
DBD, DNA binding domain;
cHMGA, Chironomus
HMGA protein;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
CK2, casein kinase 2;
Cdc2, cyclin-dependent cell cycle 2;
PKC, protein kinase C;
HPLC, high pressure liquid chromatography;
MOPS, 4-morpholinepropanesulfonic acid;
acrylodan, 6-acryloyl-2-dimethylaminonaphthalene;
IFN, interferon-
;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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