From the Dipartimento di Biochimica, Biofisica e
Chimica delle Macromolecole, Università di Trieste, 34127 Trieste, the ¶ Centro di Riferimento Oncologico, Aviano (PN),
33081 Pordenone, and the
Dipartimento di Scienze
Microbiologiche, Genetiche e Molecolari, Università di Messina,
98100 Messina, Italy
Received for publication, October 18, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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Nuclear phosphoprotein HMGA1a, high mobility
group A1a, (previously HMGI) has been investigated during apoptosis. A
change in the degree of phosphorylation of HMGA1a has been observed
during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus
type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a
have been ascertained by liquid chromatography-mass spectrometry.
Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule)
occurs at the early apoptotic stages and is probably related to HMGA1a
displacement from DNA and chromatin release from the nuclear scaffold.
De-phosphorylation (one phosphate or no phosphate groups/HMGA1a
molecule) accompanies the later formation of highly condensed chromatin
in the apoptotic bodies. We report for the first time a direct link
between the degree of phosphorylation of HMGA1a protein and apoptosis
according to a process that involves the entire amount of HMGA1a
present in the cells and, consequently, whole chromatin. At the same
time we report that variously phosphorylated forms of HMGA1a protein
are also mono-methylated.
Among nonhistone nuclear proteins of mammalian cells, a family of
three proteins called HMGA1a, HMGA1b, and HMGA2 (previously termed
HMGI, HMGY, and HMGI-C,
respectively)1 have aroused
great interest in many laboratories over the last few years, due to the
variety of biological processes in which they are involved (Refs. 1-4
and references therein).
HMGA1a and HMGA1b are very similar, differing by only 11 amino acid
residues, because they are the splicing products of the same gene (5),
whereas HMGA2 is the product of another gene (6). These proteins are
composed of about a hundred residues, and all contain three
characteristic short basic regions, called AT-hooks, that
interact with AT-rich stretches of DNA in the minor groove
(7, 8).
A property that is characteristic not only of the three proteins under
discussion, but also of other HMG proteins, is a C-terminal domain
having a very high content of acidic residues (1, 9). In HMGA1a,
HMGA1b, and HMGA2 the acidic C-terminal domain is constitutively phosphorylated in vivo by CK2 (10-12), but additional sites
for cell-cycle-dependent phosphorylation by other kinases
have also been reported (13-15).
High levels of HMGA1a, HMGA1b, and HMGA2 proteins have been
found in embryonic cell lines or tissues as well as in neoplastic cell
lines and tumors, but they are absent or expressed at very low levels
in normal cells (1, 12, 16-23). These findings support the hypothesis
that the three proteins could be linked to growth, differentiation, and
neoplastic transformation.
Given the importance of HMGA1a, HMGA1b, and HMGA2 proteins in chromatin
dynamics, we addressed the question of chromatin assembly/disassembly by studying their post-translational modifications during apoptosis, a
process where profound changes in chromatin structure occur. Apoptosis
has been induced in four leukemic cell lines (HL60, K562, NB4, and
U937), and the study has been focused on the HMGA1a protein,
that is the prevalently spliced form in this type of cells. For the
first time we demonstrate that the HMGA1a protein is subject to both
hyper-phosphorylation and de-phosphorylation during apoptosis, the two
events being time-dependent. We show that a new and fully
de-phosphorylated form of HMGA1a is the main form present in the highly
condensed chromatin of apoptotic bodies, whereas hyper-phosphorylation
is characteristic of early stages of apoptosis and is related to the
onset of DNA fragmentation and nuclear envelope degradation. We also
report for the first time that HMGA1a is mono-methylated during
apoptosis and that this modification is independent of the degree of phosphorylation.
Cell Cultures and Treatments--
The four cell lines HL60,
K562, NB4, and U937 were cultured in RPMI 1640 medium containing 10%
fetal bovine serum at 37 °C. Induction of apoptosis was carried out
by three different procedures. All cell lines were treated with
etoposide (VP-16, Calbiochem), 68 µM, for different times
to have a considerable amount of apoptotic cells: 4.5 h (HL60,
NB4), 24 h (U937), and 48 h (K562). HL60 cells were also
treated with 0.1 µg/ml camptothecin (Calbiochem) for 4.5 h
following synchronization with 1 µg/ml aphidicolin (Calbiochem) for
15 h (24). U937 cells were also induced to apoptosis by herpes
simplex virus type-1 (HSV-1) (25).
The percentage of apoptotic cells was evaluated by double fluorescence
emission of cells treated with both propidium iodide (PharMingen) and
annexin-V fluorescein isothiocyanate-conjugated (PharMingen) with a
FACScan from Becton Dickinson (Franklin Lakes, NJ).
The time course experiment on HL60 cells was carried out with a
procedure similar to that of Morana et al. (26).
Apoptosis inhibition was carried out by treating HL60 cells with 10 or
25 µM Z-VAD-fmk (Calbiochem) in culture medium for 1 h, after which etoposide 136 µM was added and cells were
left to incubate for 30 min. Following etoposide incubation,
cells were washed and left in culture medium containing Z-VAD-fmk for another 2 h.
Protein Extraction, Electrophoretic Analysis, and Western
Blotting
DNA Fragmentation Assay
HPLC and LC-MS Measurements
Trypsin Treatment An Electrophoretic Retarded Band of HMGA1a Protein Appears in
Leukemic Cells during Apoptosis--
Protein extracts from four
leukemic cell lines (K562, HL60, NB4, and U937) have been analyzed by
acetic acid/urea electrophoresis, which showed, in the extract from
apoptotic cells, a double band at the migration position of the HMGA1a
protein, in comparison with the single band obtained from control cells
(Fig. 1A). To verify if the
retarded band could be due to any new apoptotic form(s) of HMGA1a, the
regions comprising HMGN2 and HMGA1a of the acid/urea first dimension
(Fig. 1A) were analyzed by SDS two-dimensional analysis. The pattern obtained was blotted for Western
identification using a polyclonal antibody raised against an N-terminal
peptide common to both HMGA1a and HMGA1b (5, 23) (Fig. 1B).
From the result reported in Fig. 1B, it is evident that the
antibody identified both normal HMGA1a and HMGA1b proteins
as well as the retarded band, which can consequently be considered as
one or more new forms of HMGA1a appearing during apoptosis.
Both Hyper-phosphorylation and De-phosphorylation of HMGA1a Takes
Place in Leukemic Cells during Apoptosis--
To understand the nature
of the HMGA1a form(s) generated during apoptosis, the same extracts of
Fig. 1 have been separated by HPLC, and the eluted protein was detected
by both UV absorption (Fig. 2,
A and B) and LC-MS total ion count (Fig. 2,
C and D). LC-MS molecular masses of HMGA1
components have been determined (Fig. 2, E and
F): control K562 cells contain two forms of HMGA1a protein
having masses of 11,745.9 and 11,826.0 Da, respectively. The predicted
mass of human HMGA1a protein without modifications (i.e. 106 amino acid residues, no initial methionine) is 11,544.8 Da, which
becomes 11,586.8 Da if N-terminal acetylation at the first serine is
assumed, as found in preceding studies. It is possible to infer this
also from both the mass spectrometric data recently reported by Reeves
and coworkers (11, 19, 29) and by tryptic digestion experiments as
reported below. Because one phosphate group increases the mass of a
protein by 80 Da, the two forms of HMGA1a protein found in control K562
can be considered as the di-phosphorylated (found, 11,745.9 Da;
calculated, 11,746.8 Da) and the tri-phosphorylated (found, 11,826.0 Da; calculated, 11,826.8 Da) modifications. This result is in agreement
with preceding data that reported in vitro and in
vivo phosphorylation of the three last serines at the C-terminal
of both HMGA1a and HMGA1b proteins (i.e. serines 98, 101, and 102) as consensus sites for CK2 (10-12). The extract from K562
apoptotic cells contains, in addition to the di-phosphorylated and
tri-phosphorylated forms present in the control cells, other forms
whose molecular masses are consistent with hyper-phosphorylated forms
(four phosphate groups, 11,905.4 Da and five phosphate groups, 11,986.0 Da) and with de-phosphorylated forms (one phosphate group, 11,665.4 Da and zero phosphate group, 11,585.5 Da). Assuming that LC-MS peak intensity is approximately proportional to the amount of protein, it is
possible to estimate that the sample from K562 control cells of Fig.
2E contains about 50% of di-phosphorylated HMGA1a protein and about 50% of tri-phosphorylated, whereas in the apoptotic sample
(Fig. 2F) the main form is tri-phosphorylated, all other forms being present at lower percentage.
LC-MS data shown in Fig. 3A
indicate that in all four analyzed cell lines the main forms of HMGA1a
are those that are di-phosphorylated and tri-phosphorylated. The same
data from apoptotic cells (Fig. 3B) show both
hyper-phosphorylated and de-phosphorylated forms. However, inspection
of the data indicates that the degree of phosphorylation is not only
cell type-dependent but also related to the percentage of
apoptotic cells present in the analyzed cell sample. In fact, di-phosphorylated and tri-phosphorylated forms of HMGA1a are the main
molecules in both HL60 and NB4 cells, which contain 36% of apoptotic
cells, whereas in K562, having 77% of apoptotic cells, the
tri-phosphorylated molecule is the main form accompanied by a
considerable amount of de-phosphorylated forms, and in U937, containing
97% of apoptotic cells, the main form of HMGA1a is without phosphate.
Thus the data of Fig. 3 allow us to conclude that, in the control cell
lines studied, HMGA1a is mainly present as the di- and
tri-phosphorylated forms. During apoptosis induced by etoposide, two
different processes take place, one hyper-phosphorylates the di- and
tri-phosphorylated forms, the other de-phosphorylates all
phosphorylated molecules. Very similar results were obtained for
camptothecin-induced HL60 cells (data not shown). Fig. 3 also shows
that, on the right side of most of the variously phosphorylated peaks,
there is a shoulder that in U937 apoptotic cells resolves into a
definite peak having a constant increase of about 14 Da. It seems
likely that this increase is due to methylation concomitant with the
phosphorylation/de-phosphorylation processes. Methylation of HMGA1b
protein (the other spliced protein) has also been reported by Reeves
and coworkers (29) in MCF-7 cells in which up to four methyl groups
have been identified by tryptic peptide mapping. No methylation has,
however, been reported previously for HMGA1a that on the contrary
results mono-methylated in the cell lines of this study.
To obtain further confirmation that the identified forms of HMGA1a
protein are due to phosphorylation, HPLC-purified protein samples from
both control and apoptotic K562 cells were alkaline phosphatase-treated. This removed phosphate groups from all
phosphorylated forms of HMGA1a and gave only the unphosphorylated,
N-terminally acetylated molecule, having molecular masses of 11,586.2 Da (control) and 11,585.9 Da (apoptotic) (data not shown).
Tryptic Digestion of HMGA1a from both Control and Apoptotic K562
Cells: Identification of Phosphorylated Peptides--
Tryptic
fragments of HPLC-purified HMGA1a from both control and apoptotic K562
cells were analyzed by LC-MS spectrometry. 26 different peptides,
spanning the entire HMGA1a sequence, have been identified and are
reported in Fig. 4A. For each
of the identified peptides, a search for the phosphorylated forms was
carried out in both control and apoptotic samples, and the following
conclusions were reached: (i) in both control and apoptotic cells
N-terminal fragments were acetylated; (ii) in control K562 cells the
main phosphorylated fragment was the 88- to 106-animo acid peptide present as the di- or tri-phosphorylated forms; (iii) in apoptotic K562
cells the 88- to 106-amino acid peptide was mainly
unphosphorylated; one phosphate group has been detected in fragments
1-23, 73-83, and 74-83; mono- and di-phosphorylated forms have been
identified for the fragments 26-54, 30-54, and 30-57. This means
that the additional phosphates in the hyper-phosphorylated forms are
located inside the protein molecule and that the de-phosphorylation
process starts by removing the phosphate groups from the C-terminal
side.
On the basis of literature data (10-14, 29, 30), we have drawn the
scheme shown in Fig. 4B, where all the putative sites for
hyper-phosphorylation of the HMGA1a protein are shown together with the
constitutive C-terminal modified serines. Data summarized in Fig.
4B indicate that it could be possible to find HMGA1a
molecules bearing up to seven phosphate groups at a time. LC-MS data
shown in Fig. 3B clearly indicate up to five phosphate
groups; however, very low amounts of HMGA1a having six or seven
phosphate groups have been detected (data not shown). In any case, it
must be pointed out that phosphorylation and de-phosphorylation
processes could partially overlap.
Hyper-phosphorylation of HMGA1a Precedes its De-phosphorylation
during Apoptosis--
From preceding data, results show that the
hyper-phosphorylation of HMGA1a and its de-phosphorylation should be
related to different events that take place at different times during
apoptosis and concern different regions of the molecule. To clarify
this point, we have carried out a time course at the beginning of
apoptotic induction. To this end, HL60 cells have been induced with 136 µM etoposide for 30 min, then the drug was removed and
washed cells were left to proceed toward apoptosis far up to 3 h.
Proteins were PCA extracted and LC-MS analyzed at four different times (after 30-min treatment and 1, 2, and 3 h from washing). In Fig. 5A, the result of the time
course is reported grouping together de-phosphorylated HMGA1a forms
(0P + 1P), di- and tri-phosphorylated forms (2P + 3P), and hyper-phosphorylated forms (4P + 5P). The time
course shows that hyper-phosphorylation of HMGA1a starts as soon as
etoposide treatment begins, reaching a maximum 1 h after drug
removal when the forms having one or no phosphate groups begin to
appear. Phosphate groups introduced at an early stage of the apoptotic
process are added to the two or three phosphate groups already present
at the C-terminal of the molecule and produce the 4P and 5P (or more
phosphorylated) modifications. De-phosphorylation starts with the
removal of the constitutive phosphate groups, i.e. C-terminal phosphates (serines 98, 101, and 102)
probably when phosphorylation inside the HMGA1a molecule is still
active. This is the reason why di- and tri-phosphorylated forms can
have a constitutive origin (i.e. control cells or
cells at very early stages of the process) or an apoptotic origin,
deriving from the removal of constitutive phosphate groups
from molecules bearing four or five (or more) phosphates
(i.e. hyper-phosphorylated forms). Further support for the
hypothesis that hyper-phosphorylation of HMGA1a protein should be
related to the early events of the apoptotic process derives from the
electrophoretic analysis of the DNA reported in Fig. 5B
where the maximum level of HMGA1a phosphorylation observable after
1 h does correspond to high molecular weight digested DNA, whereas
the subsequent de-phosphorylation process correlates with the formation
of low molecular weight DNA fragments.
Caspase Inhibition Evidences HMGA1a Hyper-phosphorylation as an
Early Event of Apoptosis--
It is well known that topoisomerase I
and II poisons such as etoposide and camptothecin cause apoptosis
through cell cycle block and activation of a group of cysteine
proteases called caspases (31-35). Inhibition of caspase activity
slows down or stops the advance of the apoptotic process and could
allow one to obtain information on its very early events. Therefore, we
used the caspase inhibitor Z-VAD-fmk (35-38) on HL60 etoposide-treated
cells and both DNA fragmentation and HMGA1a phosphorylation were
analyzed. To understand to which nuclear morphologic change
phosphorylation of HMGA1a protein could be related during apoptosis, we
carried out microscopic observation of aliquots of the same cells
stained with 4,6-diamidino-2-phenylindole. Fig.
6A shows that using 25 µM Z-VAD-fmk (lane 2) DNA fragmentation is
almost blocked, and small differences in the chromatin status
are observable comparing representative cells from the control to the
25 µM Z-VAD-fmk-treated cells (Fig. 6B,
1 and 2). Moreover, mass data (Fig.
6C, 2) show that hyper-phosphorylated forms
(4P and 5P) have been produced. If a lower
Z-VAD-fmk concentration is used (10 µM, Fig.
6A, lane 3), an increased digestion of DNA is
observable, but it is lower, however, than the substantial nucleosomal
cleavage reached after 2 h of etoposide treatment in the absence
of Z-VAD-fmk (Fig. 6A, lane 4). Consistently, an
increased level of hyper-phosphorylation has been detected in this
protein sample: note that the 4P peak in mass spectrum
3 (Fig. 6C) has the same intensity as the 2P peak. At this stage, in which a clear beginning of DNA
fragmentation is seen (Fig. 6A, lane 3),
chromatin starts to condense showing a typical alteration of nuclear
organization (Fig. 6B, 3) but not yet forming
well defined apoptotic bodies as those observed after reported 2 h
treatment of cells without Z-VAD-fmk (Fig. 6B,
4). In this last sample both hyper-phosphorylation and
de-phosphorylation are observable (Fig. 6C, 4),
because only about 30% of cells are definitively apoptotic (see Fig.
5A), while the remaining cells are still running through the
preceding steps of the apoptotic process. It is then obvious that
protein mixtures extracted from such a composite system would contain
both hyper-phosphorylated forms (i.e. early stages of
apoptosis) and de-phosphorylated forms (i.e. late stage of
apoptosis). In conclusion, we think that inhibition experiments using
Z-VAD-fmk clearly show that hyper-phosphorylation of HMGA1a is an early
apoptotic event and, at the same time, demonstrate that the level of
phosphorylation of this protein is truly linked to the apoptosis of
leukemic cells.
U937 Leukemic Cells Induced to Apoptosis by Herpes Simplex Virus 1 (HSV-1) Show Degrees of HMGA1a Phosphorylation Similar to That Found in
Cells Induced by Nonviral Agents--
To verify that the degree of
phosphorylation of HMGA1a during apoptosis is independent of the agent
capable of triggering the signal pathway that leads to apoptosis, we
analyzed protein extracts from apoptotic U937 cells induced by herpes
simplex virus type-1 (HSV-1) (25). In Fig.
7, LC-MS data of the HMGA1a protein from
U937 apoptotic cells are compared with those of control mock cells, and
it is possible to see that a massive de-phosphorylation of HMGA1a takes
place during apoptosis. Moreover, it is noteworthy that methylation of
HMGA1a as revealed in drug-induced leukemic cells (Fig. 3B)
is also observable in virus-induced U937 cells. We conclude that both
alteration of phosphorylation and methylation are related to the
apoptotic process per se rather than to the agent used to
induce apoptosis.
This report concerns the study of post-translational modifications
of HMGA1a protein during apoptosis induced in four leukemic cell lines
(HL60, K562, NB4, and U937). These cell lines show constitutive
expression of the two proteins HMGA1a and HMGA1b, whereas the HMGA2 has
not been detected; HMGA1a is the predominant species as compared with
HMGA1b. In fact, from Coomassie Blue-stained electrophoretic patterns
we evaluated that the ratio HMGA1a/HMGA1b in both HL60 and K562 cells
is about 10:1 (data not shown). The present paper deals only with the
most abundant protein (i.e. HMGA1a), but a similar behavior
was ascertained also for HMGA1b during apoptosis, from both a careful
inspection of the Western analysis shown in Fig. 1 and mass data not
shown. Electrophoretic patterns have been also used to evaluate the
molecular ratio between histone H1 and HMGA1a that indicated a ratio of
about 20:1 in control HL60 cells (data not shown). This means that in
these cells there is on average one HMGA1a molecule for every 20 nucleosomes, assuming about one histone molecule is bound to the linker
DNA of each nucleosome (39).
What could the function for this high amount of HMGA1a protein be? It
is not conceivable that it is entirely involved in the formation of
specific protein entities that regulate transcription of specific genes.
Involvement of HMGA1a protein has been reported not only at promoter
regions of specific genes, where a limited amount of protein appears to
be necessary, but also at more global nuclear structures related to
higher order chromatin bound to the nuclear matrix and forming distinct
nucleoproteic loops. Such structures have been called MARs (matrix
attachment regions) or SARs (scaffold attached regions), and it has
been shown that they contain specialized AT-rich DNA regions
with high unwinding aptitude termed BURs (base-unpairing regions)
(40-42). BURs specifically bind HMGA1a and HMGA1b proteins (43),
although this should involve DNA regions different from those bound to
histone H1. In fact, it has been reported that HMGA1a displaces histone
H1 from chromatin and nuclease-sensitive chromatin releases HMGA1a,
HMGA1b, and HMGA2 proteins but not histone H1 (41, 44, 45). At the same
time, in an immunocytochemical study we have demonstrated that
topoisomerase II As reported under "Results," in addition to the C-terminal
constitutive phosphates due to CK2, HMGA1a protein bears other phosphate groups that could derive from the action of different kinases. The constitutive C-terminal phosphorylation due to CK2 is not
directly involved in DNA binding alteration, although an indirect
effect could be elicited by affecting protein tertiary structure (53).
On the other hand, hyper-phosphorylation of HMGA1a protein could be
related to its displacement from DNA; this results in a more open
chromatin structure that is a more accessible substrate for nucleases,
which produce large DNA fragments at the very early stages of
apoptosis, together with lamin degradation. DNA fragmentation may have
the release of MARs from nuclear scaffold as a first step followed by
chromatin unfolding that allows progressive DNA digestion. Large DNA
fragments (20-50 kbp) should thus be related to HMGA1a
hyper-phosphorylation and initial chromatin condensation as shown in
Fig. 6. Further DNA fragmentation generates DNA ladders that are
characteristic of the highly condensed chromatin of apoptotic bodies.
This last event is related to de-phosphorylated HMGA1a protein as shown
in Fig. 3B for U937 cells. It is worthwhile to mention that
at neither the initial stage of apoptosis nor the later stage of
apoptotic bodies formation is there loss of HMGA1a protein (data not
shown). We wish to point out that the phosphorylation/de-phosphorylation process evidenced for HMGA1a involves the total amount of protein present in the cell and, consequently, chromatin as a whole. Very recently, phosphorylation of
both histones H2B and H2AX has been reported during apoptosis of HL60
cells induced by etoposide (54, 55). Phosphorylation of these histones,
detected by 32P autoradiography, concerns only a fraction
of the total protein (about 5-10% in the case of histone H2B) and has
been related to the early phase of DNA fragmentation during apoptosis.
These results are consistent with our data on hyper-phosphorylation of
HMGA1a as an early event of apoptosis in which some histones, HMGA1a
and HMGA1b proteins, and other proteins of the nuclear scaffold are
substrates of a programmed process of phosphorylation that initiates
cell death. However, apoptotic hyper-phosphorylation of HMGA1a is a
quantitatively more important event both for the number of new
phosphorylation sites (at least four) and the involvement of the entire
amount of protein. Moreover, the following massive de-phosphorylation,
leading to a completely de-phosphorylated form (here observed for the
first time), could be one of the events required for an irreversible
chromatin condensation, just as the final committed apoptosis is. The
de-phosphorylation of HMGA1a protein parallels another
de-phosphorylation process recently described for H1 histone during
apoptosis of HL60 cells. In fact, Kratzmeier et al. (56)
reported that histone H1 sub-types become rapidly de-phosphorylated
upon apoptosis induction and interpret this phenomenon as an important
event for the process of chromatin condensation and/or chromatin
fragmentation. Our data are consistent with the apoptotic
de-phosphorylation of histone H1 and demonstrate, for the first time,
that phosphorylation/de-phosphorylation of HMGA1a is involved in the
apoptotic process and that HMGA1a could be considered as a structural
element in the chromatin of leukemic cells. Moreover, a characteristic
mono-methylation of HMGA1a has been evidenced that could reserve
further information on stress-exposed cells.
We have demonstrated that, at least in the cells studied, alteration in
the degree of phosphorylation of HMGA1a is independent of the agent
that induces apoptosis, i.e. drug or virus. However, it is
necessary to recall that HMGA1a protein is not present or present at
very low levels in normal cells, whereas its expression is increased in
transformed cells. Therefore, the link between alteration of
constitutive HMGA1a phosphorylation, chromatin, and apoptosis should
refer only to cells characterized by high levels of this protein. If,
on one hand, this limits the extension of the observed phenomenon to
all cells, on the other hand, it constitutes an interesting difference
between normal and neoplastic cells, which could provide a possible way
to induce or at least to influence apoptosis only in the latter. This
aspect of the question, concerning the differences of nuclear
organization and matrix protein composition in cancer and normal cells,
is a promising area for application in both cancer diagnosis and
prognosis (57).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Total HMG proteins and histone H1 were selectively
extracted by treating cells with 5% perchloric acid (PCA) (w/v) as
previously reported (17, 18). Electrophoretic analysis was carried out on 15% polyacrylamide gels in acetic acid/urea or in SDS/Tris/Tricine buffer as described previously (12). For Western blot analysis, a
rabbit polyclonal antibody raised against an N-terminal peptide of
HMGA1a protein was used (23).
DNA from control or apoptotic cells
was extracted essentially according to Zhu and Wang (27). DNA fragments
were analyzed on 2% agarose gel in Tris-acetate-EDTA buffer and
visualized with ethidium bromide under UV light.
Reverse-phase HPLC
chromatography of PCA extracts was carried out with a PerkinElmer Life
Sciences apparatus (series 200 LC pump and 785A UV-visible detector)
using a Vydac Protein C4 column (2.1 × 150 mm). Protein was
eluted by a water/acetonitrile gradient, and the chromatogram was
obtained by absorbance detection at 220 nm. At the same time, an
aliquot of the eluted solution was directly injected into an interfaced mass spectrometer (PE SCIEX, API 1), which gave an equivalent chromatogram by counting total ions (TIC) that reach the mass detector
(28). Search for ion composition was carried out on each TIC peak, and
a succession of m/z+ values was
obtained for every protein form contained in that peak. The
identification of all successions of
m/z+ allowed to obtain reconstructed
mass spectra (±1 Da) that gave the molecular composition of the
analyzed peak. The same procedure was employed for the analysis of
tryptic fragments, the only difference being that a Waters Delta-Pak
C18 column (2 × 150 mm) was used. LC-MS then was used to identify
both different forms of the whole protein molecule and different forms
of tryptic fragments.
HPLC-purified HMGA1a (15 µg) from both
K562 control and apoptotic cells was treated for 15 h at 37 °C
in 50 µl of Tris/HCl, 100 mM, pH 8.5, containing 0.75 µg of sequencing grade trypsin (Roche Diagnostics), and the reaction
was stopped by freezing. Thawed tryptic samples were directly injected
into the HPLC apparatus interfaced with the mass spectrometer, at room temperature.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electrophoretic and Western analyses of
PCA-extracted proteins from K562 cells show new form(s) of HMGA1a
protein during apoptosis. A, first-dimension
electrophoretic analysis in acetic acid/urea 15% polyacrylamide gels
of PCA-extracted protein samples from control and etoposide-induced
apoptotic K562 cells (Coomassie Blue staining). B, the
regions comprising HMGN2 and HMGA1a proteins in the first dimension
acetic acid/urea were analyzed in a second SDS dimension, then blotted
for Western analysis. Protein identification was carried out using a
polyclonal antibody raised against an N-terminal peptide common to both
HMGA1a and HMGA1b and proteins detected by the ECL + Plus procedure
(Amersham Pharmacia Biotech). Before carrying out the second SDS
electrophoretic analysis, to prevent protein mixing by diffusion in the
course of the run, HMGA1a and the retarded band have been separately
excised and a piece of blank gel interposed between them.
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Fig. 2.
HPLC elution profiles and reconstructed
masses of PCA-extracted proteins from K562 cells show changes in the
degree of phosphorylation of HMGA1a protein during apoptosis.
Reverse phase HPLC elution profiles of PCA extracted proteins from
control (A, C) and etoposide-induced apoptotic
(B, D) K562 cells detected as absorbance at 220 nm (A, B) or as total ion count (C,
D). Dotted lines in A and B
refer to the percentage of acetonitrile (solvent B) in the gradient
used for the HPLC elution. Proteins contained in each peak are labeled
in A and B. Composition of HMGA1a peaks from
control (E) and apoptotic (F) K562 cells was
obtained by mass spectrometry, and results are reported as
reconstructed masses (Da). In E and F
the identified mass values (Da) are reported; from these
values the number of phosphate groups (OP, 1P,
2P, 3P, 4P, and 5P) borne
by HMGA1a protein were deduced.
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Fig. 3.
Both hyper-phosphorylated and
de-phosphorylated forms of HMGA1a protein are present in different
apoptotic leukemic cell lines. Proteins from both control
(A) and etoposide-induced apoptotic (B) leukemic
cell lines (HL60, NB4, K562, and U937) were PCA-extracted and
LC-MS-analyzed. Reconstructed masses were obtained for the HMGA1a peak
of each extract. A, mass values (Da) of
bi-phosphorylated (2P) and tri-phosphorylated
(3P) forms are shown. These forms resulted as constitutive
modifications of HMGA1a protein in the four studied cell lines.
B, reconstructed mass values (Da) are reported
for each sample together with the deduced number of phosphate groups
(from OP up to 5P). The percentage of apoptotic
cells was obtained by FACScan analysis and shown in square
brackets. Underlined mass values refer to
phosphorylated HMGA1a forms that are also methylated.
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Fig. 4.
Constitutive and hyper-phosphorylation
suggested sites of HMGA1a protein in leukemic cells. A,
tryptic fragments of HMGA1a protein identified by LC-MS of
HPLC-purified protein samples from both control and apoptotic K562
cells. Identified phosphorylated fragments (1-23, 25-54, 30-54,
30-57, 73, 74, 88) are evidenced. B, the
results of LC-MS analyses on whole molecule and tryptic fragments from
both control and etoposide-induced apoptotic K562 cells are summarized.
The sequences of the identified phosphorylated peptides are shown, and
suggested modified residues are indicated. AT,
AT-hooks.
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Fig. 5.
Time course of etoposide-induced HL60 cells:
Hyper-phosphorylation of HMGA1a protein precedes its de-phosphorylation
and is related to initial DNA fragmentation, whereas de-phosphorylation
accompanies formation of inter-nucleosomal fragments.
A, HL60 cells were treated with 136 µM
etoposide for 30 min and after washing left to proceed toward apoptosis
quantified by FACScan (%).The degree of phosphorylation of HMGA1a
protein was evaluated at the beginning of the experiment
(Control, 0 h), after 30 min of etoposide treatment,
and after 1, 2, and 3 h from washing. Phosphorylated forms are
reported as follows: 0P + 1P ( ); 2P + 3P (
); 4P + 5P (
). All
reported values are the results of three independent experiments.
B, DNA fragmentation in the time course of apoptosis of HL60
cells was analyzed on 2% agarose gel and visualized with ethidium
bromide.
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Fig. 6.
Use of the caspase inhibitor Z-VAD-fmk slows
down the etoposide-induced apoptosis of HL60 cells and allows
observation of hyper-phosphorylation as an early event associated with
initial chromatin condensation. HL60 cells were preincubated with
the caspase inhibitor Z-VAD-fmk for 1 h, then 136 µM
etoposide was added for 30 min and, after washing, cells were left for
other 2 h in the presence of the inhibitor. 1, HL60
control; 2, Z-VAD-fmk (25 µM); 3,
Z-VAD-fmk (10 µM); 4, HL60 apoptotic control
sample (i.e. 136 µM etoposide, no inhibitor).
A, DNA fragmentation was determined by electrophoresis on
2% agarose gel, and bands were visualized by ethidium bromide.
B, under each electrophoretic pattern microscope images of
4,6-diamidino-2-phenylindole-stained representative nuclei are
shown. C, LC-MS mass data (reconstructed masses) of HMGA1a
protein from each cell sample are reported, and the number of phosphate
groups is indicated.
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Fig. 7.
LC-MS experiments demonstrate that U937 cells
induced to apoptosis by HSV-1 contain de-phosphorylated forms similar
to those found in cells induced by nonviral agents.
Proteins were selectively PCA-extracted from both control
(A) and virus-induced U937 cells (B) and
LC-MS-analyzed. Reconstructed masses were obtained for the HMGA1a peak
of each extract. Mass values (Da) of variously
phosphorylated forms are shown (0P, 1P,
2P, 3P) together with the values
(underlined) of the mono-methylated species.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HMGA1a-HMGA1b proteins colocalize in the
interphase nucleus of HeLa cells (46). The mutually exclusive
localization of histone H1 and HMGA1a protein could account for a
different involvement of these two factors in the processes of
chromatin condensation/de-condensation, which are in turn related to
the phosphorylation of these proteins, both well known substrates for
cyclin-dependent kinases p34-Cdc2 and Cdk2 (13, 14, 47). H1
phosphorylation due to these kinases appears to be related to mitosis
(48-52) rather than to apoptosis, whereas data from this report
indicate a relationship of HMGA1a phosphorylation with apoptosis and
preceding data associated HMGA1a phosphorylation by p34-Cdc2 with
mitosis (13, 14).
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FOOTNOTES |
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* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Milano, Italy, by Ministero della Ricerca Scientifica e Tecnologica, Roma, Italy (Grants 9706274625 and 9806279300), by ASI (Grants ARS-99-47 and I/R/129/00), and by the Università di Trieste, Italy.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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Università degli Studi di Trieste, Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Via Licio Giorgieri, 1-34127 Trieste, Italy. Tel.: 39-040-6763676; fax: 39-040-6763694; E-mail: giancot@bbcm.univ.trieste.it.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009521200
1 The nomenclature of the high mobility group (HMG) proteins has been recently revised (see the Chromosomal Proteins Nomenclature on the Web). In this report we use the new nomenclature, but we report the old one in parenthesis. The abbreviations used are: HMGA1a, high mobility group A1a (HMGI); HMGA1b, high mobility group A1b (HMGY); HMGA2, high mobility group A2 (HMGI-C); HMGN1, high mobility group N1 (HMG 14); HMGN2, high mobility group N2 (HMG 17); CK2, casein kinase 2; Z-VAD-fmk, n-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PCA, perchloric acid; LC-MS, liquid chromatography-mass spectrometry; TIC, total ion counts; p34-Cdc2, p34-cycle-dependent cyclin 2 kinase; Cdk2, cyclin-dependent kinase 2; MARs, matrix attachment regions; SARs, scaffold attached regions; BURs, base-unpairing regions; kbp, kilobase pair(s); HSV-1, herpes simplex virus type-1; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography.
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