From the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sp1 sites can mediate growth/cell cycle induction
of dihydrofolate reductase in late G1 (Jensen,
D. E., Black, A. R. Swick, A. G., and Azizkhan, J. C. (1997) J. Cell. Biochem. 67, 24-31). To
investigate mechanisms underlying this induction, effects of serum
stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells,
serum stimulation did not affect Sp1 synthesis or the relative binding
of Sp1 family members to DNA; however, it did result in a rapid,
~2-fold increase in Sp1 levels and an ~3-fold increase in specific
Sp1 phosphorylation in mid-G1. In normal human diploid
fibroblasts, serum stimulation also increased Sp1 phosphorylation in
mid-G1 but did not affect Sp1 levels. Therefore, Sp1
phosphorylation is regulated in a growth/cell
cycle-dependent manner which correlates temporally with
induction of dihydrofolate reductase transcription. Further studies
revealed a kinase activity specifically associated with Sp1 in a
growth-regulated manner. This activity is distinct from purified
kinases previously shown to phosphorylate Sp1 in vitro and
phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a
region also phosphorylated in mid-G1 in vivo.
Therefore, this study indicates that phosphorylation of the C terminus
of Sp1 may play a role in the cell cycle regulation of its
transcriptional activity.
Expression of a large number of genes associated with DNA
synthesis, such as dihydrofolate reductase
(DHFR),1 is tightly regulated
with cell growth and the cell cycle. Many of these genes have promoters
which lack a TATAA element but contain binding sites for the
transcription factors Sp1 and E2F (1). Although the role of E2F sites
in growth/cell cycle regulation of transcription and the regulation of
E2F by retinoblastoma protein (pRB) and related pocket proteins have
been extensively characterized (see Refs. 2-4, for review), a role for
Sp1 sites in growth/cell cycle regulation of transcription is only
beginning to emerge (e.g. Refs. 5 and 6). We have determined
that Sp1 and E2F sites have distinct roles in the growth/cell cycle
regulation of the hamster DHFR promoter (6). Although complete
repression of DHFR transcription in G0 and early
G1 requires E2F sites, its induction in late G1
is mediated by Sp1 sites. A direct role of Sp1-dependent
transcription in growth regulation of transcription is supported by
targeting of Sp1 by viral oncoproteins (e.g. Refs. 7-9),
down-regulation of Sp1 expression, and/or DNA binding activity upon
differentiation in some systems (10), and increased Sp1 expression
during events associated with transformation (11).
Sp1 is a ubiquitous, 778-amino acid transcription factor that
recognizes GC-rich sequences present in many promoters (see Refs. 1 and
2, for review). Although Sp1 has been viewed as a constitutive
transcriptional activator which acts as a basal factor for TATAA-less
promoters, an increasing number of studies indicate that
Sp1-dependent transcription is regulated in response to a
variety of signals. For example, in addition to their role in
growth/cell cycle regulation of transcription, Sp1 sites are involved
in induction of DHFR transcription in response to methotrexate (12),
induction of CYP11A transcription in response to cyclic AMP (13), and
transforming growth factor Regulation of Sp1-dependent transcription could be effected
by changes in Sp1 abundance, DNA binding activity, and/or
transactivation activity. Sp1 is O-glycosylated and
phosphorylated, and both of these modifications are likely to be
important in its regulation. Phosphorylation has been implicated in
changes in Sp1 binding and transcriptional activation (15-17), and
changes in O-glycosylation alter the stability of Sp1
in vivo (18) and its interaction with other factors (19). An
additional mechanism that could underlie the regulation of gene
expression through Sp1 sites has come to light with the finding that
Sp1 is a member of a multigene family. Of the four known members of the
family, Sp2 does not recognize the same sequence as Sp1 and Sp4
expression is restricted to the brain. Sp3, on the other hand, is
ubiquitously expressed and recognizes the same sequences as Sp1 but has
a more complex transcriptional activity. Whereas Sp1 appears to be
almost exclusively an activating transcription factor, Sp3 contains a
transcriptionally repressive domain and can act as a transcriptional
activator or repressor, dependent on the promoter and cell type
(20-25). A role for Sp3 in regulation of Sp1
site-dependent transcription is seen in its mediation of
p21 induction during keratinocyte differentiation (26).
To determine the mechanism(s) underlying growth/cell cycle-regulated
induction of Sp1 site-dependent transcription, we
characterized changes in Sp1 following serum stimulation of quiescent
cells. Serum stimulation leads to an increase in Sp1 levels and
phosphorylation, with the increased phosphorylation correlating with
induction of Sp1-dependent activation of the DHFR promoter
in mid-late G1. We also determined that this change in
phosphorylation is accompanied by changes in the association of Sp1
with a novel Sp1 kinase activity which phosphorylates the C terminus of Sp1.
Cell Culture and Synchronization--
Balb/c 3T3 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.), 10% calf serum (Colorado Serum Co.), penicillin/streptomycin
(Life Technologies, Inc.) at 37 °C in a 10% CO2
atmosphere (6). Normal human diploid fibroblasts (NHDF) from Clonetics
were maintained in Dulbecco's modified Eagle's medium, 10% fetal
bovine serum (Life Technologies, Inc.), penicillin/streptomycin at
37 °C in a 10% CO2 atmosphere (27) and used at less
than 30 doublings. Cells were synchronized by serum starvation in
medium containing 0.5% serum for 24 h (Balb/c 3T3) or 0.2% serum
for 60-72 h (NHDF) and induced to re-enter the cell cycle by addition
of serum to 10-20% (6, 27).
In Vivo Labeling and Western Blot Analysis--
For
[35S]methionine labeling, cells were rinsed with 20 mM HEPES, pH 7.2, 150 mM NaCl and placed in
methionine-free Dulbecco's modified Eagle's medium containing
appropriate concentrations of dialyzed fetal bovine serum. After 30 min, medium was removed and cells were labeled for 1 h in the same
medium containing 7.5 µCi/ml [35S]methionine.
32PO4 labeling was carried out similarly except
that cells were incubated in phosphate-free medium for 2 h prior
to being labeled in phosphate-free medium containing 7.5-15 µCi/ml
32PO4 for 2 h. At the indicated times
following serum stimulation, cells were rinsed with phosphate-buffered
saline (135 mM NaCl, 4 mM KCl, 10 mM Na2PO4,, pH 7.4) and lysed
directly in boiling 10 mM Tris-HCl, pH 7.2, 1% SDS,
reboiled and DNA was sheared. Following addition of 2.2 volumes of
ice-cold 15 mM Tris-HCl, pH 7.2, 7.5 mM EDTA,
150 mM sodium fluoride, 230 mM NaCl, 1.5% Triton X-100, 0.75% Nonidet P-40, 100 mM
Electrophoretic Mobility Shift Assay of Sp1 Family
Members--
Balb/c 3T3 cells were harvested at the indicated times
following serum stimulation and nuclear extracts were prepared as
described (28). Electrophoretic mobility shift assay of binding to a
double-stranded 32P-labeled probe corresponding to the
first Sp1 site in the hamster DHFR promoter was performed as described
(28, 29). Assays were conducted with equal amounts of nuclear extract
protein from serum-starved and serum-stimulated extracts. Specificity
of binding was confirmed by competition with a 50-fold excess of
unlabeled probe.
Analysis of ATP Pools--
Balb/c 3T3 cells were labeled with
32PO4, washed with phosphate-buffered saline,
and harvested by scraping in 1 M formic acid. Non-acid
soluble material was removed by centrifugation and free 32PO4 was removed and samples were neutralized
as described (30). Nucleotides were separated on a 3-µm Supelcosil
LC-18 column using a 15-min linear gradient of 0.1 M
potassium dihydrogen phosphate, pH 6.0, 8 mM
tetrabutylammonium, 0.1 M potassium dihydrogen phosphate, pH 6.0, 8 mM tetrabutylammonium, 30% methanol at a flow
rate of 1.5 ml/min (31). Eluted nucleotides were detected by their
absorbance at 254 nm using a Perkin-Elmer UV95 uv/visible in-line
detector and radioactivity was detected by counting Cerenkov radiation with an INSUS Systems Inc. Phosphoamino Acid Analysis--
Sp1 was immunoprecipitated from
serum-stimulated 32PO4-labeled cells, separated
by 8% SDS-PAGE, and transferred to polyvinylidene difluoride membrane.
The region of the membrane containing the 32P-labeled Sp1
(as determined by autoradiography) was excised and phosphoamino acid
analysis was performed by two-dimensional thin layer electrophoresis in
the presence of unlabeled phosphoserine, phosphothreonine, and
phosphotyrosine standards as described (32).
Assay of Promoter Activity--
Balb/c 3T3 cells were
transfected with 20 µg of DNA (5 µg of reporter constructs and 10 µg of Flag-Sp1 constructs) by the calcium phosphate co-precipitation
method and chloramphenicol acetyltransferase activity was assayed as
described (6). Luciferase activity was assayed 24 h after serum
stimulation using the Promega Luciferase Assay System. Correction for
differences in transfection efficiency between precipitates used the
activity of a co-transfected Rous sarcoma virus long-term
repeat-chloramphenicol acetyltransferase construct which is unaffected
by Sp1 overexpression. DHFR In Vitro Kinase Assays--
NHDF, serum-starved or
serum-stimulated, were extracted in 5 volumes of 100 mM
HEPES, pH 7.4, 500 mM KCl, 5 mM
MgCl2, 0.5 mM EDTA, 5 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and freeze/thawed 3 times. Particulate matter was removed by
centrifugation (13,000 × g, 10 min) and extracts were
stored at GST-Sp1 Constructs and Expression of GST Proteins--
GST-Sp1
and GST-Sp1(1-612), in which Sp1 cDNA was fused to glutathione
S-transferase cDNA in pGEX1, were kind gifts from Dr. J. Horowitz and are described elswhere (27). Other GST-Sp1 constructs were
generated using standard cloning techniques by deletion between the 5'
and 3' end of the cDNA and the internal XmnI and
Eco47III sites of Sp1. Bacteria, freshly transformed with
GST-Sp1 expression vector, were grown to mid-log and protein was
induced for 4 h with 1 mM isopropyl
In Vitro Phosphorylation of GST Fusion Proteins--
GST fusion
proteins were bound to glutathione-Sepharose for 30 min at 4 °C and
unbound proteins were removed by washing the beads with RIPA (4 × 1 ml) and kinase buffer (3 × 0.5 ml). Glutathione-Sepharose bound
GST-fusion proteins were incubated in 50 µl of kinase buffer containing 5 µl of 1 M NaCl extract of Sp1
immunoprecipitates and 10 µCi of [ Analysis of Phosphorylation of Flag-Sp1 Fusion
Proteins--
Flag-Sp1 fusion constructs were generated by subcloning
Sp1 cDNA into pFlag-CMV2 (Kodak). pFlag-Sp1(1-612) and
pFlag-Sp1(612-778) were produced likewise except that 5' and 3'
deletions of the Sp1 cDNA were generated using the internal
BamHI site in Sp1 cDNA. Balb/c 3T3 cells were
transfected with these plasmid constructs (20 µg), serum-starved and
-stimulated as described (6). Cells were harvested 9 h after serum
stimulation and Flag fusion proteins were immunoprecipitated from the
pre-cleared extracts with a mouse monoclonal anti-Flag antibody (M2;
Kodak) and protein G-Sepharose as described above. Proteins were
analyzed by SDS-PAGE and autoradiography. Expression of Flag fusion
proteins was confirmed by Western blot analysis with a rabbit anti-Flag
antibody (Santa Cruz).
Comparison of Sp1 from G0 and G2 Balb/c 3T3
Cells--
To elucidate mechanisms underlying the growth/cell cycle
regulation of Sp1-dependent transcription, changes in
various aspects of Sp1 regulation were examined in serum-starved Balb/c
3T3 cells and in these cells following 8 h of serum stimulation
(i.e. cells in G0 and late G1,
respectively (6)). Levels of expression, rate of synthesis, and
phosphorylation of Sp1 were examined by labeling equal numbers of cells
with [35S]methionine or 32PO4,
followed by immunoprecipitation of Sp1 and SDS-PAGE/autoradiography and
Western blotting. Serum stimulation resulted in an ~2-fold (1.9 ± 0.4, n = 8) increase in Sp1 levels (Fig.
1A), whereas Sp1 synthesis
remained essentially unchanged (G1/G0 = 0.8 ± 0.1, n = 4; Fig. 1B).
Incorporation of 32P into Sp1 increased ~6-fold (6.1 ± 1.5, n = 4) with serum stimulation (Fig.
1C). Since the increase in phosphorylation was greater than the increase in the level of protein, the increase in 32P
incorporation not only reflects the increased amounts of Sp1 but also
an increase (3.1 ± 0.4-fold, n = 4) in the
specific activity of Sp1 molecules.
Growth factor stimulation of quiescent Swiss 3T3 cells leads to a rapid
increase in phosphate uptake, ATP turnover, and ATP pool size (33, 34).
To determine if the changes in incorporation of 32P into
Sp1 were due to differences in the specific activity of ATP pools,
cells were labeled as above and ATP specific activities were measured
(31). In Balb/c 3T3 cells, serum stimulation led to a 1.8-fold increase
in cellular ATP pools, but only a slight (1.3-fold) increase in the
specific activity of ATP (Table I). Since
the change in specific activity of ATP is significantly lower than the
increase in specific activity of Sp1, there must be an increase in Sp1
phosphorylation during the G0 to S phase transition.
To determine if the ratio of Sp1 family members changed following serum
stimulation, electrophoretic mobility shift assays of nuclear extracts
were performed. These assays detected no change or a slight decrease in
Sp1 family binding activity following serum stimulation (Fig.
1D). This apparent inconsistency with the Western blot data
(Fig. 1A) arises from different methods of normalization.
Western blots were normalized to cell number and, therefore, reflect a
change in the level of Sp1 per cell; however, electrophoretic mobility
shift assays were normalized to the amount of protein in the extracts,
which increases on a per cell basis following serum stimulation.
Western blot analysis of nuclear extracts indicated that, when
normalized to total nuclear protein, the relative levels of Sp1 in
G1 extracts were also the same or slightly less than those
in G0 extracts (data not shown). Thus, although the level
of Sp1 per cell increases following serum stimulation, its specific
binding activity remains unaltered. These electrophoretic mobility
shift assay results also indicate that the relative levels of Sp1 and
Sp3 do not change significantly following serum stimulation (Fig.
1D, arrowhead and arrows). Therefore, of the
parameters tested, only changes in Sp1 levels and/or phosphorylation could account for the induction of Sp1-dependent
transcription seen upon serum stimulation.
Time Course for Serum Induction of Sp1 Levels and
Phosphorylation--
Since serum induction of
Sp1-dependent transcription from the DHFR promoter occurred
in late G1 (6), Sp1 levels and phosphorylation were
assessed in Balb/c 3T3 cells at various times following serum stimulation. As seen in Fig.
2A (lower panel),
induction of Sp1 levels was apparent by 2 h and was essentially
maximal by 4-6 h following serum stimulation. In keeping with the
increased levels of Sp1, 32P incorporation into Sp1 also
increase by 2 h; however, a clear difference between the timing of
the increase in Sp1 levels and its increased phosphorylation became
apparent when the changes in Sp1 levels were taken into account (Fig.
2B). Following serum-stimulation, specific Sp1
phosphorylation changed little for the first 4 h, but increased to
maximal levels by 6-8 h. Since S-phase occurs 10-12 h following serum
stimulation of these cells, Sp1 phosphorylation is induced in
mid-G1 and, therefore, occurs concomitant with or slightly
precedes the induction of Sp1-dependent DHFR transcription (6).
The delayed induction of DHFR following serum stimulation would
indicate that it is relatively insensitive to Sp1 levels. This is
supported by experiments in which overexpression of Sp1 had little
(1.34 ± 0.44-fold, n = 4) effect on
DHFR
To determine if growth regulation of Sp1 phosphorylation is unique to
Balb/c 3T3 cells, a similar experiment to that in Fig. 2A
was performed using NHDF which enter S-phase 10-14 h after serum
stimulation (27). These cells were chosen because (a) they
represent primary human cells, and (b) Sp1 levels do not change significantly during the G0 to S phase transition in
these cells (Ref. 27 and Fig.
3A). Serum stimulation of NHDF
led to an increase in Sp1 phosphorylation (2.4 ± 0.4-fold,
n = 4) while neither Sp1 levels
(G1/G0 levels = 1.2 ± 0.1, n = 7) nor the specific activity of ATP pools was
affected (Fig. 3A, Table I). Thus, induction of Sp1
phosphorylation in response to growth stimulation is not cell
type-specific and can be seen in the absence of significant changes in
Sp1 protein levels. Induction of Sp1 phosphorylation was also delayed
following serum stimulation of NHDF, with increased phosphorylation
first seen 6-8 h following addition of serum.
The vast majority of Sp1 phosphorylation is on serine in cycling HeLa
cells (35). To determine if this was the case following serum
stimulation of serum-starved cells, phosphoamino acid analysis was
performed on 32P-labeled Sp1 from late G1
cells. Since only phosphoserine was detected (Fig. 3B), the
increased phosphorylation of Sp1 seen in mid- to late G1
must be due to serine phosphorylation.
Identification of a Growth-regulated Sp1-associated Kinase
Activity--
It has been proposed that association of kinases with
their substrate proteins can be an important factor for their
specificity and activity (36); therefore, the possibility that
increased Sp1 phosphorylation could be due to its association with a
cellular kinase was investigated. Sp1 was immunoprecipitated from
extracts of G0 or G1 cells and the
immunoprecipitates were subjected to an in vitro kinase
reaction. As seen in Fig. 4A,
kinase activity immunoprecipitated with Sp1. Furthermore, the kinase
activity associated with Sp1 was 2-3-fold higher in G1
cells than in G0 cells. Similar results were obtained when
Sp1 was immunoprecipitated from whole cell extracts (Fig.
4A) or nuclear
extracts2 and were seen with
extracts from NHDF (Fig. 4) and Balb/c 3T3 cells.3 In these reactions,
although a phosphorylated band corresponding in size to Sp1 was
sometimes seen (Fig. 4A, arrow), this was not the major
phosphoprotein detected. The low level of Sp1 phosphorylation is not
unexpected since (a) antibody, rather than Sp1, represents the major protein component of the immunoprecipitates (c.f.
Fig. 1A), and (b) Sp1 that is associated with the
kinase activity is likely to be already phosphorylated and would not,
therefore, represent a substrate in these reactions. The identity of
other bands phosphorylated in these extracts is unknown, but they are likely due to phosphorylation of proteins in the antibody preparations used for immunoprecipitation and possibly Sp1-associated proteins. Nevertheless, the data clearly demonstrate that a kinase activity does
co-immunoprecipitate with Sp1 and that this activity is
growth-regulated.
To determine if the Sp1-associated kinase activity could phosphorylate
Sp1, it was eluted from Sp1 immunoprecipitates and used in an in
vitro kinase reaction containing bacterially expressed glutathione
S-transferase Sp1 fusion protein (GST-Sp1). The 1 M NaCl extraction eluted the majority of the kinase
activity from the immunoprecipitates (Fig. 4A) and the
eluted kinase was able to phosphorylate GST-Sp1 in a growth-regulated
manner (Fig. 4B). The specificity of the association of this
Sp1 kinase activity with Sp1 was confirmed, since very little
phosphorylation of GST-Sp1 could be detected when a blocking peptide
(recognized by the anti-Sp1 antibody) was added to the cell extracts
prior to immunoprecipitation of Sp1, and no activity was detected when
anti-Sp1 antibody was omitted from the immunoprecipitation (Fig.
4C). Phosphorylation did not occur in the GST portion of the
fusion protein since the activity was unable to phosphorylate GST (data
not shown) or some of the GST-Sp1 truncation mutants tested below
(c.f. Fig. 6). Phosphorylation of Sp1 by a contaminating
bacterial kinase can also be excluded since kinase reactions performed
in the absence of eluate from Sp1 immunoprecipitations revealed no
phosphorylation of GST-Sp1 (see Fig. 6A, lane 1). In these
reactions, a band which migrated below GST-Sp1 at ~80 kDa could be
seen; this band was seen to varying extents in different preparations
of GST proteins and is presumably due to a bacterial kinase.
The Sp1-associated Kinase Activity Represents a Novel Sp1
Kinase--
Sp1 can be phosphorylated in vitro by protein
kinase CK2 (15, 37), DNA-dependent protein kinase (35, 38),
and protein kinase A (PKA) (17). Therefore, various substrates and
inhibitors were used to determine if the Sp1-associated kinase activity
corresponded to any of these enzymes. CK2 can utilize GTP as a
phosphate donor essentially as efficiently as ATP (39). However, GTP
was a very poor phosphate donor for the Sp1-associated kinase (Fig.
5, lanes 1 and 2);
therefore it is unlikely to represent CK2. DNA-dependent protein kinase requires Sp1 and the kinase to be bound to DNA (38).
Since disruption of protein-DNA interaction with 50 µg/ml ethidium
bromide (40) did not affect Sp1 phosphorylation (Fig. 5, lane
3), the Sp1-associated kinase activity cannot be due to DNA-dependent protein kinase. Finally, the ability of
staurosporine to inhibit the Sp1-associated kinase activity was tested.
Staurosporine is a competitive inhibitor of ATP binding to a number of
kinases and has a Ki for PKA of 5 nM
(41). Although staurosporine was able to inhibit the Sp1-associated
kinase activity (Fig. 5, lanes 4 and 5), the
IC50 of this inhibition was 40-50 nM under conditions where the IC50 for PKA would be <10
nM; therefore, the Sp1-associated kinase activity also
differs from PKA. Inhibition of the activity by 50 nM
staurosporine further argues against the kinase being CK2 which has a
Ki of 6 µM for staurosporine (41).
Thus, the growth-regulated Sp1-associated kinase activity differs from
kinases known to phosphorylate Sp1 and presumably represents a novel
Sp1 kinase.
Consistent with the physiological relevance of the Sp1-associated
kinase activity, treatment of serum-stimulated cells with 100 nM staurosporine for 6 h led to a 58% (±5%,
n = 5) reduction in activity of DHFR G1 Phosphorylation of the C Terminus of Sp1--
Since
Sp1 is multiply phosphorylated (35), the region of Sp1 preferentially
phosphorylated in G1 was investigated by comparing the
region phosphorylated by the Sp1-associated kinase with phosphorylation of Sp1 mutants in vivo. In addition to phosphorylating
full-length GST-Sp1 (GST-Sp1(1-778)), the Sp1-associated kinase
activity could phosphorylate C-terminal truncations of Sp1 which
contained amino acids 1-658 or 1-687 (Fig.
6A). However, no
phosphorylation of the fusion protein containing amino acids 1-612 of
Sp1 was detected, indicating that the phosphorylation is in the
C-terminal region of the protein. The band seen in the 1-612 lane
migrates below GST-Sp1(1-612) and corresponds to the band produced by
bacterial kinase seen in lane 1 (Fig. 6A,
arrowhead). When N-terminal truncation mutants were examined, the
kinase was able to phosphorylate GST-Sp1(612-778), consistent with the
phosphorylation occurring within this C-terminal region. Since no
phosphorylation of GST-Sp1(687-778) was detected, the sites
phosphorylated by the Sp1-associated kinase activity appear to lie
between amino acids 612 and 687 of Sp1. Interestingly, the activity
could phosphorylate both GST-Sp1(658-778) and GST-Sp1(1-658) indicating that at least two sites are phosphorylated by this kinase
activity in these in vitro assays.
Studies of Sp1 phosphorylation in vivo used Sp1 truncation
mutants containing amino acids 1-612 or 612-778 since they have been
shown to correctly localize to the nucleus (35). N-terminal Flag-tagged
Sp1 (Flag-Sp1), Flag-Sp1(1-612), or Flag-Sp1(612-778) were
transfected into Balb/c 3T3 cells and their phosphorylation was
determined using an anti-Flag antibody to immunoprecipitate the
proteins from 32PO4-labeled late G1
cells. SDS-PAGE/autoradiographic analysis clearly detected
phosphorylation of not only full-length Flag-Sp1 and Flag-Sp1(1-612)
but also of Flag-Sp1(612-778) (Fig. 6B). Although phosphorylation of Flag-Sp1(612-778) in late G1 was less
than that of full-length Flag-Sp1 protein, it was not possible to
determine how the phosphorylation of these transfected proteins varies
with serum stimulation because Western blot analysis has revealed that their expression (which is driven by the cytomegalovirus promoter) is
very low in serum-starved cells (data not shown). However, the data
clearly demonstrate that the C-terminal portion of Sp1 is
phosphorylated in late G1 cells in vivo and
support the physiological relevance of the growth-regulated
phosphorylation of this region seen in vitro.
To elucidate possible mechanisms underlying the serum induction of
Sp1 site-dependent transcription, we have examined the levels, synthesis, phosphorylation, and DNA binding of Sp1. Serum stimulation of Balb/c 3T3 cells leads to an increase in Sp1 levels and
phosphorylation, while neither Sp1 synthesis nor its specific DNA
binding activity change. The increase in Sp1 phosphorylation was
greater than that of protein, indicating that it results, at least in
part, from an increase in the level of phosphorylation of individual
Sp1 molecules. This was confirmed in NHDF where increases in Sp1
phosphorylation were seen with no significant increase in protein
levels. The ratios of the different Sp1 site binding complexes were not
affected by serum stimulation, indicating that the induction of
transcription is not simply due to changes in the relative binding of
stimulatory (Sp1) and inhibitory (Sp3) factors to the Sp1 sites.
Therefore, of the parameters tested, only changes in Sp1 levels and
phosphorylation could account for the growth-related changes in
Sp1-dependent transcription.
Analysis of the kinetics of these events revealed that induction of Sp1
protein levels occurs rapidly following serum stimulation of Balb/c 3T3
cells (Fig. 2A), whereas the increase in phosphorylation is
delayed (Figs. 2B and 3). Previously, we have reported that the kinetics of serum induction of Sp1-dependent
transcription is promoter dependent in Balb/c 3T3 cells (6), occurring
either with delayed kinetics (the DHFR promoter) or rapidly
(5XSp1 While the current study was ongoing, it was reported that serum
stimulation of CHO K1 cells leads to a delayed induction of Sp1 levels
and that this change correlates with the increase in Sp1-dependent transcription of DHFR (43). The discrepancy
between this study and our findings presumably resides in the different cell types and/or promoters used (the studies in Chinese hamster ovary
cells used a truncated DHFR promoter containing only one Sp1 site which
has shown very low activity in other studies (28)). Although the
possible contribution of phosphorylation to the induction seen in
Chinese hamster ovary cells was not addressed, it appears that
Sp1-dependent transcription may be regulated at multiple levels dependent on the promoter and cell type studied. This is supported by the finding that Sp1 sites play a greater role in growth
regulation of the rep3 promoter than the DHFR promoter in
NIH 3T3 cells (5), and that Sp1 levels and/or binding activity do not
change following serum stimulation of NIH 3T3 cells or human
fibroblasts (8, 27, 44, 45).
Although not examined here, another potential mechanism for regulation
of Sp1 is through O-glycosylation which has been shown to
play a role in the association of Sp1 with other factors (19) and in
regulation of its degradation (18). Our findings are suggestive of a
possible role for O-glycosylation since Sp1 levels change in
Balb/c 3T3 cells without concomitant changes in Sp1 synthesis,
indicating that its levels may be regulated by changes in degradation.
In keeping with the serum stimulation of the phosphorylation of Sp1, we
have observed an Sp1-associated kinase activity, whose interaction with
Sp1 is growth-regulated. Although rigorous proof that the
Sp1-associated kinase activity represents a physiologically relevant
Sp1 kinase will require identification of the kinase(s) involved and
determination of the effects of its inhibition in vivo (46)
(and is, therefore, beyond the scope of the present study), several
factors argue in favor of its physiological relevance. The level of the
activity is growth-regulated, indicating that either the activity
and/or association with Sp1 of the relevant kinase(s) is likewise
growth-regulated. Furthermore, at least the majority of the activity is
specifically associated with Sp1 (Fig. 4B). The specificity
of the interaction is reinforced by the finding that this activity
binds strongly to GST-Sp1 (it remains bound following extensive washing
of glutathione-Sepharose-bound GST-Sp1 which had been preincubated with
eluate from Sp1 immunoprecipitates2). Finally, the
Sp1-associated kinase activity phosphorylates Sp1 in its C-terminal
domain, a region phosphorylated in late G1 cells in
vivo.
Although these studies have identified an Sp1-associated kinase that is
likely to be involved in regulation of its transcription, the identity
of the kinase is unknown at present. Sp1 has been shown to be
phosphorylated in vitro by CK2, PKA, and
DNA-dependent protein kinase; however, the Sp1-associated
kinase differs from these kinases in its ability to utilize GTP and its
sensitivity to staurosporine and ethidium bromide, respectively.
Although it is possible that the Sp1-associated kinase activity
represents more than one kinase (it phosphorylates at least two sites
on Sp1 in vitro), the low level of its GTP usage, minimal
inhibition by low concentration of staurosporine, and insensitivity to
ethidium bromide indicate that, if it contains any of the above
kinases, they represent only a small proportion of the total activity. During the preparation of this manuscript, it was reported that DNA-dependent protein kinase leads to an increase in Sp1
activity through phosphorylation of serine 131 in the N terminus of Sp1 (47). Although this phosphorylation could be enhanced by HIV-TAT, it
remained dependent on the presence of DNA. Since the Sp1-associated kinase did not phosphorylate the N terminus of Sp1 and was insensitive to disruption of protein-DNA complexes by ethidium bromide, these findings further emphasize that the kinase identified here is distinct
from DNA-dependent protein kinase. Therefore, the
Sp1-associated kinase activity identified here contains a novel
growth-regulated Sp1 kinase(s).
Consistent with the data obtained in vitro, it is also
unlikely that CK2, PKA, or DNA-dependent protein kinase are
directly responsible for the increased phosphorylation of Sp1 in
G1 in vivo. Phosphorylation of Sp1 by CK2 leads
to a reduction in Sp1 DNA binding and occurs on threonine (15, 37).
Therefore, since G1 phosphorylation of Sp1 is on serine and
no differences in DNA binding were observed between G0 and
G1 cells, CK2 is unlikely to be directly responsible for
the growth/cell cycle-regulated phosphorylation of Sp1. Although PKA
activation leads to activation of Sp1 site-dependent
transcription at a number of promoters, its precise role in this
phenomenon is unclear. PKA phosphorylates Sp1 in vitro and
this phosphorylation leads to increased Sp1 DNA binding (17). In
contrast, activation of PKA in vivo leads to enhanced Sp1
site-dependent transcription of the sgk promoter without changes in Sp1 levels or DNA binding activity (48), and
enhanced binding to Sp1 sites at the NF-L promoter following PKA
activation is due to a factor other than Sp1 (49). The increased DNA
binding induced by PKA and the presence of only threonine (Thr366) in the PKA consensus site in Sp1 preclude it from
being the kinase responsible for the G1 phosphorylation
seen here. DNA-dependent protein kinase requires DNA ends
for activity and has been implicated in responses to DNA damage (50);
therefore, it is unlikely to be relevant to the G1
phosphorylation of Sp1. The involvement of kinases other than
DNA-dependent kinase in phosphorylation of Sp1 is supported
by its phosphorylation in G1 SCID mouse
fibroblasts2 which lack DNA-dependent kinase
activity (51). Thus, in keeping with the presence of Sp1 sites in
multiple promoters, Sp1 is phosphorylated at multiple sites by multiple
kinases which are likely to be important in regulation of its activity
in response to diverse signals. Ongoing studies are aimed at
identifying the Sp1-associated kinase(s) and determining the precise
physiological roles of its phosphorylation of Sp1.
Sp1 is multiply phosphorylated and migrates as a doublet on SDS-PAGE
due to different mobilities of the phosphorylated and unphosphorylated
forms (35) (Fig. 1A). Interestingly, serum stimulation did
not change the ratio of these forms in either Balb/c 3T3 cells or NHDF
(Figs. 1-3 and Ref. 27). Therefore, the enhanced phosphorylation of
Sp1 in mid-late G1 presumably reflects increased
phosphorylation of only one or a small subset of the Sp1
phosphorylation sites. In keeping with this, the Sp1-associated kinase
phosphorylated a limited region of Sp1 within its C terminus (Fig. 6).
Furthermore, in addition to phosphorylation of the N terminus of Sp1,
our studies revealed phosphorylation in the C-terminal 167 amino acids
of Sp1 in G1 cells in vivo. Unfortunately, due to low expression of transfected proteins in serum-starved cells, it
was not possible to determine how phosphorylation of this fragment is
related to the up-regulation of Sp1 phosphorylation in G1. Interestingly, while studies in asynchronously growing HeLa cells readily detected phosphorylation of the N-terminal portion of Sp1, they
failed to detect significant phosphorylation of the C-terminal domain
(35). Although this discrepancy may reflect cell type differences,
together with the phosphorylation of the C terminus of Sp1 by a
growth-regulated, Sp1-associated kinase activity, it further argues
that the C terminus of Sp1 is preferentially phosphorylated in
G1 cells.
The region of Sp1 phosphorylated in vitro lies in the
N-terminal half of the DNA-binding domain of the protein. This region contains only 3 serines (Fig. 5C); however, due to the
relaxed kinase substrate specificity observed in vitro, it
is not possible to say which of these are physiological sites of
phosphorylation. Nonetheless, these studies delineate the region of Sp1
which can be phosphorylated by the Sp1-associated kinase activity. The
N-terminal zinc finger contributes weakly to the high affinity binding
of Sp1 to DNA (52), which may account for the finding that enhanced Sp1
phosphorylation in G1 does not correlate with significant changes in the specific DNA binding activity of Sp1.
Lack of correlation between DHFR transcription and increased levels or
DNA binding of Sp1 indicate that the enhanced Sp1 activity is likely to
involve alterations in its association with other proteins. With regard
to enhanced phosphorylation of Sp1 in G1, it is noteworthy
that the C terminus of Sp1 has been implicated in its interaction with
other transcription factors. This region of Sp1 is involved in
synergistic activation with sterol regulatory element-binding protein
(53, 54) and mediates its interaction with TAFII55 (55) and
with both YY1 and E2F1 (27, 56), two factors which can mediate serum
induction and cell cycle regulation of transcription (2, 57). Indeed,
the region of Sp1 that interacts with E2F1 maps precisely to the region
phosphorylated by the Sp1-associated kinase
activity.4 Since interaction
with YY1 or E2F1 can affect Sp1-dependent transcription in
the absence of their direct binding to DNA (27, 56), modulation of
these interactions by phosphorylation of Sp1 in mid-late G1 could mediate changes in Sp1 site-dependent transcription.
Sp1 has also been shown to physically or functionally interact with other cell cycle regulatory proteins such as retinoblastoma protein and
p107 (58-61). Current efforts are directed at determining how mutation
of this C-terminal region of Sp1 affects its interaction with other
factors and the growth/cell cycle regulation of
Sp1-dependent transcription.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
induction of p15 (14).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-glycerophosphate, 15 mM sodium pyrophosphate, 400 µM Na2VO3, 2 mM
phenylmethylsulfonyl fluoride, 20 µM leupeptin, 10 µg/ml aprotinin, particulate material was removed by centrifugation
(13,000 × g, 10 min). Supernatants were precleared
with normal rabbit serum and protein A-Sepharose and Sp1 was
immunoprecipitated with anti-Sp1 antibody (PEP2; Santa Cruz) and
protein A-Sepharose. Immunoprecipitates were washed 4 times with RIPA
(phosphate-buffered saline containing 1% (w/v) Ipegal CA-630, 0.5%
(w/v) sodium deoxycholate, and 0.1% (w/v) SDS) and separated by 8%
SDS-PAGE, transferred to nitrocellulose, and subjected to
autoradiography. Membranes were then blocked in 5% non-fat dried milk,
20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05%
Tween 20, and subjected to Western blotting with anti-Sp1 (PEP2)
antibody and anti-rabbit horseradish peroxidase secondary antibody as
prescribed by the manufacturers (Santa Cruz and Promega). Detection
utilized the Pierce Supersignal system.
-RAM in-line radiation detector. Levels of ATP in samples were estimated by comparison of peak height with that
obtained with unlabeled standard ATP. Relative specific activity of ATP
was determined by dividing radioactive peak height by absorbance peak height.
E2F and 5XSp1
53MLP constructs and
adenovirus major late promoter constructs were as described (6, 27)
except where the chloramphenicol acetyltransferase cDNA was
replaced with that of firefly luciferase.
90 °C. For imunoprecipitation, extracts were diluted
with 9 volumes of 20 mM HEPES, pH 7.4, 0.624% Nonidet
P-40, 10 µM leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride and precleared with normal
rabbit serum and Protein A-Sepharose. Sp1 was then immunoprecipitated
with anti-Sp1 and Protein A-Sepharose and the beads were washed 4 × 5 min in 20 mM HEPES, pH 7.4, 100 mM KCl, 1 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA. Where indicated, an equal weight of PEP2 peptide
(Santa Cruz) was added to the extracts prior to the addition of
anti-Sp1 (PEP2) antibody. For direct kinase assay of the
immunoprecipitates, kinase reactions were carried out in 50 µl of
kinase buffer (125 mM HEPES, pH 7.4, 1% (w/v) glycerol, 10 mM NaCl, 1 mM MgCl2, 0.5 mM MnCl2) containing 10 µCi of
[
-32P]ATP (3000-6000 Ci/mmol) for 30 min at 37 °C.
Phosphorylated proteins were analyzed by 8% SDS-PAGE and
autoradiography. For 1 M NaCl extraction of Sp1-bound
kinase, immunoprecipitates were extracted with 20 mM HEPES,
pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 1 M NaCl for 1 h at
4 °C, centrifuged (13,000 × g, 10 min), and the
supernatant was stored at
90 °C.
-D-thiogalactopyranoside. Cells were harvested by
centrifugation and extracted with 6 M urea. Following
removal of particulate material by centrifugation, proteins were
renatured by removing urea through dialysis against 10 mM
Tris-HCl, pH 7.5, 0.2 M NaCl, 50 µM
ZnCl2, 5% (w/v) glycerol, 1 mM
2-mercaptoethanol, and stored at
90 °C.
-32P]ATP or
[
-32P]GTP (6000 Ci/mmol) for 30 min at 37 °C. Where
indicated, inhibitors were added to the GST fusion proteins prior to
addition of 1 M NaCl extracts and ATP. Phosphoproteins were
analyzed by SDS-PAGE and autoradiography.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (63K):
[in a new window]
Fig. 1.
Analysis of differences in Sp1 regulation in
G0 and G1 cells. Equal numbers of Balb/c
3T3 cells were serum-starved for 32 h (G0) or
serum-starved for 24 and refed with 20% serum for 8 h
(G1). A, Western blot analysis of Sp1 levels in
extracts of G0 or G1 cells.
Arrowheads indicate the migration of molecular weight
standards (203, 116, 83, and 48 kDa). Lines show the
migration of phosphorylated (upper) and non-phosphorylated
(lower) Sp1. B, G0 and G1
cells were labeled with [35S]methionine for 2 h
prior to harvest and extraction. Sp1 was immunoprecipitated from
cellular extracts and the levels of 35S incorporation into
(synthesis of) Sp1 were determined by 8% SDS-PAGE and autoradiography.
C, G0 and G1 cells were labeled with
32PO4 for 2 h prior to harvest and the
levels of 32P labeling of Sp1 were determined as for
35S incorporation. D, electrophoretic mobility
shift assay of Sp1 site binding material in nuclear extracts from
G0 and G1 cells. The 32P-labeled
probe was a double-stranded oligonucleotide corresponding to the first
GC box in the hamster DHFR promoter. The arrowhead and
arrows indicate the migration of Sp1 and Sp3 containing
complexes, respectively, as shown by others (e.g. Ref. 24)
and confirmed by separate antibody supershift assays. Specificity of
binding was confirmed by competition with unlabeled probe (not
shown).
Specific activity of ATP pools in 32PO4-labeled
Balb/c 3T3 cells
View larger version (43K):
[in a new window]
Fig. 2.
Analysis of Sp1 levels and phosphorylation in
Balb/c 3T3 cells. A, time course for serum induction of
Sp1 levels and phosphorylation in Balb/c 3T3 cells. Equal numbers of
Balb/c 3T3 cells were serum-starved for 24 h prior to re-addition
of serum to 10%. Cells were labeled with 32PO4
for 2 h prior to harvest at the indicated times (in hours) after
addition of serum (0 indicates no re-addition of serum). Cells were
extracted, Sp1 was immunoprecipitated, and subjected to 8% SDS-PAGE
and transferred to nitrocellulose. Incorporation of 32P
into Sp1 was detected by autoradiography (upper panel) and
Sp1 levels were determined by Western blot analysis (lower
panel). Arrowheads indicate the migration of molecular
weight standards (116 and 83 kDa). B, increase in specific
phosphorylation of Sp1. Levels of Sp1 protein and phosphorylation were
quantified by densitometric analysis of autoradiograms and Western blot
data as above. Specific phosphorylation was calculated by dividing the
densitometric values for the band corresponding to Sp1 in each lane of
the autoradiograms by those from the Western blots and are plotted
relative to specific phosphorylation seen in serum-starved cells. Data
are the mean (± S.E.) of values from two independent
experiments.
E2F-luciferase activity in serum-stimulated cells, whereas it
led to a 2.89 ± 0.69-fold induction of transcription from a
promoter containing 5 Sp1 sites upstream of a TATAA box
(5XSp1
53MLP). The higher sensitivity of the latter promoter to Sp1
levels is consistent with its more rapid induction following serum
stimulation of Balb/c 3T3 cells (6).
View larger version (63K):
[in a new window]
Fig. 3.
Analysis of Sp1 levels and phosphorylation in
NHDF. A, time course for serum induction of Sp1
phosphorylation in NHDF. Equal numbers of normal human diploid
fibroblasts were serum-starved for 72 h prior to re-addition of
serum to 10%. Cells were labeled with 32PO4
and Sp1 levels and incorporation of 32P into Sp1 was
determined as described in the legend to Fig. 2. Arrowheads
indicate the migration of molecular mass standards (116 and 83 kDa).
B, phosphoamino acid analysis of late G1
phosphorylated Sp1. 32P-Labeled Sp1 from normal human
diploid fibroblasts which had been serum stimulated for 14 h was
immunoprecipitated, subjected to 8% SDS-PAGE, transferred to
polyvinylidene difluoride, and subjected to acid hydrolysis as
described under "Materials and Methods." Material released from the
membrane was analyzed by two-dimensional thin layer electrophoresis and
autoradiography. Circled areas indicate the migration of
unlabeled phosphoamino acid standards (detected by ninhydrin staining)
which were co-electrophoresed with the samples and of free
32PO4 released during hydrolysis.
View larger version (50K):
[in a new window]
Fig. 4.
Association of Sp1 with a kinase
activity. A, association of Sp1 with a growth-regulated
kinase activity. Equivalent numbers of NHDF were serum-starved for
74 h (G0) or serum-starved for 60 h prior to
re-addition of serum to 10% for 14 h (G1). Cells were
then extracted and Sp1 was immunoprecipitated as described under
"Materials and Methods." Immunoprecipitates were extensively washed
and kinase activity was assayed by incubation in the presence of
[ -32P]ATP followed by 8% SDS-PAGE/autoradiography
(Unextracted). Alternatively, kinase reactions were
performed with the immunoprecipitates which were extensively washed and
extracted with 1 M NaCl (Extracted). Reactions
were analyzed by 8% SDS-PAGE and autoradiography. The arrow
indicates the migration of Sp1 and the numbers to the
right indicate the migration of molecular weight standards.
B, phosphorylation of GST-Sp1 by the Sp1-associated kinase
activity. The presence of kinase activity capable of phosphorylating
Sp1 in a 1 M NaCl eluate of Sp1 immunoprecipitates from
G0 or G1 cells was assessed in in
vitro kinase reactions containing glutathione-Sepharose-purified,
bacterially expressed GST-Sp1 with 5 µl of eluate and 10 µCi of
[
-32P]ATP. Reactions were analyzed by 8% SDS-PAGE and
autoradiography. The left panel shows autoradiographic
detection of 32P-labeled protein and the right
panel shows the Coomassie Blue-stained gel. C, as in
B, except that immunoprecipitation reactions from
G1 cells were carried out in the presence of anti-Sp1
antibody (
Sp1), anti-Sp1 antibody and a blocking peptide
recognized by the antibody (
Sp1 + peptide) or in the
presence of no antibody (No antibody).
View larger version (46K):
[in a new window]
Fig. 5.
Characterization of the Sp1-associated kinase
activity. In vitro kinase assays of the Sp1-associated
kinase activity were carried out as described in the legend to
Fig. 4B, except that [ -32P]GTP was used as
a phosphate donor in lane 1 (GTP); reactions shown in other
lanes used [
-32P]ATP as a phosphate donor. Ethidium
bromide (EtBr) or staurosporine (Staur.) were
added to reactions at the indicated final concentration prior to
addition of the eluate from Sp1 immunoprecipitations.
E2F-luciferase.
However, it is not possible to attribute this effect to direct
inhibition of the Sp1-associated kinase since this level of
staurosporine inhibits a broad range of kinases (41), including
cyclin-dependent kinases which are involved in cell-cycle progression.
View larger version (52K):
[in a new window]
Fig. 6.
Region of Sp1 phosphorylated by the
Sp1-associated kinase activity. A, in vitro labeling of
GST-Sp1 truncation mutants. Autoradiogram and Coomassie-stained 10%
SDS-PAGE gel of in vitro kinase assays of the Sp1-associated
kinase activity carried out as in the legend to Fig. 4B. In
lane 1 (1-778, no eluate), kinase reactions were carried
out with GST-Sp1 but without addition of eluate from Sp1
immunoprecipitations. The substrates in the other kinase reactions
(which contained eluate from Sp1 immunoprecipitations) were full-length
GST-Sp1 (1-778) or various truncation mutants of Sp1 fused to GST. The
numbers above the lanes indicate the amino acids of Sp1
present in these fusion proteins. The arrowhead indicates
the migration of a phosphoprotein due to nonspecific phosphorylation.
B, in vivo labeling of Sp1 truncation mutants. Flag-tagged,
full-length Sp1 (Flag-Sp1), and Flag-tagged truncation mutants
containing the N-terminal 612 (Flag-Sp1(1-612)) or C-terminal 167 (Flag-Sp1(612-778)) amino acids of Sp1 were transfected into Balb/c
3T3 cells prior to serum starvation for 24 h. Cells were then
serum stimulated for 9 h with 32P labeling for 2 h prior to harvest. Flag-tagged proteins were immunoprecipitated from
cell extracts with anti-Flag antibody and 32P incorporation
into the proteins was determined by 8% (Flag-Sp1 and Flag-Sp1(1-612))
or 15% (Flag-Sp1(612-778)) SDS-PAGE and autoradiography. For clarity,
panels show only the region of the autoradiogram where the fusion
proteins migrate. With the exception of material trapped at the top of
the 15% gel, no other bands were apparent on the autoradiograms.
C, amino acid sequence of the C-terminal region of Sp1.
Underlined regions delineate the zinc fingers of Sp1, while
the bold letters show the region phosphorylated by the
Sp1-associated kinase in the in vitro assays. The 3 serines
in the labeled region are shown in bold italics.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
53MLP) following serum stimulation. In vivo
footprinting data demonstrating that the DHFR promoter is
constitutively occupied during the G0-S phase transition
(42) suggest that Sp1 is not limiting at this promoter. The finding
that the 5XSp1
53MLP promoter shows greater sensitivity to Sp1
overexpression than the DHFR promoter (see "Results") supports this
notion and indicates that Sp1 levels are more limiting at the more
rapidly induced 5XSp1
53MLP promoter. This leads to a model for
differential regulation of promoter activity by Sp1: at promoters where
Sp1 levels are limiting, the rapid induction of Sp1 levels by serum
would lead to rapid induction of transcription, whereas with promoters
which are regulated by Sp1 phosphorylation and where Sp1 levels are not
limiting, induction would be delayed.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank other members of the Azizkhan Laboratory, in particular Dr. Sanja Pajovic and Dr. Jennifer D. Black for helpful input to this work and Dr. Alexander Maccubbin for help with the ATP pool analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by American Cancer Society Grant CB196 and Institute Core Grant CA 16056 from the National Cancer Institute.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: The Wistar Institute, 36th and Spruce
Streets, Philadelphia, PA 19104.
§ Present address: Graduate School of Biomedical Sciences, The University of Texas-Houston, Houston, TX 77030.
¶ To whom correspondence should be addressed. Tel.: 716-845-3563; Fax: 716-845-8857; E-mail: azizkhan{at}sc3101.med.buffalo.edu.
The abbreviations used are: DHFR, dihydrofolate reductase; PKA, protein kinase A; NHDF, normal human diploid fibroblasts; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
2 A. R. Black and J. C. Azizkhan, unpublished data.
3 S. Pajovic and J. C. Azizkhan, unpublished data.
4 L-W. Guo, A. R. Black, and J. C. Azizkhan, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|