(Received for publication, January 11, 1996; and in revised form, March 22, 1996)
From the
sgk is a novel member of the serine/threonine protein kinase gene family that is transcriptionally regulated by serum and glucocorticoids in mammary epithelial cells. To functionally determine if the sgk promoter is regulated by the p53 tumor suppressor protein in mammary cells, a series of sgk promoter fragments with 5`-deletions were linked to the bacterial chloramphenicol acetyltransferase gene (sgk-CAT) and transiently cotransfected into nontumorigenic NMuMG or transformed Con8Hd6 mammary epithelial cells with p53 expression plasmids. Wild-type p53, but not mutant p53, strongly stimulated sgk promoter activity in both mammary epithelial cell lines. These effects were mediated by specific regions within the sgk promoter containing p53 DNA-binding sites. The sgk p53 sequence at -1380 to -1345 (site IV) was sufficient to confer p53-dependent transactivation to a heterologous promoter, and p53 was capable of binding to this sequence in vitro as assessed by gel shift analysis. In the nontumorigenic NMuMG epithelial cell line, cotransfection of wild-type p53 strongly stimulated the activities of both the sgk promoter and the well characterized p53-responsive p21/Waf1 promoter, whereas in Rat-2 fibroblasts, wild-type p53 repressed the basal activities of both promoters, revealing that sgk and p21/Waf1 are similarly regulated in a cell type-specific manner. Taken together, these results demonstrate that sgk is a new transcriptional target of p53 in mammary epithelial cells and represent the first example of a hormone-regulated protein kinase gene with a functionally defined p53 promoter recognition element.
An intricate network of protein kinases and phosphatases
propagates various extracellular growth and differentiation signals
from the plasma membrane into the nucleus, leading to changes in the
phosphorylation status and the function of discrete sets of
transcription factors. The catalytic activities of most protein kinases
are regulated by specific interactions with regulatory
proteins(1, 2, 3) and/or by
phosphorylation(4, 5) . Recent studies have uncovered
a newly emerging subfamily of serine/threonine protein kinase genes,
including snk, sgk, plk, and fnk,
that are predominantly regulated at the transcriptional level by
hormone- and/or mitogen-induced
pathways(6, 7, 8, 9, 10, 11, 12, 13) .
Our previous studies have identified the sgk (serum- and
glucocorticoid-inducible protein kinase) serine/threonine protein
kinase gene, which is transcriptionally regulated by serum and/or
glucocorticoids in mammary epithelial cells and Rat-2
fibroblasts(12, 13) , as the second member of this
subfamily of transcriptionally regulated protein kinase genes. sgk encodes a 49-kDa putative protein kinase that shares 45-55%
homology with the catalytic domain of protein kinase C, the
cAMP-dependent protein kinase A, the rac protein kinases, and
the ribosomal protein S6 kinase(13) . We have documented that sgk transcripts are expressed in a variety of adult rat
tissues, with the highest expression in the thymus, lung, and ovary and
detectable levels in the mammary gland and several other
tissues(13) . The cellular and tissue context strongly
influences the expression of sgk since, depending on the cell
type, different extracellular stimuli affect sgk transcription, or the same signals can regulate sgk expression with different kinetics. For example, in Rat-2
fibroblasts, induction of sgk gene transcription is an
immediate-early response to serum that returns to near basal levels
4 h after serum stimulation, whereas in mammary epithelial cells, sgk transcript levels remain at the induced levels for at
least 48 h after their rapid induction by serum (12, 13) . In granulosa cells of the rat ovary, sgk expression is regulated by a combination of testosterone
and follicle-stimulating hormone(14) , while in rat brain
tissue, sgk expression is induced following injury to the
central nervous system(15) .
The cellular mechanisms
governing the expression of sgk by diverse sets of
extracellular stimuli are not well understood. To define the molecular
details of this process and to uncover the transcriptional regulatory
factors involved in modulating sgk gene expression, 4 kb ()of the sgk promoter region upstream of the
transcriptional start site was cloned from a rat genomic
library(13) . Sequence analysis of the sgk promoter
region revealed a glucocorticoid response element (GRE), at
approximately -1.0 kb, that by functional analysis is responsible
for the glucocorticoid-stimulated transcription of sgk(13) . The sgk promoter also contains a
TATA box and Sp-1 elements as well as putative binding sites for a
variety of transcriptional regulators such as the AP-1 complex,
CCAAT/enhancer-binding protein, NF-
B, GATA, and Ets-2, (
)which in other systems have been found to be important for
transducing proliferation and/or differentiation
signals(16, 17, 18, 19, 20) .
A striking feature of the sgk promoter is the presence of
multiple putative binding sites for the p53 tumor suppressor protein,
which implicates the sgk gene as a direct transcriptional
target of p53. The p53 protein is a transcriptional regulator (21, 22) that plays an important role in cell cycle
control, cellular differentiation, apoptosis, genomic stability, and
response to DNA
damage(23, 24, 25, 26) .
The p53 nuclear phosphoprotein can either positively or negatively regulate transcription in a gene- and tissue-specific manner. For example, the promoters of a variety of cellular genes associated with diverse p53-mediated responses are transcriptionally activated by p53 through p53-responsive elements in their promoter regions(27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . In contrast, p53 represses transcription of several genes that lack p53-responsive elements(39, 40, 41, 42, 43, 44, 45, 46, 47) . This repression response requires both the amino and carboxyl termini of p53 (48) as well as the oligomerization domain (49) and is likely due to interactions with components of core transcriptional machinery(44, 50, 51) . Although not well characterized, for a few genes, transcriptional repression by p53 may involve its specific DNA binding capacity(42, 45) . Additionally, p53 has also been shown to regulate activity of certain genes either positively or negatively depending on specific cell type(27, 52) , suggesting the involvement of cell-specific factors or coregulators in p53-mediated transcriptional regulation.
In rat Con8Hd6 mammary tumor epithelial cells, from which the sgk protein kinase gene was cloned, treatment with glucocorticoids and serum suppresses cell growth and stimulates sgk expression(13) . Thus, it appears that in these mammary tumor cells, the regulated transcription of sgk occurs concomitantly with suppression of growth. Recent studies are now emerging that strongly support a role for p53 in the homeostasis of mammary epithelial cell growth, although the cellular targets for p53 in mammary cells in the physiological context are largely unknown(53) . Given the role of p53 as a transcription factor that inhibits cell cycle progression and the presence of multiple p53 DNA-binding sites in the sgk promoter, it is tempting to consider that sgk transcription may be regulated by p53. Besides the Con8Hd6 mammary tumor cells, sgk transcripts are also expressed and induced by glucocorticoids in nontumorigenic NMuMG mammary epithelial cells(13) , thus providing a transformed (Con8Hd6) and a nontumorigenic (NMuMG) mammary epithelial cell line to compare the effects of p53. In this study, we show that p53 transactivates the sgk promoter in both NMuMG and Con8Hd6 mammary epithelial cells via a p53-responsive element located within the sgk promoter, thus demonstrating for the first time that a hormone-responsive protein kinase gene is a direct transcriptional target of the p53 tumor suppressor protein.
Radiolabeling of 5`-ends of the appropriate
oligonucleotides was carried out in the presence of equal amounts (10
pmol) of sense and antisense strands,
[-
P]ATP (7000 Ci/mmol; ICN Biomedicals
Inc.), and T4 polynucleotide kinase (Boehringer Mannheim) at 37 °C
for 30 min, followed by annealing of the labeled strands. The annealing
procedure for generating either labeled or unlabeled DNA involved
adding 0.1 M NaCl (0.1 volume) to equal amounts of sense and
antisense strands, heating for 10 min at 70 °C, and gradually
cooling to room temperature. The free unincorporated nucleotides and
single-stranded DNA were separated from the end-labeled double-stranded
DNA by native polyacrylamide gel electrophoresis (8% gel). The labeled
double-stranded oligonucleotide was excised and eluted in 400 µl of
TE buffer (10 mM Tris, 1 mM EDTA) and 40 µl of 3 M sodium acetate, pH 5.0, for 3 h, followed by ethanol
precipitation, rinsing in 70% ethanol, and resuspension in TE buffer.
The radioactive oligonucleotide probes were stored at -70 °C.
The DNA binding reactions (20 µl) contained nuclear extract
proteins (10 µg), 0.5 ng of P-labeled (5
10
cpm) DNA probe, poly(dI-dC) (500 ng), and 7 µl of 2
binding buffer (20% glycerol, 20 mM Hepes, pH 7.9, 50
mM KCl, 6.25 mM MgCl
, 0.5% Nonidet P-40,
0.2 mM EDTA, 4 mM spermidine) and were allowed to
incubate at 4 °C for 20 min. For competition experiments, a
100-fold excess of the indicated unlabeled double-stranded
oligonucleotides was added prior to the addition of radiolabeled DNA
probe. In some cases, the reaction mixtures were preincubated with 100
ng of specific anti-p53 antibodies (PAb421) for 15 or 60 min before the
addition of radiolabeled probes. The protein-DNA complexes were
resolved on a 4% native polyacrylamide gel (80:1
acrylamide/bisacrylamide) in 0.25
TAE running buffer (0.04 M Tris acetate, 1 mM EDTA, pH 7.4) containing 1
mM EDTA and 0.05% Nonidet P-40 at 4 °C at 180 V. The gels
were usually prerun for 2 h at 4 °C. The protein-DNA complexes in
the dried gels were visualized by autoradiography using Amersham
Hyperfilm.
Figure 1:
Transcriptional activation of the sgk promoter by p53 in nontumorigenic NMuMG and transformed
Con8Hd6 mammary epithelial cells. NMuMG cells (upper panel) or
Con8Hd6 cells (lower panel) were transfected with 10 µg of
-1428sgk-CAT reporter plasmid alone or along with 10
µg of expression plasmids encoding wild-type p53 (wt p53)
as described under ''Experimental Procedures.`` Transfected
cells were treated with (Dex) or without (none) 1
µM dexamethasone for 24 h and assayed for CAT activity by
quantitation of the conversion of [H]acetyl-CoA
into [
H]acetylchloramphenicol by the two-phase
fluor diffusion assay(61) . CAT activity was normalized to
protein levels, and the data represent means ± S.D. obtained
from at least three separate transfections, each carried out in
triplicate.
Parallel sets of mammary cells were transfected with a GRE-CAT reporter plasmid that contains six copies of GREs fused upstream of the CAT reporter gene as a control for glucocorticoid responsiveness or with the CMV-CAT reporter plasmid, which is known to be transcriptionally repressed by p53 in a variety of cell types(46) . As shown in Fig. 2, dexamethasone strongly stimulated GRE-CAT activity in both cell types (left panel), while cotransfection of wild-type p53 strongly repressed CMV-CAT reporter gene activity (right panel) under conditions in which p53 induced sgk promoter activity. Taken together, our results show that wild-type p53 strongly activates the glucocorticoid-responsive sgk promoter in both nontumorigenic (NMuMG) and transformed (Con8Hd6) mammary epithelial cells in a dexamethasone-independent manner.
Figure 2: Transactivation and transrepression of control reporter plasmids by dexamethasone and wild-type p53 in NMuMG and Con8Hd6 cells. NMuMG or Con8Hd6 cells were transfected with 10 µg of synthetic GRE-CAT reporter plasmid containing six copies of GREs linked to a minimal promoter (left panel), and the total amount of DNA was adjusted appropriately with the empty CAT vector plasmid pBLCAT3 as described under ''Experimental Procedures.`` Following transfection, cells were treated either with (+) or without(-) dexamethasone (Dex; 1 µM) for 24 h. Both cell types were also transfected with the CMV-CAT reporter plasmid (10 µg) alone(-) or together with (+) 10 µg of expression plasmids for wild-type p53 (wp53; right panel). Forty hours post-transfection, cells were harvested for measurement of CAT activity and calibrated to protein levels as described for Fig. 1. Values denote means ± S.D. from triplicate samples derived from three separate experiments.
Figure 3: p53-mediated activation of sgk promoter-CAT deletions in Con8Hd6 and NMuMG cells. A, the upper panel shows the sequences of the four putative p53 recognition elements within the -1428-bp sgk promoter fragment and are designated as sites I-IV. The base pairs underlined denote nucleotides sharing homology with the p53 consensus sequence. The lower panel illustrates the location of the putative p53-binding sites and the GRE in the context of the -1428sgk-CAT reporter construct. The arrows and corresponding numbers refer to the 5`-end points of the sgk promoter deletions incorporated into the -1428sgk-CAT, -681sgk-CAT, -303sgk-CAT, -236sgk-CAT, and -190sgk-CAT reporter plasmids. B, the indicated sgk-CAT reporter plasmids (10 µg) were transfected alone or cotransfected with 10 µg of expression vectors for either wild-type p53 (wt p53) or mutant p53 (mt p53) into either Con8Hd6 cells (left panel) or NMuMG cells (right panel) as described in the text. The 5`-sgk promoter fragments used are shown on the far right, and the corresponding numbers refer to the 5`-end points of deletions of the various constructs. Forty hours post-transfection, cells were assayed for CAT activity as described for Fig. 1, and the CAT activity was normalized to protein levels. Data represent means ± S.D. obtained from at least three separate transfections, each carried out in triplicate.
Figure 4: sgk p53 site IV (-1380 to -1345) confers positive regulation by p53 to a heterologous promoter. NMuMG cells were transfected with the sgk p53tk-CAT reporter plasmid (10 µg) containing sgk p53 sequences (-1380 to -1345) linked to a heterologous thymidine kinase minimal promoter, with the tk-CAT vector lacking these sequences, with the PG13-CAT reporter construct containing 13 copies of the p53-responsive element, or with the MG15-CAT reporter plasmid containing 15 copies of mutated p53 sequences either in the absence or presence of cotransfected expression plasmids for wild-type p53 (wt p53; 10 µg) or mutant p53 (mt p53; 10 µg). CAT activity was determined in cell lysates as described for Fig. 1(upper panel) and expressed with respect to protein content. Experiments were performed in triplicates, and the reported values denote means ± S.D. derived from three separate transfections.
Figure 5:
Gel shift analysis of binding of p53 with
the sgk p53 site IV sequence. Nuclear extracts (10 µg)
prepared from NMuMG cells were incubated with P-end-labeled double-stranded DNA probes representing
region -1380 to -1345 of the sgk promoter (site
IV). The indicated reaction mixtures contained no extract; no unlabeled
competitor DNA (No competitor); a 100-fold molar excess of
double-stranded oligonucleotides corresponding to sgk p53 site
IV, the consensus p53-binding site (Con p53), an unrelated DNA
(nonspecific DNA), or a mutant p53-binding site (Mut p53); or a
100-fold molar excess of unlabeled competitor DNA sgk p53 site
I (-235 to -205), sgk p53 site II (-285 to
-255), or sgk p53 site III (-1155 to -1125).
Binding reactions were preincubated for 1 h with 100 ng of anti-p53
antibodies (PAb421). The protein-DNA complexes formed were separated by
4% native polyacrylamide gel electrophoresis and visualized by
autoradiography. Arrows indicate the positions of protein-DNA
complexes and free probe.
To functionally test for the presence of p53 in nuclear extracts, a parallel set of gel shift experiments was carried out using a radiolabeled oligonucleotide corresponding to the consensus p53 DNA-binding site. In contrast to the three protein-DNA complexes detected with the sgk DNA site IV probe in NMuMG extracts (Fig. 5), incubation of the p53 consensus probe with these nuclear extracts yielded a single specific complex (Fig. 6, No competitor). Competition experiments established the specificity of binding. For example, the addition of excess specific unlabeled Con p53 fragment completely abolished the single band, whereas excess unrelated nonspecific DNA or DNA encoding a mutated p53-binding site was incapable of competing off the protein-DNA complex (Fig. 6). More importantly, the specific complex using radiolabeled Con p53 was competed off with excess unlabeled sgk p53 site IV DNA. When the nuclear extracts were preincubated with p53 antibodies for a short time (15 min), a partial supershift of the specific complex was observed, whereas upon longer incubation (60 min) with p53-specific antibodies, a completely supershifted complex was obtained. The presence of p53 in these nuclear extracts was analyzed by Western blotting. As also shown in Fig. 6, detectable amounts of p53 were present in these nuclear extracts, accounting for the specific protein-DNA complex formation. These results show that p53 present in the nuclear extracts of NMuMG epithelial cells can bind specifically to the sgk p53 site IV sequence or the consensus p53 DNA-binding site. In addition, the different putative sgk p53-binding sequences (sites III, II, and I) are all homologous to some extent to the site IV sequence as these three sites can efficiently compete for specific binding of p53 with the radiolabeled sgk p53 site IV DNA probe.
Figure 6:
p53 present in nuclear extracts as
determined by Western blotting interacts with p53 consensus sequences
in NMuMG cells as assessed by gel shift analysis. Nuclear extracts (10
µg) prepared from NMuMG cells were incubated with P-end-labeled double-stranded DNA probes representing the
p53 consensus sequence (Con p53). The indicated reaction mixtures
contained no extract; no unlabeled competitor DNA (No
competitor); or a 100-fold molar excess of double-stranded
oligonucleotides corresponding to sgk p53 site IV, the
consensus p53-binding site (Con p53), an unrelated DNA (nonspecific
DNA), or a mutant p53-binding site (Mut p53). Binding reactions were
preincubated for 15 or 60 min with 100 ng of anti-p53 antibodies
(PAb421). The protein-DNA complexes formed were separated by 4% native
polyacrylamide gel electrophoresis and visualized by autoradiography. Arrows indicate the positions of protein-DNA complexes and
free probe. Nuclear protein extracts (30 µg) from NMuMG cells were
electrophoretically fractionated, blotted onto nitrocellulose filters,
and analyzed for p53 protein by Western blotting as described under
''Experimental Procedures`` (inset). The protein
molecular mass standards are shown on the left. The arrow indicates the position of the p53-specific
band.
Figure 7: Cell type-dependent activation or inhibition of the sgk and p21/Waf1 promoters by p53. NMuMG cells or Rat-2 fibroblasts were transfected either with the -1428sgk-CAT reporter plasmid alone (left panel) or with the WWp-Luc reporter plasmid alone (right panel) or cotransfected with expression plasmids encoding either wild-type p53 (wt p53) or mutant p53 (mt p53). CAT or luciferase activity was estimated in cell extracts as described in the text and normalized to protein levels. Data shown are means ± S.D. obtained from three separate transfections, each carried out in triplicate.
The p53 tumor suppressor protein has been proposed to mediate
its diverse biological effects in response to various external stimuli
by stimulating or repressing the transcription of particular sets of
target genes. Our results demonstrate that, both in NMuMG
nontumorigenic mammary epithelial cells and Con8Hd6 mammary tumor
cells, one such transcriptional target of wild-type p53 is the sgk serum/glucocorticoid-inducible protein kinase gene. In both
mammary cell types, wild-type p53 strongly stimulated basal sgk promoter activity in transiently transfected cells in a manner
independent of glucocorticoids. Cotransfection of wild-type p53 reduced
the dexamethasone responsiveness of the sgk promoter, which
was likely due to near maximal stimulation of sgk-CAT reporter
plasmid activity by p53. In Con8Hd6 mammary tumor cells, dexamethasone
induces a G cell cycle arrest under conditions in which sgk gene expression is rapidly stimulated(13) ,
implicating a role for sgk in the growth suppression response.
The transcriptional stimulation of sgk promoter activity by
p53 further supports this idea and represents the first example of a
hormone-regulated protein kinase gene that contains a functionally
defined p53-responsive element in its promoter region.
Deletion analysis of the sgk promoter revealed that the p53-responsive region observed in both transfected mammary epithelial cell lines harbors two putative p53-binding sites, sgk p53 site IV (-1380 to -1345) and sgk p53 site III (-1155 to -1125). Transfection assays demonstrated that the 35-bp fragment corresponding to sgk p53 site IV alone was functional and that p53 specifically interacted with these sequences in vitro. We have also observed specific binding of p53 to the sgk p53 site III sequence (data not shown). Previous studies have defined two copies of the 10-bp motif 5`-RRRC(A/T)(T/A)GYYY-3` separated by 0-13 bp (63) as the p53 consensus sequence. The sgk p53 site IV sequence (5`-ccTGCCCAACTCAGGCTGCCTCCTGCGACTTGCCT-3`) as well as the site III sequence (5`-agagGGGCAGGCATCAGGGCAAGGGTATTGACTg-3`) exhibit strong homology to the p53 consensus sequence compared with the other two p53-binding sites, I and II, outside of the functionally defined p53-responsive region. These two sites may account for the minor activation of sgk-CAT reporter plasmids containing shorter regions of sgk promoter activity in transfected NMuMG cells. A wide range of degeneracy in p53 recognition sequences exists as a variety of p53-activated promoters contain p53-binding sites different from the p53 consensus sequence. For instance, the p53-responsive element within the human T-cell leukemia virus type I enhancer is composed of a GC-rich element (5`-GCCCTGACGTGTCCCC-3`) (65) . Other p53-responsive elements that differ from the consensus sequence include the two copies of the weak p53-binding element (5`-GGGCGGAGTTA-3`) within the SV40 origin of replication (66) and the p53 sequences defined from mouse genomic clones (5`-GACACTGGTCACACTTGGCTGCTTAGGAAT-3`)(67) . In this regard, although sgk p53 site I and II sequences are not strongly related to the p53 consensus sequence, p53 was capable of specifically binding to these sequences in vitro (data not shown), and these sequences efficiently competed with the sgk p53 site IV sequence for binding as assessed by gel shift assays.
In contrast to the strong p53-dependent stimulation of sgk promoter activity in mammary cells, wild-type p53 evoked a repression of sgk promoter activity in Rat-2 fibroblasts. More importantly, wild-type p53 regulated the activity of the p21/Waf1 gene promoter, a well characterized transcriptional target of p53(38) , in a cell type-specific manner generally similar to the effects observed on sgk promoter activity. This result further establishes the sgk gene as a biologically relevant transcriptional target of p53. The exact mechanism involved in the p53-mediated down-regulation of cellular promoters is not well understood, although it has been proposed that repression by p53 occurs by interaction with components of the general transcriptional machinery (44, 50, 51) and, in some cases, may entail binding of p53 to specific DNA sequences(42, 45) . In fact, the cell type-specific positive or negative regulation of genes by p53 appears to be confined to genes containing p53-binding sites. For example, the creatine kinase gene and proliferating cell nuclear antigen gene promoters, which contain functional p53-binding sites within their promoter regions, are also either positively or negatively regulated in a cell type-specific manner(27, 29, 52) . In contrast, a CMV-CAT reporter that lacks p53-binding sites and is known to be repressed by p53 (46) was suppressed by p53 in both mammary epithelial cells and Rat-2 fibroblasts (data not shown). A variety of viral and cellular proteins (68, 69) and also certain transcription factors (70) have been shown to interact with p53 and to modulate its function(71, 72, 73, 74) . Thus, depending on the cellular context, p53 is able to affect either transactivation or transrepression of the sgk gene, or the p21 gene, presumably in conjunction with cell type-specific regulatory factors.
Many transcriptional target genes of p53 are involved in
important cellular processes such as growth regulation (e.g.
fos, jun, myc, cyclin A, and p21/Waf1) (38, 40, 75, 76) and apoptosis (e.g. bcl-2 and bax) (33, 42) . The cell type-specific growth suppression
effects of p53 appear to be related to the ability of p53 to act as a
specific transcriptional activator and, in certain tissues, its ability
to interact with the MDM2 protein(77) . Many studies have
implicated p53 to be important for the growth control of mammary
epithelial cells. For example, approximately one-fourth of all sporadic
breast cancers express mutant forms of p53 containing deletions or
mutations(78) , whereas other mammary tumors are characterized
as having an allelic loss of the short arm of chromosome 17, which
includes p53(79, 80) . Consistent with a role for p53
in the evolution of mammary tumors, a deficiency of p53 accelerates
mammary tumorigenesis in Wnt-1 transgenic mice(81) . Other
evidence suggests that p53 mutations are important in the early
preneoplastic stages of mammary tumorigenesis(77) . In
addition, loss of p53 protein in certain types of mammary cells exposed
to -irradiation (53) or as a result of human
papillomavirus E6-induced degradation (82) is accompanied by
immortalization of these cells and failure to arrest in the G
phase of the cell cycle.
In our study, wild-type p53, but not mutant p53, strongly stimulated sgk promoter activity in both nontumorigenic and transformed mammary epithelial cells. In this context, functional characterization of a positive p53 recognition element within the promoter of this protein kinase gene implicates that a subset of p53-mediated responses in mammary cells, such as on the control of cell proliferation or programed cell death after exposure to DNA-damaging agents(83) , may be mediated by positive or negative changes in cellular phosphorylation cascades and/or activities of potential sgk substrates. In mammary tumor epithelial cells, we propose that sgk may be involved in the glucocorticoid-regulated growth suppression response and that activation of sgk by p53 may represent a distinct, but complementary, growth regulatory pathway. A key focus of our future approaches will be to understand the functional roles and biological context by which p53 targets the sgk gene in mammary epithelial cells and if activation of the sgk promoter requires mammary-specific nuclear factors or coactivators to interact with the p53-DNA complex.