(Received for publication, February 21, 1996)
From the
The insulin-response element from the prolactin gene is
identical to the Ets-binding site, and dominant-negative Ets protein
inhibits insulin-increased prolactin gene expression. Immunoblotting
identified the Ets-related transcription factor GABP in nuclear
extracts from GH cells. Expression of GABP and GABP
1
squelches insulin-increased prolactin gene expression. GABP
and
GABP
1 bind the insulin-response element of the prolactin promoter,
and anti-GABP
and anti-GABP
1 antibodies supershift a species
seen with nuclear extracts from GH cells. GABP
immunoprecipitated
from insulin-treated,
P-labeled GH cells was
phosphorylated 3-fold more than GABP
from control cells. There was
no increase in phosphorylation of GABP
in response to insulin.
Mitogen-activated protein (MAP) kinase activity is increased 10-fold in
insulin-treated GH4 cells. MAP kinase immunoprecipitated from control
cells does not phosphorylate GABP
while MAP kinase
immunoprecipitated from insulin-treated cells shows substantial
phosphorylation of GABP
. These studies suggest that GABP mediates
insulin-increased transcription of the prolactin gene. GABP may be
regulated by MAP kinase phosphorylation.
The activation of gene transcription by hormones that function
through protein-tyrosine kinase receptors is not well understood in
comparison with that mediated by other classes of hormones. The
receptors for the steroid-thyroid hormones are transcription factors
that are activated by hormone binding(1) . G protein-coupled receptors activate gene transcription following
hormonal initiation of a cascade ending in the phosphorylation of
CREB/ATF transcription factors(2) . Recently, numerous
cytokines have been shown to activate transcription via phosphorylation
of cytosolic ISGF-3 proteins that then become nuclear localized
transcription factors(3) . The responsiveness of genes to each
of these classes of hormones is dependent on the presence in the gene
of the appropriate DNA sequence to which the activated transcription
factor binds. Neither the hormone-responsive DNA element nor the
transcription factors activated by protein-tyrosine kinase receptors
are known.
Recently, we have identified an insulin-response element
in the prolactin promoter that is identical to the binding site for the
Ets-related transcription factors(4) . This element also
mediates the insulin sensitivity of the thymidine kinase and
somatostatin promoters in both HeLa and GH4 cells and confers insulin
responsiveness to the mammary tumor virus promoter when it is added to
that promoter at -88. Further, the increase in the transcription
of these genes in insulin-treated cells was inhibited by expression of
a dominant-negative Ets protein(5) . These studies identify the
predominant Ets-related protein of GH4 cells, GABP, and suggest
that GABP mediates insulin-increased prolactin gene expression.
Phosphorylation of GABP by MAP (
)kinase may regulate its
activity.
Immunoblot analysis of GH4 cell nuclear extracts was
performed to determine which of the Ets-related transcription factors
might mediate the effects of insulin on prolactin gene expression (Fig. 1). A pan-Ets antibody was used to visualize Ets-related
proteins in GH4 cell nuclear extracts. This antibody was raised against
the conserved DNA binding domain of Ets-1, and it demonstrates broad
cross-reactivity with Ets family proteins. One band of approximately 51
kDa was visible using either 1 or 3 µg of nuclear extract. This is
the same size as the previously identified Ets-related protein
GABP. GABP
is a subunit of the heteromeric transcription
factor, GABP. The other subunit is GABP
, a notch-related
protein(12) . GABP was originally identified as the
transcription factor that binds to a purine-rich cis-regulatory element required for VP16-mediated activation
of herpes simplex virus immediate early gene (13) . A separate
set of filters was therefore analyzed using antibodies against
GABP
or GABP
1. One band, identical in size to that seen using
the pan-Ets antibody, is seen with anti-GABP
. Anti-GABP
1
antibody reveals two bands. The lower, more intense band migrates with
an apparent molecular mass of 43 kDa and thus likely represents
GABP
1. The levels of GABP
and GABP
1 are not
significantly different in nuclear extracts from control and
insulin-treated cells (data not shown).
Figure 1:
Analysis
of Ets-related proteins in GH4 cell nuclear extracts by immunoblotting. Lane 1, 1 µg, and lane 2, 3 µg of GH4 cell
nuclear extract blotted with an antibody prepared against the conserved
DNA binding domain of the c-Ets-1 (Santa Cruz). Lanes 3 and 4 are two nuclear extracts blotted against a polyclonal
antibody to GABP (Dr. S. L. McKnight). Lanes 5 and 6 are the same nuclear extracts blotted with a polyclonal antibody
to GABP
1 (Dr. S. L. McKnight). The position of migration of
molecular weight markers ovalbumin (46,000 kDa) and bovine serum
albumin (68,000 kDa) is indicated. The arrow on the left indicates the position of GABP
while the arrow on
the right indicates GABP
1.
Experiments were performed
to determine if GABP might be involved in the response of the prolactin
gene to insulin. Cotransfection of small amounts of GABP
expression vector inhibited the insulin-induced increase in prolactin
gene expression (Fig. 2) to 25% of that seen in control
transfections (22-fold) or in transfections with expression vector
(21-fold). Cotransfection with an expression vector for GABP
1
resulted in a 2-fold increase in the insulin-stimulated prolactin-CAT
expression (45-fold). This suggests that levels of GABP
1 may be
limiting in GH cells. GABP
1 was shown to increase the affinity of
GABP
binding to DNA(12) . Thus, it might be expected that
a cotransfection with both GABP
and GABP
1 would further
affect insulin-increased prolactin-CAT expression. Coexpression of
vectors for both GABP
with GABP
1 completely eliminated any
effect of insulin (Fig. 2) but has no significant effect on
basal or EGF-increased prolactin gene expression. Basal expression of
prolactin-CAT was not affected in these experiments (1.15 ±
0.25% acetylation/10 µg of protein in control cultures versus 1.07 ± 0.44% acetylation/10 µg of protein in GABP
+ GABP
-cotransfected cells). Further, EGF-increased
expression of prolactin-CAT was also not affected by cotransfection
with GABP. EGF results in a 13-fold increase in control cells (15.12
± 0.7% acetylation/10 µg of protein) and a 12-fold increase
in GABP
- and GABP
-cotransfected cells (13.6 ± 0.66%
acetylation/10 µg of protein).
Figure 2:
Effect of expression of GABP on
insulin-increased prolactin-CAT transcription. In control
transfections, GH4 cells were cotransfected with 15 µg of
Prl-(-173/+75) CAT (10) and with 5 µg of an
expression vector for the human insulin receptor, pRT3HIR2 (J.
Whittaker, Stony Brook) alone or with 10 µg of a cytomegalovirus
expression vector pRK5. Vectors expressing GABP and/or GABP
1
(C. C. Thompson, Carnegie Institute) or Elk-1 and SRF (R. Treisman,
Imperial Cancer Research Fund, London, United Kingdom) under control of
the cytomegalovirus promoter were included at 5 µg in the
designated experiments. The average percent acetylation/mg of protein
in control and insulin-treated cultures was determined, and the insulin
incubations were compared with control levels to determine the -fold
stimulation by insulin (Fold-Control). The results are from
three separate experiments done in
duplicate.
Similar results were seen previously with SRF and Elk-1(14) . The effects of Elk-1 and SRF were attributed to squelching caused by titration of limiting components or formation of non-functional transcription complexes(14) . The squelching of prolactin gene expression due to overexpressed GABP appears to be specific since overexpression of SRF and Elk-1 has no effect on prolactin gene expression in GH cells (Fig. 2) although the Elk-1 binding site of c-Fos is similar to the insulin-response element of the prolactin gene.
Since expression
of GABP was able to block insulin responsiveness of the prolactin
promoter, the binding of GABP and GABP
1 to the
insulin-response element of the prolactin gene was examined. Nuclear
extracts from GH cells produced a characteristic mobility shift pattern (Fig. 3, lane 1). This gel-shift pattern was identical
with that previously shown to be inhibited by low levels of
non-radioactive competitor(10) . Gel-mobility shift experiments
performed with bacterially expressed GABP
showed three retarded
bands with the prolactin insulin-response element (Fig. 3, lane 2). The upper band is a complex formed with a bacterially
expressed protein since it was present in extracts from unprogrammed
bacteria (not shown) The other two bands are complexes containing
GABP
. Similar complexes were previously reported to be formed
between DNA and GABP
monomer and GABP
dimer(12) .
Only the bacterial protein band is seen with bacterially produced
GABP
1 (lane 3). This was expected since previous studies
had shown that GABP
1 is not a DNA binding protein(12) .
When GABP
and GABP
1 were added together (lane 4),
the bands corresponding to GABP
were no longer visible and an
abundant, more slowly migrating band was seen. This band corresponds to
an abundant band seen with nuclear extracts from GH cells. The identity
of this band as consisting of GABP
and GABP
1 is confirmed in
the experiment shown in Fig. 3(right). Lanes 1 and 2 show the gel-mobility shift pattern with nuclear
extract alone (lane 1) and nuclear extract with normal rabbit
serum (lane 2). Antibodies to GABP
, lane 3,
GABP
1 (lane 4), or antibodies to both GABP
and
GABP
1 (lane 5) shifted this complex to one with a slower
migration.
Figure 3:
Binding of GABP to the prolactin promoter. Left, the proteins complexed with the P-labeled
Prl-(-106/-87) were: lane 1, nuclear extract; lane 2, GABP
; lane 3, GABP
1; and lane
4, GABP
and GABP
1. The positions of migration of the
GABP
monomer, dimer, and the GABP are indicated on the left. The migration of a nonspecific bacterial protein complex
is also indicated. Right, all reactions contained 2 µg of
GH4 cell nuclear extract. In addition, lane 2 contained 1
µl of normal rabbit serum, lane 3 contained a polyclonal
antibody to GABP
(Dr. S. L. McKnight), lane 4 contained a
polyclonal antibody to GABP
1 (Dr. S. L. McKnight), and lane 5 contained both antibodies. The position of GABP and the supershift
are indicated on the left.
Since insulin receptor is a tyrosine-protein kinase that
is activated by insulin binding, it is thought that activation of gene
transcription by insulin may be the end product of a phosphorylation
cascade. Therefore, we examined the phosphorylation of GABP in response
to insulin in P-labeled GH cells. The phosphorylation of
GABP
was increased 3-fold in 1 h in insulin-treated cells as
compared with control cells (Fig. 4A). GABP
co-immunoprecipitated with GABP
in this experiment shows no
increase in response to insulin. Immunoprecipitation with
anti-GABP
1 confirms this observation. GABP
1 phosphorylation
was not significantly increased by insulin treatment (20% above
control) while the co-immunoprecipitated GABP
is increased 3-fold
by insulin.
Figure 4:
A, immunoprecipitation of GABP and
GABP
from
P-labeled GH4 cells. GH cells were labeled
with
P and incubated with insulin for 1 h as described
under ``Experimental Procedures.'' Labeled proteins were then
precipitated with an antibody to GABP
(lanes 1 and 2) or an antibody to GABP
1 (lanes 3 and 4). Immunoprecipitations with control lysates are in lanes
1 and 3 while insulin-treated cell lysates are in lanes 2 and 4. The migration of GABP
and
GABP
is indicated on the left while the migration of
molecular weight markers is shown on the right. This
experiment was repeated twice with similar results. B, insulin
activation of MAP kinase. GH cells were transfected with 1 µg of a
vector expressing a human influenza hemagglutinin-tagged MAP kinase and
with 5 µg of pRT3HIR2. After a 24-h incubation in insulin-depleted
serum containing medium, the cultures were incubated with insulin for 5
min or left untreated as controls. The cells were harvested and
immunoprecipitated with anti-hemagglutinin antibody (Boehringer
Mannheim) as described under ``Experimental Procedures.'' The
kinase activity of the immunoprecipitated MAP kinase was assayed as
described under ``Experimental Procedures'' using myelin
basic protein (Sigma) as a substrate. Lane 1, MAP kinase from
control cells; lane 2, MAP kinase from insulin-treated cells. C, MAP kinase phosphorylation of GABP
. Lanes
1-4 used immunoprecipitated MAP kinase from insulin-treated
cells while lanes 5-8 contained MAP kinase
immunoprecipitated from control cells. The even lanes contained GST-GABP
, and the odd lanes are GST
protein. Lanes 3, 4, 7, and 8 show
assays performed without MgCl
and with 2 mM EDTA.
The arrow to the left indicates the position of
GST-GABP
. The arrows on the right indicate the
migration of molecular weight standards. An immunoblot of the MAP
kinase in the immunoprecipitates used for this experiment is shown below each lane.
MAP kinase activation was shown to be required for
several types of insulin responses in numerous systems(15) .
Further, Elk-1, an Ets-related transcription factor, was shown to be
activated by MAP kinase phosphorylation(16) . Pointed-P2, an
Ets-related protein from Drosophila, is phosphorylated by MAP
kinase in the sevenless signal transduction pathway(17) . Our
studies ()suggest that insulin activation of prolactin gene
expression in GH cells is MAP kinase-dependent since all factors that
inhibit insulin-increased prolactin gene expression also inhibit MAP
kinase activation. Therefore, MAP kinase activated by insulin might
phosphorylate GABP
. The representative increase in MAP kinase
activity in cell lysates from insulin-treated cells is shown (Fig. 4B). Multiple experiments show an increase of 10
± 0.8-fold in MAP kinase activity in insulin-treated cells.
GABP
contains three potential MAP kinase phosphorylation sites
near the DNA binding domain. Therefore, GST-GABP
fusion protein
containing the three MAP kinase phosphorylation sites was prepared and
used in a kinase assay with MAP kinase immunoprecipitated from control
or insulin-treated GH4 cells. GABP
was phosphorylated only by MAP
kinase immunoprecipitated from insulin-treated cells (Fig. 4C). This shows that GABP
is a substrate for
MAP kinase and suggests that MAP kinase phosphorylation of GABP may be
functionally important.
GABP was shown to be important for enhancement of transcription of the herpes simplex virus immediate early gene, but its physiological role in uninfected cells is unknown. Our studies show that GABP mediates the insulin response of the prolactin gene. Since GABP is widely distributed, these results could be significant to understanding insulin regulation of other genes. Analysis of 22 insulin-responsive promoters has identified potential Ets-response elements in all of these. For some of these, the Ets-response element is in a region defined by deletion analysis to be important for the effects of insulin (5) . The insulin-mediated increase in the transcription of all three genes, prolactin, somatostatin, and thymidine kinase, that we have studied is inhibited by dominant-negative Ets protein. This indicates that GABP may be implicated in the regulation of other insulin-responsive genes.
Ets-related transcription factors such as GABP are often found in large complexes with other transcription factors. For example, Ets-1 and Sp-1 interact to synergistically activate the human T-cell lymphotrophic virus long terminal repeat(18) . Although this report demonstrates that GABP is necessary to the insulin effect, it may not be sufficient. The insulin responsiveness of the prolactin gene can be eliminated by mutation of two Ets motifs at -96/-87 and -76/-67 of the prolactin promoter(4) . These mutations have little effect on basal prolactin gene transcription. However, mutation of -101/-92 of the prolactin promoter eliminates the effect of insulin and reduces basal prolactin gene expression by >100-fold. Clearly, another protein(s) interacts at this sequence and is important both for basal prolactin gene expression and the effect of insulin. It is likely that GABP is complexed with this protein(s) in the prolactin promoter and that this complex is important to the increase in prolactin gene expression seen in insulin-treated cells.