Reconstitution of the Protein Kinase A Response of the Rat Prolactin Promoter: Differential Effects of Distinct Pit-1 Isoforms and Functional Interaction with Oct-1
Scott E. Diamond,
Matt Chiono and
Arthur Gutierrez-Hartmann
Department of Medicine and Department of Biochemistry
and Molecular Genetics Program in Molecular Biology, and
Colorado Cancer Center University of Colorado Health Sciences
Center Denver, Colorado 80262
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ABSTRACT
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PRL gene transcription is primarily regulated by
dopamine, which lowers cAMP levels and inhibits protein kinase A (PKA)
activity. Current data indicate that the cAMP/PKA response maps to the
most proximal Pit-1/Pit-1ß binding site footprint I (FP I) on the rat
PRL (rPRL) promoter. Pit-1, a POU-homeo domain transcription factor, is
specifically expressed in the anterior pituitary and is required both
for the normal development of anterior pituitary cell types,
somatotrophs, lactotrophs, and thyrotrophs, and for the expression of
their hormones: GH, PRL, and TSHß. Pit-1 has been shown to
functionally interact, via FP I, with several transcription factors,
including Oct-1, a ubiquitous homeobox protein, and thyrotroph
embryonic factor, which is found in lactotrophs, to activate
basal rPRL promoter activity. Pit-1ß/GHF-2, a distinct splice isoform
of Pit-1, acts to inhibit Ras-activated transcription from the rPRL
promoter, which is mediated by a functional interaction between Pit-1
and Ets-1 at the most distal Pit-1 binding site (FP IV). In this
manuscript we show 1) that the Pit-1ß isoform not only fails to block
PKA activation, but is, in fact, a superior mediator of the PKA
response; 2) that the PKA response requires intact POU-specific and
POU-homeo domains of Pit-1; and 3) that Oct-1, but not thyrotroph
embryonic factor, functions as a Pit-1-interacting factor to mediate an
optimal PKA response.
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INTRODUCTION
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Pit-1 is a POU-homeo domain transcription factor that is
specifically expressed in the anterior pituitary, where it is required
both for the normal development of three cell types, somatotrophs,
lactotrophs, and thyrotrophs (1, 2, 3), and for proper expression of the
PRL, GH, and TSHß genes (1, 2). The 33-kDa Pit-1 protein is encoded
by six exons (Fig. 1A
) that encode a
number of important and separable subdomains. On the N terminus two
exons make up the 80-amino acid (AA) transactivation domain (TAD),
which has been shown to be sufficient for transactivation in mammalian
cells (Fig. 1B
) (4, 5). On the C terminus, the POU-specific domain (AA
128198) and POU-homeo domains (AA 213273) are necessary and
sufficient for DNA binding and homodimerization (Fig. 1B
) (5, 6, 7, 8).

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Figure 1. Genomic and Protein Domain Organization of Pit-1
A, Pit-1 genomic organization. Six exons of Pit-1 with AA endpoints and
subdomains they encode are shown as open rectangles, 5'-
and 3'-untranslated regions (UTRs) as black rectangles,
and alternatively spliced ß-exon as a gray rectangle.
B, Pit-1/Pit-1ß protein domain organization. The functional domains
of Pit-1 and Pit-1ß proteins, the TAD, the POU-specific, and the
POU-homeo domain, are delimited by brackets and AA
endpoints. Also, Pß and HDß represent the
POU-specific and POU-homeo domain basic domains, and 14 and
13 represent the POU-specific and POU-homeo domain -helices.
Hinge is the region between the TAD and the bipartite DNA-binding
domain, and FL indicates the 15 AA flexible linker between the
POU-specific and POU-homeo domain regions.
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Pit-1ß is a splice isoform of Pit-1 that differs only in the
ß-domain, a 26-AA insertion at position 48 in the TAD caused by the
use of an alternate 3'-splice acceptor at the end of the first intron
(Fig. 1
) (9, 10, 11). The AA sequence of the ß-domain has been conserved
among vertebrates from teleost fish to aves to humans and is present in
the predominant Pit-1 isoform found in teleost fish and aves (9, 10, 11, 12, 13, 14),
suggesting a conservation of function (reviewed in Ref. 15). Moreover,
the wild-type AA sequence of the ß-domain is required for cell
type-dependent display of dominant-negative properties of Pit-1ß
(15). In pituitary cells Pit-1ß represses basal and Ras/Raf-induced
rat PRL (rPRL) promoter activity (9, 10, 11, 16, 17), while in nonpituitary
cells Pit-1ß activates basal rPRL promoter activity as well as Pit-1
per unit protein (15). Thus, the sequence conservation of the
ß-domain, and the distinct transcription properties that it confers
upon Pit-1ß, suggest a distinct and conserved role for Pit-1ß in
the regulation rPRL promoter activity.
The cAMP-protein kinase A (PKA) pathway is important to the regulation
of rPRL gene expression in lactotrophs. Dopamine, which lowers cAMP
levels and inhibits PKA activity, down-regulates rPRL promoter activity
(18, 19, 20), while agents such as forskolin, cAMP analogs, or PKA
expression vectors, which increase cAMP and activate PKA activity,
stimulate rPRL gene transcription in cultured GH4 rat pituitary tumor
cells (21, 22, 23, 24, 25). Negative regulation by dopamine requires dopamine D2
but not D4 receptors (26) and acts through G(i)
2 to block a
positive cAMP and PKA-dependent signaling cascade directed at Pit-1
(19). An additional G(o)
-dependent (and cAMP-independent) pathway
may also inhibit PRL gene expression, although that pathway has not
been fully characterized (27, 28).
The cAMP-PKA signal is most often transduced by the nuclear
transcription factor cAMP-response element-binding protein
(CREB), which binds to a specific DNA-regulatory element, the cAMP
response element (CRE) (TGACGTCA) (29). However, cAMP-PKA
signaling to the rPRL promoter appears to act through a different
transducer. The proximal rPRL promoter contains no CRE consensus sites
and binds neither affinity-purified CREB from GH4 cells nor
recombinant CREB from bacteria (25, 30). In contrast, cAMP-PKA
signaling to the rPRL promoter requires a DNA binding site for Pit-1,
footprint I (FP I) (25, 30, 31), and PKA synergizes with Pit-1 to
activate rPRL promoter activity in a FP I-dependent manner in a HeLa
nonpituitary cell transfection-reconstitution assay developed in this
laboratory (31). That Pit-1 is not a direct nuclear target of PKA (32, 33) suggests that a FP I- or Pit-1-associated cofactor is the actual
PKA substrate (25, 30, 31, 34).
Pit-1 functionally interacts via FP I with several transcription
factors, including Oct-1, a ubiquitous homeobox protein, and thyrotroph
embryonic factor (TEF), which is found in lactotrophs, to activate
basal rPRL promoter activity (35, 36). These results suggest that Oct-1
or TEF might be the factor that functionally interacts with Pit-1 to
mediate PKA signaling through FP I of the rPRL promoter.
To better understand how Pit-1 transduces the PKA signal to the rPRL
promoter, we 1) examined whether the Pit-1ß isoform differs from
Pit-1 as a transducer of the PKA-signal; 2) used deletion mutagenesis
to identify domains of Pit-1 required for PKA-signaling; and 3) tested
TEF and Oct-1 for ability to function as PKA-signaling cofactors. We
show that Pit-1ß is a more potent transducer of the PKA response than
is Pit-1. Mapping studies with internal deletions of the Pit-1 isoform
demonstrate that the POU-specific region of Pit-1 is required for
mediation of the PKA response. Further experiments show that Oct-1, but
not TEF, acts as a PKA signaling cofactor. These data are important
because they show that the PKA signal is mediated by the POU-specific
domain, and that Oct-1 can contribute to full reconstitution of the PKA
response.
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RESULTS
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Pit-1ß Is a More Potent Enhancer of PKA Signaling Than Is
Pit-1
We have previously demonstrated that cAMP/PKA signaling to
the rPRL promoter relies not upon the usual transducer, CREB, but
rather upon Pit-1 and another factor via FP I of the rPRL promoter (25, 30, 31, 34). Given that the Pit-1ß isoform has distinct effects on
basal and Ras-activated transcription (9, 10, 11, 16, 17), it seemed
possible that Pit-1ß might also display altered ability to transduce
PKA signaling. To address this issue, we examined the relative
abilities of Pit-1 and Pit-1ß to stimulate basal and PKA-stimulated
rPRL promoter activity in a HeLa nonpituitary cell
transfection-reconstitution assay developed in this laboratory (31).
First, both isoforms were tested for activation of basal rPRL promoter
activity. Increasing, but low and nonsaturating, doses of pRSV-Pit-1
and pRSV-Pit-1ß were introduced into HeLa nonpituitary cells by
electroporation with a rPRL-driven luciferase reporter (pA3425 rPRL
Luc) and assessed for basal transcriptional potency (Fig. 2
). The rPRL-driven luciferase reporter
alone produced about 5 relative light units (RLU), and pRSV Pit-1 DNA
doses of 0.03, 0.3, and 3 µg increased rPRL promoter activity to
1.4-, 1.9- and 11.9-fold, respectively (Fig. 2A
). Identical
pRSV-Pit-1ß DNA doses had no discernible effect upon rPRL promoter
activity. These findings are consistent with earlier work in which the
lower levels of Pit-1ß protein, expressed per unit DNA (see also Fig. 2C
), result in lower activation of basal rPRL promoter activity in
pituitary cells (10, 15).

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Figure 2. Pit-1ß Is a Superior Mediator of PKA Signaling
For panels A and B, combinations of 3 µg pA3 -425 rPRL luc and either
pRSV-Pit-1 (0.03, 0.3, or 3 µg), pRSV-Pit-1ß (0.03, 0.3, or 3
µg), and 10 µg pRSV-PKAß were introduced into HeLa nonpituitary
cells by electroporation. pRSVß-globin was added to keep the total
amount of RSV promoter-containing DNA constant. Cells were harvested
after 4448 h and total light units measured, as described in
Materials and Methods. Results for panels A and B are
expressed as the mean ± SD of a representative
experiment of three experiments done in duplicate. A, Effects of
increasing DNA doses of Pit-1- vs. Pit-1ß-expressing
plasmids on basal rPRL promoter activity. B, Effects of increasing DNA doses of Pit-1-
vs. Pit-1ß-expressing plasmids on PKA-stimulated rPRL
promoter activity. C, Analysis of Pit-1 isoform expression ± PKA.
Combinations of pA3PRLluc-425, pRSV-HA-Pit-1, pRSV-HA-Pit-1ß, and
PKAß were introduced into HeLa nonpituitary cells by electroporation.
pRSVß-globin was added to keep the total amount of RSV
promoter-containing DNA constant. Lanes were loaded with equal protein
(100 µg) from extracts of HeLa cells transfected as follows: 3 µg
of pA3PRLluc-425 (lane 1); 3 µg of pA3PRLluc-425 and 10 µg of
pRSV-PKA (lane 2); 3 µg of pA3PRLluc-425 and 10 µg of pRSV-HA-GHF1
(lane 3); 3 µg of pA3PRLluc-425, 10 µg of pRSV-PKA, and 10 µg of
pRSV-HA-GHF1 (lane 4); 3 µg of pA3PRLluc-425 and 10 µg of pRSV-GHF2
(lane 5); 3 µg of pA3PRLluc-425, 10 µg of pRSV-PKA, and 10 µg of
pRSV-GHF2 (lane 6). After 24 h, cells were harvested and analyzed
by SDS-PAGE. (a) The blot was probed with a mouse monoclonal anti-HA
antibody. BSA-HA was used as a positive control (data not shown). (b)
To demonstrate equal protein loading, the blot was stripped and probed
with a mouse monoclonal antiactin antibody.
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To assess the relative abilities of Pit-1 and Pit-1ß to enhance
stimulation of rPRL activity by PKA, the same doses of pRSV-Pit-1 and
pRSV-Pit-1ß were introduced into HeLa nonpituitary cells by
electroporation with pA3425 rPRL Luc and pRSV-PKAß, a plasmid
encoding the catalytic ß-subunit of PKA (Fig. 2B
). While PKAß alone
increased rPRL promoter activity 35-fold, the addition of 0.03, 0.3,
and 3 µg DNA doses of pRSV-Pit-1 further increased promoter activity
to 164-, 306- and 148-fold, respectively (Fig. 2B
). Identical DNA doses
of pRSV-Pit-1ß increased promoter activity to 73-, 344-, and
535-fold, respectively. Surprisingly, the highest dose of Pit-1 in the
presence of PKA had a smaller effect on promoter activity than did the
two lower doses (Fig. 2B
). Moreover, the maximal fold induction was
greater for Pit-1ß/PKAß (535-fold) than for Pit-1/PKAß
(306-fold). The blunting of the response of the rPRL promoter at high
Pit-1 doses in the presence of PKAß is a novel observation. Indeed,
previous reports have shown that increasing Pit-1 DNA input results in
a consistent dose-dependent increase in basal rPRL promoter activity,
even at 10-fold higher Pit-1 DNA inputs (15). Furthermore, no such
blunting of the response to Pit-1ß by PKAß has been observed, at
these or even at 10-fold higher Pit-1ß DNA doses (15). These two
points indicate that the blunting of the PKA response at the highest
Pit-1 DNA dose is not simply due to squelching.
It is possible that PKAß might affect the transcriptional
potency of Pit-1 or Pit-1ß by altering Pit-1/Pit-1ß protein
expression. To rule this out, we performed Western blot analysis on
extracts from HeLa cells transfected with combinations of Pit-1,
Pit-1ß, and PKAß. We first attempted to visualize Pit-1 isoforms
with a rabbit polyclonal anti-Pit-1 antibody, but found that it lacked
sufficient sensitivity to detect Pit-1ß protein. To increase the
sensitivity of the Western blot assay, Pit-1 and Pit-1ß were
N-terminally tagged with the influenza hemagglutinin (HA) epitope, to
allow use of the more sensitive monoclonal anti-HA antibody. The HA tag
has no effect on the ability of Pit-1 or Pit-1ß to activate rPRL
promoter, alone or with known cofactors (15). HA-Pit-1 and HA-Pit-1ß
were not detected in mock-transfected HeLa cells (Fig. 2C
, lane 1) or
in HeLa cells transfected with pRSV-PKAß only (Fig. 2C
, lane 2).
Easily detectable and constant levels of HA-tagged Pit-1 protein were
found in HeLa cells transfected with pRSV-HA-Pit-1 without or with pRSV
PKAß (Fig. 2C
, lanes 3 and 4), and lower, but still constant, levels
of HA-tagged Pit-1ß protein were detected in HeLa cells transfected
with pRSV-HA-Pit-1ß without or with pRSV PKAß (Fig. 2C
, lanes 5 and
6). The blot was reprobed with a monoclonal antiactin antibody to
demonstrate that all lanes were loaded with equal amounts of cell
lysate (Fig. 2C
). These data demonstrate that while Pit-1ß protein is
indeed expressed at a lower level per unit input DNA than is Pit-1, the
addition of PKAß does not affect these protein expression levels.
Taken together, the data presented in Fig. 2
reveal that Pit-1ß is a
superior transducer of PKA signaling, reaching levels of induction
above those reached by Pit-1 at any DNA dose. Moreover, the observation
that Pit-1ß protein is expressed at lower levels per unit DNA input
than is Pit-1 would indicate that the PKAß response may be
underestimated for this isoform, since the PKAß-fold was not
normalized to the amount of HA Pit-1 protein expressed.
This laboratory has shown that it is, in fact, the AA sequence of the
ß-domain that determines the unique properties of Pit-1ß (15). Here
we have shown that Pit-1ß is a superior transducer of the PKA signal
and thus implicated the ß-domain as one domain of Pit-1 intrinsic PKA
signal-transducing properties. However, the ability of the Pit-1
isoform to transduce the PKA signal means that there are other
domain(s), shared by both isoforms, with intrinsic PKA-modulating
properties. For example, the TAD alone is sufficient, when fused to the
LexA DNA-binding domain (DBD), to mediate the dopamine
repression of a target promoter through a reduction of PKA signaling
(37).
The POU-Specific and POU-Homeo Domains of Pit-1 Are Required for
PKA Signaling
To identify the shared region(s) of Pit-1 and Pit-1ß that are
required for efficient transduction of the PKA signal to the rPRL
promoter, wild-type and internally deleted versions of Pit-1 were
introduced into HeLa nonpituitary cells by electroporation in the
presence or absence of PKAß and assessed for basal transcriptional
potency, for the ability to mediate PKA signaling (Fig. 3A
), and for protein expression level
(Fig. 3B
). Specific DNA doses that result in equivalent Pit-1 protein
levels were determined for each construct, as in previous studies (Fig. 3B
) (15). However, four constructs,
245,
4873,
209252, and
255291, failed to yield equivalent protein
expression regardless of DNA input.

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Figure 3. Deletion Mapping of Functionally Important Regions
of Pit-1
A, Effects of internal deletions on Pit-1 transcription potency.
Combinations of pA3PRLluc-425 (3 µg), pRSV-PKAß (10 µg), Pit-1 (3
µg), Pit-1 245 (15 µg), Pit-1 4873 (15 µg),
Pit-1 72125 (2 µg), Pit-1 124201 (3 µg), Pit-1 178201
(10 µg), Pit-1 200211 (3 µg), Pit-1 209252 (10 µg), and
Pit-1 255291 (10 µg) were introduced into HeLa nonpituitary cells
by electroporation. Specific DNA doses that result in essentially
equivalent Pit-1 protein levels were determined for each construct as
in previous studies (15 ). pRSVß-globin was added to keep the total
amount of RSV promoter-containing DNA constant. Cells were harvested
after 48 h, total light units measured, and Pit-1 potency
expressed as percent of wild-type (100% = 6-fold); PKA response was
calculated as described previously. The domain structure of
Pit-1 is shown at the top. The TAD is represented by the
solid black box; the DNA-binding domains, the
POU-specific (POU), and the POU-homeo (HOMEO) are repesented by the
hatched boxes, and a region with several negatively
charged residues is shown by the checked box. The
regions deleted by the various Pit-1 internal deletions are indicated
in the schematic diagram, and the AA end points of the
deleted areas are shown to the right. These data are
expressed as the mean ± SD of a representative
experiment of three experiments done in duplicate. B, Western analysis
of Pit-1 deletion mutants expressed in HeLa cells. Lanes were loaded
with equal protein (100 µg) from extracts of HeLa cells transfected
as follows: 3 µg of pA3PRLluc-425 (lane 1); 10 µg of pRSV-PKAß
(lane 2); 3 µg of pRSV-Pit-1 (lane 3); 15 µg of Pit-1 245 (lane
4); 15 µg of Pit-1 4873 (lane 5); 2 µg of Pit-1 72125 (lane
6); 3 µg of Pit-1 124201 (lane 7); 10 µg of Pit-1 178201
(lane 8); 3 µg of Pit-1 200211 (lane 9); 10 µg of
Pit-1 209252 (lane 10); 10 µg of Pit-1 255291 (lane 11); GH3
pituitary cell extract (lane 12); and RSET-Pit-1 (a bacterially
produced Histidine-tagged version of Pit-1) (lane 13). The blot was
probed with a rabbit polyclonal anti-Pit-1 antibody specific for AA
3652 and 214230 (BAbCO).
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In Fig. 3A
, the basal transcription potency of
each internally deleted Pit-1 construct is shown. The basal
transcription potency of full-length Pit-1, which activated rPRL
promoter activity 6-fold, was normalized to 100% (Fig. 3A
). Two
internal deletion constructs,
245 and
4873, which remove
portions of the TAD, displayed 3-fold reductions in transcription
potency. However, under these experimental conditions, their levels of
protein expression were also lower than wild type (Fig. 3B
). Internal
deletion construct
72125, which removes the hinge region between
the TAD and POU-specific domain, displayed a greater than 10-fold
increase in basal transcription potency, but also a higher level of
protein expression. Two internal deletion constructs,
124201 and
178201, which remove portions of the POU-specific domain,
displayed 2- to 3-fold reductions in basal transcription potency, yet
were expressed at higher protein levels than wild type. Also, internal
deletion
200211, which deletes a region between the POU-specific
and POU-homeo domains, displayed basal transcription potency similar to
wild type, but was expressed at higher levels than wild type. Two
internal deletion constructs,
209252 and
255291, which
remove portions of the POU-homeo domain, displayed lower levels of
basal transcription potency and protein expression. Because differences
of basal transcription potency not corresponding to equivalent
differences of protein expression level are meaningful, the negative
effects of internal deletion constructs,
124201 and
178201,
which remove the POU-specific domain, and
200211, which removes a
region between the POU-specific and POU-homeo domains, on basal
transcription potency are significant. The negative effects of the
POU-homeo domain deletions,
209252 and
255291, while
accompanied by a decreased protein expression level, may also be due,
in part, to loss of DNA-binding function of the homeo domain.
Nevertheless, the pattern of basal expression by these deletion
constructs is consistent with that noted previously (4).
The PKA responsiveness of each internally deleted Pit-1 construct is
shown in Fig. 3A
. This responsiveness is derived from the ratio of fold
activation with and without PKAß and, thus, takes into account the
protein expression level of each construct. PKAß increased the
transcription potency of full-length Pit-1 by about 2-fold. The
TAD-specific internal deletion constructs,
245 and
4873,
displayed an increased response to PKA (4- to 5-fold). The
hinge-specific internal deletion construct,
72125, was as
responsive as wild type to PKA. The two POU-specific internal deletion
constructs,
124201 and
178201, were insensitive to PKA
(1.5- and 1.1-fold, respectively), while the
200211 construct was
more responsive to PKA than wild type (4.3-fold). The two POU-homeo
domain-specific internal deletion constructs,
209252 and
255291, were relatively insensitive to PKA signaling. Because the
level of protein expression of Pit-1 is not affected by PKA (Fig. 2D
),
any decreased sensitivity to PKAß is significant. Thus, loss of the C
terminus (or all) of the POU-specific domain reduces the ability of
Pit-1 to transduce the PKA signal, as does loss of the POU-homeo
domain. Of note, deletion of either half of the TAD or of the region
between the POU-specific and POU-homeo domains increased responsiveness
to PKA signaling.
TEF Is Not the Pit-1/PKA Coactivator
Pit-1, although an important transducer of PKA signaling, is not a
direct nuclear target of PKA (32, 33). Thus, a FP I- or
Pit-1-associated coactivator is likely to be the actual substrate of
PKA (25, 30, 31, 34). TEF and Oct-1, both of which bind to FP I (35, 36), seemed likely candidates for a PKA-signaling coactivator.
To determine whether TEF acts as a Pit-1 coactivator to transduce the
PKA signal to FP I, combinations of pRSV-Pit-1, pRSV-Pit-1ß,
pRSV-FLAG-TEF, and pRSV PKAß were introduced into HeLa nonpituitary
cells by electroporation and assessed for the ability to mediate PKA
signaling (Fig. 4A
). In these
experiments, PKAß alone stimulated rPRL promoter activity 5-fold. TEF
further increased the stimulation of rPRL promoter activity to 17-fold.
Pit-1 and Pit-1ß increased rPRL promoter activity to 33- and 96-fold,
respectively. TEF and either Pit-1 or Pit-1ß worked in a merely
additive manner; the combination of TEF and Pit-1 stimulated promoter
activity to 79-fold, and the combination of TEF and Pit-1ß stimulated
promoter activity to 163-fold. Western blot analysis of HeLa cell
extracts with a mouse monoclonal anti-FLAG antibody shows that neither
PKA nor Pit-1 or Pit-1ß alter the levels of FLAG-tagged TEF
expression (Fig. 4B
). These data show no evidence of synergy between
TEF and Pit-1 or Pit-1ß in mediating the PKA signal and thus do not
support a role for TEF as a Pit-1/PKA coactivator.

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Figure 4. TEF Has Little Effect on the PKA Response
A, Effects of TEF on PKA-activated rPRL promoter activity in
HeLa. Plasmid pA3PRLluc-425 (3 µg) and combinations of pRSV-PKAß
(10 µg), pCMV-FLAG-hTEF (0.05 µg), pRSV-Pit-1 (3 µg), and
pRSV-Pit-1ß (3 µg) were introduced into HeLa nonpituitary cells and
harvested as described in Materials and Methods. Fold
activation of basal rPRL promoter activity was calculated by dividing
the mean RLU obtained in the presence of exogenous transcription
factors by the mean RLU obtained in the absence of exogenous
transcription factors. These data represent the mean ±
SD of two experiments performed in triplicate. B, Western
analysis of TEF expression in HeLa cells. Lanes were loaded with equal
protein (100 µg) from extracts of HeLa cells transfected as follows:
3 µg of pA3PRLluc-425 (lane 1); 3 µg of pA3PRLluc-425 and 10 µg
of pRSV-PKAß (lane 2); 3 µg of pA3PRLluc-425 and 3 µg of
pCMV-FLAG-hTEF (lane 3); 3 µg of pA3PRLluc-425, 3 µg of
pCMV-FLAG-hTEF, and 10 µg of pRSV-PKAß (lane 4); 3 µg of
pA3PRLluc-425, 3 µg of pCMV-FLAG-hTEF, and 3 µg of pRSV-Pit-1 (lane
5); 3 µg of pA3PRLluc-425, 3 µg of pCMV-FLAG-hTEF, and 3 µg of
pRSV-Pit-1ß (lane 6). Total plasmid amount was maintained constant
with pRSVß-globin DNA. The blot was probed with mouse monoclonal
anti-FLAG antibody as described in Materials and
Methods.
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Oct-1 Is a Pit-1/PKA Coactivator
To determine whether Oct-1 acts as a Pit-1 coactivator to
transduce the PKA signal to FP I, combinations of pRSV-Pit-1,
pRSV-Pit-1ß, pRSV-HA-Oct-1, and pRSV-PKAß were introduced into HeLa
nonpituitary cells by electroporation with pA3425 rPRL Luc and
assessed for ability to mediate PKA signaling (Fig. 5A
) and basal transcriptional potency
(Fig. 5B
). In these experiments, PKAß alone stimulated rPRL promoter
activity 5-fold. Oct-1 further increased the stimulation of rPRL
promoter activity to 38-fold. Pit-1 and Pit-1ß increased rPRL
promoter activity to 33- and 96-fold, respectively. Oct-1 and either
Pit-1 or Pit-1ß worked in a synergistic manner; the combination of
Oct-1 and Pit-1 stimulated promoter activity to 449-fold, and the
combination of Oct-1 and Pit-1 stimulated promoter activity to
458-fold.

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Figure 5. Synergistic Interaction between Oct-1 and Pit-1 in
PKA Signaling
A, Effects of Oct-1 on basal rPRL promoter activity in HeLa cells.
Plasmid pA3PRLluc-425 (3 µg) and combinations of pCGN-Oct-1 (0.05
µg), pRSV-Pit-1 (3 µg), and pRSV-Pit-1ß (3 µg) were introduced
into HeLa nonpituitary cells and harvested as described in
Materials and Methods. Fold activation of basal rPRL
promoter activity was calculated by dividing the mean RLU obtained in
the presence of exogenous transcription factors by the mean RLU
obtained in the absence of exogenous transcription factors. These data
represent the mean ± SD of two experiments
performed in triplicate. B, Effects of Oct-1 on PKA-activated rPRL
promoter activity in HeLa cells. Plasmid pA3PRLluc-425 (3 µg) and
combinations of pRSV-PKAß (10 µg), pCGN-Oct-1 (0.05 µg),
pRSV-Pit-1 (3 µg), and pRSV-Pit-1ß (3 µg) were introduced into
HeLa nonpituitary cells by electroporation. Fold activation of basal
rPRL promoter activity was calculated by dividing the mean RLU obtained
in the presence of PKA by the mean RLU obtained in the absence of PKA.
C, Western analysis of Oct-1 expression in HeLa cells. Lanes were
loaded with equal protein (100 µg) from extracts of HeLa cells
transfected as follows: 3 µg of pA3PRLluc-425 (lane 1); 3 µg of
pA3PRLluc-425 and 10 µg of pRSV-PKAß (lane 2); 3 µg of
pA3PRLluc-425 and 3 µg of pCGN-HA-Oct-1 (lane 3); 3 µg of
pA3PRLluc-425, 3 µg of pCGN-HA-Oct-1, and 10 µg of pRSV-PKAß
(lane 4); 3 µg of pA3PRLluc-425, 3 µg of pCGN-HA-Oct-1, and 3 µg
of pRSV-Pit-1 (lane 5); 3 µg of pA3PRLluc-425, 3 µg of
pCGN-HA-Oct-1, and 3 µg of pRSV-Pit-1ß (lane 6). Total plasmid
amount was maintained constant with pRSVß-globin DNA. The blot was
probed with mouse monoclonal anti-HA antibody as described in
Materials and Methods.
|
|
By contrast, without PKA, Oct-1 synergized with Pit-1 and Pit-1ß in a
much more modest manner. Oct-1 alone increased basal rPRL promoter
activity 1.5-fold (Fig. 5B
). Pit-1 increased rPRL promoter activity
3-fold, while Pit-1ß had little effect on promoter activity. Oct-1
and Pit-1 or Pit-1ß synergized to 8- and 2.5-fold, respectively.
These results are consistent with previous data showing that Oct-1 and
Pit-1 synergistically activate rPRL promoter activity (38). However, we
see a much stronger synergy in the presence of PKA. Western blot
analysis of HeLa cell extracts with an anti-HA antibody shows that
neither PKAß nor Pit-1 or Pit-1ß alter the levels of HA-Oct-1
expression (Fig. 5C
). Thus, Oct-1 is able to functionally interact with
Pit-1 and Pit-1ß to reconstitute the PKA response in HeLa cells. In
fact, given the lower level of Pit-1ß protein expression relative to
Pit-1, and the lower level of basal Oct-1:Pit-1ß synergy, the
equivalent levels of Oct-1:Pit-1 and Oct-1:Pit-1ß synergy in the
presence of PKA suggest a stronger synergy between Oct-1 and Pit-1ß
than between Oct-1 and Pit-1.
 |
DISCUSSION
|
---|
cAMP- and PKA-dependent signaling to the rPRL promoter is unusual
both for its lack of involvement of CREB and for its requirement for
Pit-1 to work through the most proximal binding site, FP I (25, 30, 31). In this paper, we show 1) that Pit-1ß is a more efficient
transducer than is Pit-1 of the PKA signal; 2) that in addition to the
ß-domain, the POU-specific and POU-homeo domain are required for
optimal PKA-signaling; and 3) that Oct-1 is a PKA coactivator.
The importance of Pit-1ß as a nuclear integrator of signals
regulating pituitary hormone gene expression is becoming increasingly
clear (15, 39). Figure 2
demonstrates that Pit-1ß not only is capable
of transducing the PKA signaling to the rPRL promoter, but acts more
efficiently than does Pit-1 (Fig. 2A
) despite lower levels of protein
expression (Fig. 2B
). While it might be argued that this differential
effect of Pit-1ß vs. Pit-1 might be due to the different
levels of protein expressed in this study, we have previously reported
that with equal levels of Pit-1ß and Pit-1 protein expressed,
Pit-1ß clearly shows an enhanced PKA response compared with Pit-1
(15). This increased efficiency of PKA signaling is, like the other
effects of the ß-domain, dependent on its precise AA sequence (15).
The ß-domain is richer in serine and threonine residues than the
surrounding region, and the threonine at site 57 of Pit-1ß is
possibly a protein kinase C phosphorylation site (10). Thus, the
ß-domain contains AA acid residues directly involved in
PKA-signaling. That Pit-1 is also able to transduce the PKA signal
shows that the ß-domain is not absolutely required for signal
transduction, but rather acts to regulate it. The differential
abilities of Pit-1 and Pit-1ß to mediate signaling by the Ras and PKA
pathways may indicate that these two isoforms serve to integrate
information from separate signaling pathways at the rPRL promoter
(15).
The reason for the drop in activation at the high dose of Pit-1
in the presence of PKA is not clear. That this effect is seen only with
the Pit-1 isoform, but not with the Pit-1ß isoform and only in the
presence of PKA, suggests that there is a "titratable" component
that interacts with Pit-1 but not with Pit-1ß, and that the
interaction is PKA dependent, and not simply squelching.
To identify Pit-1 domains common to both Pit-1 and Pit-1ß that are
required for efficient PKA signaling, we examined basal transcription
potency and ability to transduce PKA signaling for intact Pit-1 and
internal deletion mutations of Pit-1. The ß-domain insertion, which
enhances PKA signaling, falls in the middle of the TAD. It is possible
that the ß-domain eliminates an inhibitory region while inserting a
stimulatory region. This is consistent with our data, shown in Fig. 3
, whereby removal of either half of the TAD may increase PKA signaling.
Of note, the TAD has been implicated in transducing the dopamine
repression of PKA signaling (37), suggesting that this repressive
function may be inherent to the TAD. Analysis of the other internal
deletion mutant Pit-1 constructs indicate that the regions required for
PKA signaling, as well as basal transcription potency, map to the
POU-specific and POU-homeo domains, and the carboxyl-terminal region of
each may be necessary for these effects. The precise role of the TAD in
PKA signaling is less clear. One interpretation of the data is that the
TAD and the linker between the two POU domains may function as
inhibitory domains with regard to PKA signaling (Fig. 3
). That deletion
of the POU-specific and POU-homeo domains eliminates transduction of
the PKA signal is consistent with an absolute requirement for the
DNA-binding functions of Pit-1 and with the fact that Pit-1-Oct-1
interactions are mediated through these domains (38, 40).
While Pit-1 is absolutely required for PKA stimulation of the rPRL
promoter, it appears not to be a direct PKA target. Pit-1 can be
phosphorylated in intact cells or in vitro by protein
kinases (41, 42). However, two recent reports have shown that
phosphorylation of Pit-1 is not essential in mediating the PKA response
(32, 33). The lack of correlation between PKA or protein kinase C
phosphorylation of Pit-1 and rPRL activation, as well as the
PKA-independent activation of rPRL promoter, strongly suggests the
involvement of a coactivator in mediating the PKA/Pit-1 activation of
the rPRL promoter.
Pit-1 has been shown to functionally interact, via FP
I, with several transcription factors, including TEF and Oct-1, a
ubiquitous homeobox protein that is found in lactotrophs, to activate
basal rPRL promoter activity (35, 36). Since the PKA effect maps to FP
I, we were led to directly test the ability of TEF and Oct-1 to mediate
PKA signaling to the rPRL promoter. The failure of TEF to synergize
with Pit-1 or Pit-1ß in the presence of PKA implies that TEF is not
the Pit-1/PKA coactivator and that not all FP I-binding factors can
function as such. However, the ability of Oct-1 to synergize with Pit-1
and Pit-1ß, either to activate basal rPRL expression (Fig. 5B
) (38)
or to transduce the PKA signal (Fig. 5A
), demonstrates that Oct-1 is a
Pit-1/PKA coactivator. Thus, the Pit-1-Oct-1-PKA interaction is a
selective one.
 |
MATERIALS AND METHODS
|
---|
Tissue Culture
Monolayer cultures of HeLa human cervical carcinoma cells were
kindly provided by Dr. Kathryn Horwitz (University of Colorado Health
Sciences Center). Cells were maintained in DMEM, with 10% FCS (Figs. 2
and 3
) and 12.5% horse serum and 2.5% FCS (Figs. 4
and 5
), and 50
µg/ml of penicillin and streptomycin. Cells were maintained at 37 C
in 5% CO2, and the medium was changed no more than 16
h before electroporation. Cells used for transfections were harvested
at approximately 7080% confluence using 0.05% trypsin and 0.5
mM EDTA.
Plasmids
The pA3 -425 rPRL Luc reporter construct contains a 498-bp
fragment spanning positions -425 to +73 of the rPRL gene ligated
upstream to the firefly luciferase reporter gene and downstream of
three polyadenylation sites in pA3luc (20, 43, 44, 45, 46, 47). The plasmids coding
for Escherichia coli ß-galactosidase under the control of
the human cytomegalovirus immediate early promoter (pCMVß-gal) (48, 49) (CLONTECH, Palo Alto, CA) or the Simian virus-40 early promoter
(pSVß-gal) (21) were included in transfections to control for
transfection efficiency. The pRSV-PKAß plasmid encoding the
ß-isoform of the PKA catalytic subunit derived from Chinese hamster
ovary (CHO) cells was kindly provided by Dr. R. A. Maurer
(University of Oregon Health Science Center, Portland, OR). Plasmid
pRSV-Pit-1 and pRSV-Pit-1ß were generously provided by Dr. M. Karin
(University ofCalifornia, San Diego. CA) (4, 31). Plasmid
pCGN-HA-Oct-1 was a generous gift of Dr. Winship Herr (Cold Spring
Harbor, NY). Plasmid pCMV-FLAG-hTEF was a generous gift of Dr. Hunger
(University of Colorado Health Sciences Center).
The vectors pRSV-HA-Pit-1 and pRSV-HA-Pit-1ß, which encode N-terminal
influenza HA-tagged Pit-1/Pit-1ß, were constructed through PCR
mutagenesis of the Pit-1 TAD. Wild-type pRSV Pit-1/ß plasmids were
used as substrates for PCR (50), in which an HA tag was added to the
amino terminus of the Pit-1/Pit-1ß TAD by its inclusion in the
5'-oligonucleotide primer. To minimize the target sequence to be
submitted to PCR amplification, a HindIII-PpuM I
fragment encompassing nucleotides 1337 of Pit-1 or 1415 of Pit-1ß
from each PCR product was subcloned into a derivatized pGem-7Z
(Promega, Madison, WI) plasmid DNA whose SacI site had been
converted to a PpuM I site (pGem7P). Commercially
synthesized deoxyoligonucleotides (Macromolecular Resources, Fort
Collins, CO; and GIBCO/BRL, Grand Island, NY) contained the following
sequences:
5'-TAD: AAA AAG CAA GCT TCC ATG GGG TAC CCA TAC GAT GTT CCG GAT TAC GCT
AGT TGC AAC CTT TC; and
3'-TAD: GTT TGT CTG GGT GTA TC.
The presence of each introduced HA-tagged Pit-1/Pit-1ß fragment in
pGEM-7Z was tested by digestion with restriction enzymes and verified
by Sanger sequencing using reagents and protocols obtained from a
commercial kit (Sequenase; United States Biochemical Corp. Cleveland,
OH), and commercially available T7 and SP6 promoter-specific primers
(Promega; Madison, WI). Sequencing was done both in our
laboratory and through the UCHSC Cancer Center Core facility.
HA-tagged Pit-1 and Pit-1ß were excised from pGem-7P by digestion
with HindIII and PpuM I and ligated to the unique
HindIII and PpuM I sites of pRSV-Pit-1 to produce
pRSV-HA Pit-1 and pRSV-HA Pit-1ß.
Plasmid DNAs were prepared by passage over an anion exchange column
(QIAGEN, Inc., Chatsworth, CA) and quantitated by absorbance at 260 nm
or on a Dynaquant fluorimeter and by comparison with DNA standards on
agarose gels.
Transfections
DNA was introduced into HeLa cells by electroporation as
follows: approximately 23 x 106 enzymatically
dispersed cells were mixed with plasmid DNA in a sterile gene-pulse
chamber and exposed to a controlled electrical field of 500 µfarads
at 220 V, as described previously (24). Cells from individual
transfections were then maintained in DMEM, 10% FCS, and 50 µg/ml of
penicillin and streptomycin at 37 C. The nonspecific effects of the
rous sarcoma virus (RSV) promoter upon transcription factor
availability was controlled for by including amounts of pRSV ß-globin
plasmid DNA in all assays to render the total pRSV DNA concentration
constant.
Luciferase Assays
Transient transfections were performed in duplicate or
triplicate, in at least two separate experiments. After incubation for
48 h, cells were harvested with PBS containing 3 mM
EDTA, pelleted, and resuspended in 100 mM potassium
phosphate buffer (pH 7.8) 1 mM dithiothreitol. Cells were
lysed by three cycles of freeze-thawing and by vortexing for 1 min
between thaws. Cell debris was pelleted by centrifugation for 10 min at
10,000 x g at 4 C, and the supernatant was used for
subsequent assays. Luciferase activity in the supernatant was assayed
as previously described (21). Samples were measured in duplicate using
a Monolight 2010 Luminometer (Analytical Luminescence Laboratories, San
Diego, CA). Total luciferase units were normalized to
ß-galactosidase activity levels driven by the internal control SV40
promoter and assayed as described (Figs. 2
and 3
) (48, 49) or
normalized to total protein in the extract (Figs. 4
and 5
) where
protein assays were performed according to the method of Bradford (51)
using commercially available reagents (Bio-Rad, Richmond, CA).
Visualization of Proteins
Transient transfections were performed in duplicate. For Fig. 3
, HeLa cells transfected with plasmid DNAs were harvested after 24 h
incubation with PBS containing 3 mM EDTA, pelleted, and
resuspended in 100 mM potassium phosphate buffer (pH 7.8)-1
mM dithiothreitol. Cells were lysed by three cycles of
freeze-thawing and by vortexing for 1 min between thaws. Cell debris
was pelleted by centrifugation for 10 min at 10,000 x
g at 4 C, and the supernatant and pellet were separated. The
protein content of each lysis supernatant was assayed according to the
method of Lowry et al. (52), using commercially available
reagents (Bio-Rad). Pellets were then combined with equal amounts of
lysis supernatant (75 µg) and used for SDS-PAGE separation.
For Figs. 2
, 4
, and 5
, HeLa cells transfected with plasmid DNAs
were harvested after 24 h with PBS containing 3 mM
EDTA, pelleted, and resuspended in a TEA-SDS solubilization buffer (55
mM triethanolamine, 111 mM NaCl, 2.2
mM EDTA, and 0.44% SDS) (53) and a mix of protein
inhibitors (leupeptin, pepstatin A, chymostatin, aprotinin, antipain,
and bestatin, each at 6 ng/mL) at 4 C. Lysed extracts were passed
through a 25 G needle seven times. The protein content of each extract
was assayed according to the method of Lowry et al. (52),
using commercially available reagents (Bio-Rad). Equal amounts of
protein (100 µg) were used for SDS-PAGE separation.
Samples were separated on 10% SDS polyacrylamide gels and transferred
to Immobilon-P polyvinylidene fluoride membrane (Millipore,
Bedford, MA). The proteins of interest were visualized with appropriate
antibodies and dilutions, and enhanced chemiluminescence (ECL) media
(Amersham Life Sciences, Arlington Heights, IL). To demonstrate equal
loading of protein from each sample, blots were stripped with a
solution containing 100 mM ß-mercaptoethanol, 62.5
mM Tris-HCl (pH 6.8), and 2% SDS at 50 C for 30 min, and
actin was visualized with a mouse monoclonal antiactin
(Boehringer-Mannheim Corp., Indianapolis, IN) diluted 1:1000 and
secondary sheep antimouse horseradish peroxidase (HRP)-conjugated
antibodies (Amersham Life Sciences) diluted 1:10,000.
HA-tagged Pit-1 and Pit-1ß (Fig. 2D
), as well as HA-tagged
Oct-1 (Fig. 5
), were visualized with a mouse monoclonal anti-actin
(Boehringer-Mannheim Corp.) diluted 1:1000 and secondary sheep
antimouse HRP-conjugated antibodies (Amersham Life Sciences) diluted
1:10,000. Wild-type and internally deleted Pit-1 (Fig. 3
) were
visualized with a rabbit polyclonal anti-Pit-1 antibody specific for AA
3652 and 214230 (BAbCO, Richmond, CA) diluted 1:1000 and secondary
goat antirabbit HRP-conjugated antibodies (Amersham Life Sciences)
diluted 1:10,000. FLAG-tagged-TEF (Fig. 4
) was visualized with mouse
monoclonal anti-FLAG M2 antibody at 10 µg/ml (Kodak Scientific
Imaging Systems-IBI FLAG System, New Haven, CT) and secondary
sheep antimouse HRP-conjugated antibodies (Amersham Life Sciences)
diluted 1:10,000.
 |
ACKNOWLEDGMENTS
|
---|
We thank Kelley Fantle, Nicole Manning, Deirdre
Cooper-Blacketer, and Jeanette Wagner for technical assistance, and
members of the Gutierrez-Hartmann laboratory for their helpful
suggestions and comments. We also thank Andrew Bradford, John Tentler,
Dana Manning, and Phil Zeitler for critical reading and discussions of
this manuscript. Tissue culture media were prepared by the Tissue
Culture Core Facility of the Colorado Cancer Center.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Arthur Gutierrez-Hartmann, Department of Medicine, University of Colorado Health Science Center, 4200 East Ninth Avenue B-151, Denver, Colorado 80262. Email: a.gutierrezhartmann@UCHSC.edu.
This research was supported by NIH Grant DK-37667. S.E.D. was
supported by a Colorado Cancer League Postdoctoral Fellowship, National
Research Service Award F32 DK-09160, and a Postdoctoral Fellowship from
the Lalor Foundation.
Received for publication May 11, 1998.
Revision received October 14, 1998.
Accepted for publication October 15, 1998.
 |
REFERENCES
|
---|
-
Dolle P, Castrillo JL, Theill LE, Deerinck T,
Ellisman M, Karin M 1990 Expression of GHF-1 protein in mouse
pituitaries correlates both temporally and spatially with the onset of
growth hormone gene activity. Cell 60:809820[Medline]
-
Simmons DM, Voss JW, Ingraham HA, Holloway JM,
Broide RS, Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes
involve cell-specific Pit-1 mRNA translation and synergistic
interactions with other classes of transcription factors. Genes Dev 4:695711[Abstract]
-
Ryan AK, Rosenfeld MG 1997 POU domain family
values: flexibility, partnerships and developmental codes. Genes Dev 11:12071225[CrossRef][Medline]
-
Theill LE, Castrillo JL, Wu D, Karin M 1989 Dissection of functional domains of the pituitary-specific
transcription factor GHF-1. Nature 342:945948[CrossRef][Medline]
-
Ingraham HA, Flynn SE, Voss JW, Albert VR, Kapiloff
MS, Wilson L, Rosenfeld MG 1990 The POU-specific domain of Pit-1 is
essential for sequence-specific, high affinity DNA binding and
DNA-dependent Pit-1-Pit-1 interactions. Cell 61:10211033[Medline]
-
Bodner M, Karin, M 1987 A pituitary-specific
trans-acting factor can stimulate transcription from the
growth hormone promoter in extracts of nonexpressing cells. Cell 50:267275[Medline]
-
Ingraham HA, Chen RP, Mangalam HJ, Elsholtz
HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A
tissue-specific transcription factor containing a homeo domain
specifies a pituitary phenotype. Cell 55:519529[Medline]
-
Herr W, Sturm RA, Clerc RG, Corcoran LM, Baltimore
D, Sharp PA, Ingraham HA, Rosenfeld MG, Finney M, Ruvkun G, Horvitz
HR 1988 The POU domain: a large conserved region in the mammalian
pit-1, oct-1, oct-2, and
Caenorhabditis elegans unc-86 gene products. Genes Dev 2:15131516[CrossRef][Medline]
-
Theill LE, Hattori K, Domenico D, Castrillo JL,
Karin M 1992 Differential splicing of the GHF1 primary transcript gives
rise to two functionally distinct homeo domain proteins. EMBO J 11:22612269[Abstract]
-
Konzak KE, Moore DD 1992 Functional isoforms of
Pit-1 generated by alternative mRNA splicing. Mol Endocrinol 6:241247[Abstract]
-
Morris AE, Kloss B, McChesney RE, Bancroft C, Chasin
LA 1992 An alternatively spliced Pit-1 isoform altered in its ability
to trans-activate. Nucleic Acids Res 20:13551361[Abstract]
-
Delhase M, Vila V, Hooghe-Peters EL, Castrillo JL 1995 A novel pituitary transcription factor is produced by alternative
splicing of the human GHF-1/Pit-1 gene. Gene 155:273275[CrossRef][Medline]
-
Ono M, Takayama Y 1992 Structures of cDNAs encoding
chum salmon pituitary-specific transcription factor, Pit-1/GHF-1. Gene 116:275279[CrossRef][Medline]
-
Wong EA, Silsby JL, El Halawani ME 1992 Complementary DNA cloning and expression of Pit-1/GHF-1 from the
domestic turkey. DNA Cell Biol 11:651660[Medline]
-
Diamond SE, Gutierrez-Hartmann A 1996 A 26-amino
acid insertion domain defines a functional transcription switch motif
in Pit-1ß. J Biol Chem 271:2892528932[Abstract/Free Full Text]
-
Bradford A, Conrad K, Tran P, Ostrowski M,
Gutierrez-Hartmann A 1996 GHF-1/Pit-1 functions as a cell specific
integrator of Ras signaling by targeting the Ras pathway to a composite
Ets-1/GHF-1 response element. J Biol Chem 271:2463924648[Abstract/Free Full Text]
-
Haugen BR, Wood WM, Gordon DF, Ridgway EC 1993 A
thyrotroph-specific variant of Pit-1 transactivates TSHß. J Biol
Chem 268:2081820824[Abstract/Free Full Text]
-
Maurer RA 1981 Transcriptional regulation of the
prolactin gene by ergocryptine and cyclic AMP. Nature 294:9497[Medline]
-
Elsholtz HP, Lew AM, Albert PR, Sundmark VC 1991 Inhibitory control of prolactin and Pit-1 gene promoters by dopamine.
J Biol Chem 266:2291922925[Abstract/Free Full Text]
-
Elsholtz HP 1992 Molecular biology of prolactin:
cell-specific and endocrine regulators of the prolactin gene. Semin
Reprod Endocrinol 10:183195
-
Conrad KE, Gutierrez-Hartmann A 1992 The
ras and protein kinase A pathways are mutually antagonistic
in regulating rat prolactin promoter activity. Oncogene 7:12791286[Medline]
-
Day RN, Walder JA, Maurer RA 1989 A protein kinase
inhibitor gene reduces both basal and multihormone-stimulated prolactin
gene transcription. J Biol Chem 264:431436[Abstract/Free Full Text]
-
Iverson RA, Day KH, dEmden M, Day RN, Maurer RA 1990 Clustered point mutation analysis of the rat prolactin promoter.
Mol Endocrinol 4:15641571[Abstract]
-
Keech CA, Gutierrez-Hartmann A 1989 Analysis
of rat prolactin promoter sequences that mediate pituitary-specific and
3',5'-cyclic adenosine monophosphate-regulated gene expression in
vivo. Mol Endocrinol 3:832839[Abstract]
-
Keech CA, Jackson SM, Siddiqui SK, Ocran KW,
Gutierrez-Hartmann A 1992 Cyclic AMP activation of the rat prolactin
promoter is restricted to the pituitary-specific cell type. Mol
Endocrinol 6:20592070[Abstract]
-
Sanyal S, van Tol HH 1997 Dopamine D4
receptor-mediated inhibition of cyclic adenosine 3',5'-monophosphate
production does not affect prolactin regulation. Endocrinology 138:18711878[Abstract/Free Full Text]
-
Lew AM, Yao H, Elsholtz HP 1994 G(i) alpha 2- and
G(o) alpha-mediated signaling inn the Pit-1-dependent inhibition of the
prolactin promoter. Control of transcription by dopamine recptors.
J Biol Chem 269:1200712013[Abstract/Free Full Text]
-
Fischberg DJ, Bancroft C 1995 The D2 receptor:
blocked transcription in GH3 cells and cellular pathways employed by
D2A to regulate prolactin promoter activity. Mol Cell Endocrinol 111:129137[CrossRef][Medline]
-
Meyer TE, Habener JF 1993 Cyclic adenosine
3',5'-monophosphate response element binding protein (CREB) and related
transcription-activating deoxyribonucleic acid-binding proteins. Endocr
Rev 14:269290[Medline]
-
Liang J, Kim KE, Schoderbeck WE, Maurer RA 1992 Characterization of a nontissue-specific, 3',5'-cyclic adenosine
monophosphate-responsive element of the proximal region of the rat
prolactin gene. Mol Endocrinol 6:885892[Abstract]
-
Rajnarayan S, Chiono M, Alexander LM,
Gutierrez-Hartmann A 1994 Reconstitution of protein kinase A regulation
of the rat prolactin promoter in HeLa nonpituitary cells:
identification of both GHF-1/Pit-1-dependent and -independent
mechanisms. Mol Endocrinol 9:502512[Abstract]
-
Okimura Y, Howard PW, Maurer RA 1994 Pit-1 binding
sites mediate transcriptional responses to cAMP through a mechanism
which does not require inducible phosphorylation of Pit-1. Mol
Endocrinol 8:15591565[Abstract]
-
Fischberg DJ, Chen X-H, Bancroft C 1994 A Pit-1
phosphorylation mutant can mediate either basal or induced prolactin or
growth hormone promoter activity. Mol Endocrinol 8:15661573[Abstract]
-
Gutierrez-Hartmann A 1994 Pit-1/GHF-1: a
pituitary-specific transcription factor linking general signaling
pathways to cell-specific gene expression. Mol Endocrinol 8:14471449[Medline]
-
Drolet DW, Scully KM, Simmons DM, Wegner M, Chu K,
Swanson LW, Rosenfeld MG 1991 TEF, a transcription factor expressed in
the anterior pituitary during embryogenesis, defines a new class of
leucine zipper proteins. Genes Dev 5:17391753[Abstract]
-
Voss JW, Rosenfeld MG 1992 Anterior pituitary
development: short tales from dwarf mice. Cell 70:527530[Medline]
-
Lew AM, Elsholtz HP 1995 A dopamine-responsive
domain in the N-terminal sequence of Pit-1. Transcriptional inhibition
in endocrine cell types. J Biol Chem 270:71567160[Abstract/Free Full Text]
-
Voss JW, Wilson L, Rosenfeld MG 1991 POU-domain
proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can
cooperate to induce exression of the prolactin promoter. Genes Dev 5:13091320[Abstract]
-
Sanchez-Pacheco A, Pena P, Palomino T, Guell A,
Castrillo JL, Aranda A 1998 The transcription factor GHF-1, but not
the splice variant GHF-2, cooperates with thyroid hormone and retinoic
acid receptors to stimulate rat growth hormone gene expression. FEBS
Lett 422:103107[CrossRef][Medline]
-
Verrijzer CP, van Oosterhout JA, van der
Vliet PC 1992 The Oct-1 POU domain mediates interactions between Oct-1
and other POU proteins. Mol Cell Biol 12:542551[Abstract]
-
Kapiloff MS, Farkash Y, Wegner M, Rosenfeld MG 1991 Variable effects of phosphorylation of Pit-1 dictated by the DNA
response elements. Science 253:786789[Medline]
-
Steinfelder HJ, Radovick S, Wondisford FE 1992 Hormonal regulation of the thyrotropin ß-subunit gene by
phosphorylation of the pituitary-specific transcription factor Pit-1.
Proc Natl Acad Sci USA 89:59425945[Abstract]
-
Farnsworth CL, Marshall MS, Gibbs JB, Stacey DW,
Feig LA 1991 Preferential inhibition of the oncogenic form of RasH by
mutations in the GAP binding/"effector" domain. Cell 64:625633[Medline]
-
Feig LA, Cooper GM 1988 Inhibition of NIH 3T3 cell
proliferation by a mutant Ras protein with preferential affinity for
GDP. Mol Cell Biol 8:32353243[Medline]
-
Feig LA, Cooper GM 1988 Relationship among guanine
nucleotide exchange, GTP hydrolysis, and transforming potential of
mutated Ras proteins. Mol Cell Biol 8:24722478[Medline]
-
Pellegrini S, Schindler C 1993 Early events in
signalling by interferons. Trends Biochem Sci 18:338342[CrossRef][Medline]
-
Satoh T, Endo M, Nakafuku M, Akiyama T, Yamamoto T,
Kaziro Y 1990 Accumulation of p21ras-GTP in response to stimulation
with epidermal growth factor and oncogene products with tyrosine kinase
activity. Proc Natl Acad Sci USA 87:79267929[Abstract]
-
MacGregor GR, Caskey CT 1989 Construction of
plasmids that express E. coli beta-galactosidase in
mammalian cells. Nucleic Acids Res 17:2365[Medline]
-
MacGregor GR, Nolan GP, Fiering S, Roederer M,
Herzenberg LA 1989 Use of E. coli lacZ
(ß-galactosidase) as a reporter gene. In: Murray EJ, Walker JM (eds)
Gene Expression in Vivo. Humana Press Inc., Clifton, NJ, vol 7:217236
-
Jarvis T, Kirkegaard K 1992 Poliovirus RNA
recombination: mechanistic studies in the absence of selection. EMBO J 11:31353145[Abstract]
-
Bradford M 1976 A rapid and sensitive method
for the quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
-
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the folin phenol reagent. J Biol
Chem 193:265275[Free Full Text]
-
Ottaviano Y, Gerace L 1985 Phosphorylation
of the nuclear lamins during interphase and mitosis. J Biol Chem 260:624632[Abstract/Free Full Text]