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Department of Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607-7170
Address all correspondence and requests for reprints to: Dr. Lester F. Lau, Department of Genetics, University of Illinois College of Medicine, 900 South Ashland Avenue, Chicago, Illinois 60607-7170. E-mail: LFLau{at}uic.edu
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Introduction |
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The promoter sequences required for ACTH-regulated CYP21
gene expression include the binding sites for nuclear receptors such as
Nur77 and steroidogenic factor 1 (SF-1) (3). Indeed, both Nur77 and
SF-1 are able to trans-activate the CYP21
promoter through these elements in cotransfection assays (2, 4).
Similar sequences are also present in the promoters of genes encoding
several other steroidogenic enzymes, including CYP11A1,
CYP11B1, CYP17 (17-
-hydroxylase), and
CYP19 (aromatase) (5, 6). These observations suggest that
transcription factors such as Nur77 and SF-1 may act as mediators of
ACTH-induced gene expression, resulting in increased
steroidogenesis.
Nur77 is of particular interest as a potential mediator of ACTH action, since the expression of its gene is rapidly and dramatically elevated upon ACTH treatment in both the adrenal gland and the adrenocortical tumor cell line Y1 (2, 7). In contrast, SF-1 appears to be constitutively expressed in the adrenal cortex and may not be subjected to inductive regulation (8). Nur77, a member of the steroid hormone receptor superfamily with no known ligand, is encoded by a growth factor-inducible immediate early gene (9, 10, 11, 12). Nur77 has been shown to bind DNA as a monomer to sequences consisting of a half-site estrogen response element preceded by two additional 5'-adenine nucleotides (AAAGGTCA) (13, 14). When heterodimerized to RXR, however, Nur77 binds a class of retinoid response elements composed of direct repeats separated by five nucleotides (15). SF-1 binds to the sequence element CAAGGTCA, which is similar to but distinct from the Nur77 response element (14), although SF-1 can also bind the Nur77 response element with lower affinity (4, 14).
Since Nur77 is capable of activating such genes as CYP21 (2), an attractive hypothesis holds that ACTH may induce the synthesis and activity of Nur77, which, in turn, activates the transcription of steroidogenic enzyme genes. The nur77 gene itself is induced within minutes in the adrenal cortex of rodents injected with ACTH, whereas the Nur77 protein accumulates to a peak level within 1–1.5 h after treatment (7). Although Nur77 appears to be an excellent candidate as a regulator of CYP21 expression, the regulation of CYP21 messenger RNA levels in nur77/NGFI-B-deficient mice appears normal (8). This finding suggests that there may be functional redundancies in the control of steroidogenic enzyme genes. For example, the Nur77-related transcription factor Nurr1 (Nur-related) has an expression pattern similar to that of Nur77 and trans-activates promoters through the identical DNA sequences (7, 16). Such functional redundancies may also explain the finding that although Nur77 function is required for T cell receptor-mediated apoptosis in culture (17, 18), Nur77-deficient mice undergo normal thymocyte and peripheral T cell deletion (19).
Several lines of evidence suggest that Nur77 activity may also be regulated through phosphorylation. In PC12 cells, NGF treatment and membrane depolarization cause divergent biological effects that result in differential phosphorylation of Nur77 (20). Nur77 is phosphorylated in vivo on multiple sites in the amino-terminus, which is primarily responsible for the trans-activation activity (21). The carboxyl-terminus, which may regulate Nur77 subcellular localization and trans-activation, is devoid of phosphorylation (21). In the central DNA-binding domain, Nur77 is phosphorylated at Ser354, a site within a region essential for sequence-specific DNA binding (21, 22). Phosphorylation in vitro of a bacterially expressed Nur77 fragment at Ser354 decreases DNA binding affinity (23). In addition, Nur77 exhibits different patterns of phosphorylation before and after ACTH treatment in Y1 cells (7). Taken together, these observations suggest that phosphorylation may play an important role in regulating Nur77 function. However, it is heretofore unclear whether this phosphorylation affects Nur77 activity in vivo and how this phosphorylation is regulated by ACTH.
Here we present in vivo evidence for the regulatory role of Nur77 phosphorylation, in particular phosphorylation at Ser354. We show that ACTH induces the synthesis of Nur77 that is unphosphorylated at Ser354, binds DNA, and can activate transcription of target genes. In contrast, Nur77 synthesized constitutively is phosphorylated at Ser354, binds poorly to DNA, and is thus less active. Ser354 appears to be the critical phosphorylation site that affects activity; there does not appear to be critical modifications in either the N- or C-terminal domains that significantly affect Nur77 activity. Although the specific kinases that phosphorylate Nur77 are unknown, the mitogen-activated protein (MAP) kinase/pp90RSK pathway does not seem to be responsible for Nur77 phosphorylation at Ser354. Furthermore, ACTH stimulation of cells in the presence of okadaic acid (OA) results in the accumulation of Nur77 phosphorylated at Ser354, suggesting that ACTH regulates Nur77 through modulating the activities of as yet unidentified kinases and phosphatases.
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Materials and Methods |
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Plasmid constructs
pSG77 is a pSG5 (Stratagene, La Jolla, CA)-based expression
vector that drives the nur77 complementary DNA (cDNA) under
the simian virus 40 early promoter. pSG77 was also used for
PCR-mediated mutagenesis to generate the plasmids pSG77S354A,
pSG77S354D, and pSG77S354E, in which the codon for Ser354
was changed to those encoding alanine (A), aspartate (D), or glutamate
(E), respectively. Direct DNA sequence analysis confirmed these
specific mutations, and no other sequence changes were found. The
p77RE2-PRL-LUC luciferase expression vector was constructed for this
study and consists of two copies of the Nur77 response element placed
upstream of the rat PRL basal promoter (-36 to +33), driving the
luciferase reporter gene (derived from pGL2, Promega, Madison, WI).
pPGKßGal is a ß-galactosidase expression vector used in all
transient transfections as internal control for transfection
efficiencies (24). p77Gal4 is a pSG5-based expression vector of a
chimeric protein in which the Nur77 DNA-binding domain is replaced by
that of the yeast transcription factor Gal4 (25). p17MX2tkCAT is a
reporter plasmid in which the chloramphenicol acetyltransferase (CAT)
reporter gene is driven by the thymidine kinase basal promoter plus two
copies of palindromic Gal4 response elements (26). pSG-MKP is a
pSG5-based expression vector of MAP kinase phosphatase (MKP-1) (27).
pFos-CAT is a CAT reporter construct in which the CAT gene is driven by
the basal c-fos promoter, which contains the serum response
element and responds to increased MAP kinase activity (28).
Transfection
Y1 cells were transiently transfected with indicated plasmids
using the standard calcium phosphate precipitation procedure. Briefly,
1.2 x 106 cells were plated in a 60-mm dish
overnight, and medium was refreshed 2–4 h before transfection. The
total amount of DNA was brought up to 7.5 µg/60-mm dish with pSG5
vector DNA. When 100-mm dishes were used, both DNA amount and cell
number were doubled. Cells were incubated with DNA-calcium phosphate
precipitates in whole medium for 8 h, then washed with PBS and
cultured in whole medium for 48 h. When ACTH treatment was
indicated, cells were treated with ACTH for the last 8 h.
Luciferase assays, CAT assays, and ß-galactosidase assays were
carried out with reagents from Promega, according to the
manufacturer’s instructions. Expression of wild-type or mutant Nur77
proteins was detected by Western blot analysis as previously described
(7).
Gel mobility shift assay
One microgram of pSG5 vector, pSG77, pSG77S354A, pSG77S354D, or
pSG77S354E was transcribed and translated using the in vitro
reticulocyte lysate system (Promega) according to the manufacturer’s
specifications. Protein concentrations were determined by the amount of
[35S]methionine incorporation in the Nur77 band
identified by SDS-PAGE. Two microliters of in vitro
translation reaction mixture were used for each gel mobility shift
assay as previously described (21). The double stranded oligonucleotide
probe (5'-TCGAGAGATAGAAAGGTCAGACGAC) contains a single copy of the
Nur77 response element. For competition experiments, 30 min after
incubation of in vitro translated Nur77 with
32P-labeled probes, different amounts of unlabeled probes
were added and further incubated for 20 min. After gel electrophoresis
as previously described (21), a piece of paper was placed between the
dried gel and the detection screen to block signals from
35S-labeled proteins.
Phosphopeptide mapping
Y1 cells were cultured in phosphate-free DMEM (Sigma) under
serum-free conditions overnight, and then 0.5 mCi/ml
[32P]orthophosphate (New England Nuclear, Boston, MA) was
added for 8 h. In the last 1.5–2 h, ACTH or (Bu)2cAMP
was added to a final concentration of 10 nM or 1
mM, respectively. When double stimulation was required, 15
min before adding ACTH or (Bu)2cAMP, OA or CHX was added to
a final concentration of 120 µM or 10 µg/ml,
respectively. Cells were then chilled on ice, rinsed with PBS, and
lysed in 1 ml ice-cold RIPA (150 mM NaCl; 50 mM
Tris-HCl, pH 8.0; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1%
SDS; 1 mM phenylmethylsulfonylfluoride; 1% aprotinin; and
10 µg/ml leupeptin). [32P]Orthophosphate-labeled Nur77
proteins were isolated by immunoprecipitation using polyclonal
anti-Nur77 antibodies, separated on SDS-PAGE, and subjected to
two-dimensional tryptic phosphopeptide mapping as previously described
(21).
DNA affinity chromatography of Nur77
Y1 cells grown in serum-free, phosphate-free DMEM were labeled
with [32P]orthophosphate. Cells in one 100-mm plate were
left untreated, whereas those in seven other plates were treated with
ACTH. One plate of untreated Y1 cells and one plate of ACTH-treated
cells were lysed in RIPA buffer (containing 0.1 M KCl
instead of 150 mM NaCl); the phosphopeptide maps of Nur77
from these fractions are shown in Fig. 2
, A and B, respectively. The
remaining ACTH-treated Y1 cells were lysed in buffer Z [0.1
M KCl; 25 mM HEPES (K+), pH 7.8; 1
mM MgCl2; 1 mM dithiothreitol; 20%
glycerol; 0.1% Nonidet P-40; 1 mM
phenylmethylsulfonylfluoride; 1% aprotinin; and 10 µg/ml
leupeptin], and the lysates were cleared by centrifugation. The
lysates were then incubated, at 4 C with mild agitation for 2 h,
together with salmon sperm DNA and Sepharose CL-4B onto which the Nur77
response elements were coupled (7). The supernatants were collected and
saved as fraction C. The Sepharose beads were extensively washed four
times with buffer Z containing 0.1 M KCl, and the
supernatant from the last washing step was saved as fraction F. The
beads were then washed with buffer Z containing 0.3 M and
1.0 M KCl. The supernatants were saved as fractions D and
E, respectively, and were dialyzed at 4 C against RIPA buffer
containing 0.1 M KCl to decrease the salt concentration.
Nur77 proteins from fractions A through F were immunoprecipitated with
affinity-purified anti-Nur77 antibodies and purified by electrophoresis
on SDS-PAGE gel (no detectable signal from fraction F) before
phosphopeptide mapping as described previously (21).
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Results |
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). Compared with Nur77 from
untreated Y1 cells, a significant fraction of Nur77 from ACTH- or
(Bu)2cAMP-treated cells displayed slower mobility on
SDS-PAGE (Fig. 1A). On higher resolution gels, it can be discerned that
these cells contain two fractions of Nur77: one with a mobility
identical to that of Nur77 in untreated cells, and the other with a
slower mobility that was unique to cells treated with ACTH or
(Bu)2cAMP. The appearance of this new, low mobility
fraction was blocked by the presence of CHX (Fig. 1A), suggesting that
it corresponded to newly synthesized Nur77.
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, B and D). Two characteristic phosphopeptides
serve as a signature of Nur77 phosphorylated at Ser354
(indicated by arrowheads in Fig. 1); the specific loss of
these phosphopeptides when Ser354 is mutated demonstrated
that they are both derived from phosphorylated Ser354 (7).
Nur77 from both untreated Y1 cells and the high mobility fraction from
ACTH-treated cells were phosphorylated at Ser354, as shown
by the presence of these two characteristic phosphopeptides (Fig. 1, B
and D). By contrast, the low mobility Nur77 fraction, unique to
ACTH-treated cells, displayed a very different pattern of
phosphorylation (Fig. 1C). Fewer phosphopeptides were detected, and in
particular, there was a clear absence of phosphorylation at
Ser354. Thus, ACTH treatment of Y1 cells results in a
population of differentially phosphorylated Nur77, which is
unphosphorylated at Ser354.
OA is a potent inhibitor of protein phosphatases 1 and 2A, which are
the predominant cellular protein phosphatases that act on a wide range
of serine and threonine phosphoproteins (29, 30). OA treatment of Y1
cells in the presence of ACTH resulted in a low mobility form of Nur77
(Fig. 1A) that is highly phosphorylated, as demonstrated by
phosphopeptide mapping (Fig. 1E). These results indicate that during
ACTH treatment, newly synthesized Nur77 accumulates in a highly
phosphorylated state when phosphatases are blocked. When new protein
synthesis was blocked by CHX, ACTH treatment yielded Nur77
indistinguishable from that of the untreated cells, as expected (Fig. 1, A and F).
DNA-bound Nur77 is devoid of phosphorylation at
Ser354
In vitro phosphorylation of a bacterially expressed
Nur77 fragment by protein kinase A (PKA) at Ser354 resulted
in decreased DNA binding affinity, as determined by gel mobility shift
assays (23). To determine whether phosphorylation at Ser354
or other sites is important to Nur77 function in vivo, we
fractionated DNA-bound and -unbound Nur77 from Y1 cells and
characterized their phosphorylation patterns (Fig. 2). ACTH-treated Y1 cells were
metabolically labeled with [32P]orthophosphate, and whole
cell extracts were incubated with Sepharose beads onto which the Nur77
response element had been coupled. Unbound Nur77 (eluted with low salt
wash) and DNA-bound Nur77 (eluted with high salt wash) were compared by
two-dimensional phosphopeptide mapping. High salt elutions of
transcription factors from DNA affinity chromatography do not alter
their phosphorylation states (31, 32, 33, 34). The unbound fraction of Nur77
from ACTH-treated cells displayed a phosphopeptide map similar to that
of Nur77 from untreated Y1 cells (Fig. 2, A–C). In contrast, DNA-bound
Nur77 from ACTH-treated cells displayed a different phosphorylation
pattern characteristic of the ACTH-stimulated Nur77 fraction (compare
Fig. 2, D and E, with Fig. 1C). The DNA-bound fraction exhibited slower
electrophoretic mobility (data not shown) and was unphosphorylated at
Ser354 (Fig. 2, D and E). These results indicate that in Y1
cells, only the ACTH-induced Nur77, which is unphosphorylated at
Ser354, can bind DNA.
Negatively charged amino acid substitutions for
Ser354 decrease Nur77 DNA-binding and
trans-activation activities
The Nur77 fraction that can bind DNA is unphosphorylated at
Ser354 (Fig. 2), which is the only available
phosphorylation site within the Nur77 DNA-binding domain (23). A
plausible hypothesis is that phosphorylation at Ser354
places a bulky, negatively charged phosphate group in the DNA-binding
domain, rendering the protein unable to interact with DNA due to steric
hindrance or charge repulsion with the phosphate backbone of DNA. To
evaluate this hypothesis, we mutated this residue to either alanine (A)
to mimic the unphosphorylated serine at this site or to aspartate (D)
or glutamate (E) to mimic the phosphorylated form (35, 36). The
abilities of these mutant Nur77 to bind DNA in vitro (Fig. 3A) and to trans-activate
target genes in vivo (Fig. 3B) were assessed.
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, lower
panel). These proteins were subjected to gel mobility shift
analysis using a labeled oligonucleotide probe bearing the Nur77
response element (Fig. 3A). Wild-type Nur77 protein and the mutant in
which Ser354 was mutated to alanine both bound strongly to
the Nur77 response element; competition for binding to the labeled
probe was not effective until a 30-fold excess of unlabeled
oligonucleotide was added. By contrast, although both the Asp and Glu
substitutions at residue 354 bound DNA, their binding was completely
disrupted in the presence of a 5-fold excess of unlabeled
oligonucleotide. These results indicated that the wild-type Nur77 and
the alanine substitution bind to the Nur77 response element with
similar affinities, whereas the aspartate and glutamate substitution
mutants bind the Nur77 response element with significantly reduced
affinities.
To evaluate the trans-activation activities of these mutants
in vivo, expression vectors of wild-type or mutant
nur77 were transfected into Y1 cells. The p77RE2-PRL-LUC
plasmid was cotransfected as a reporter for Nur77
trans-activation activity, and the ß-gal expression
plasmid pPGKßGal was used to normalize transfection efficiencies. The
wild-type and mutant Nur77 proteins accumulated to similar levels in Y1
cells as judged by immunoblotting (Fig. 3B, lower panel),
indicating a similar overall rate of synthesis and degradation. In
these transient transfection assays, Nur77 mutants that substituted
Ser354 with a negatively charged residue (either D or E)
displayed a significantly reduced trans-activation activity,
whereas substitution to the uncharged alanine (A) had no detectable
effect (Fig. 3B, upper panel). Taken together, these results
show that substitution of Ser354 with alanine results in a
mutant that possessed full DNA binding and trans-activation
capabilities and indicate that a serine per se at residue
354 is not necessary for function. However, substitution of this
residue by a negatively charged amino acid, mimicking the effects of
phosphorylation, significantly reduces both the DNA-binding and
trans-activation activities.
Posttranslational regulation of Nur77 activities is primarily
mediated through modification of the DNA-binding domain
Although the above experiments showed that ACTH treatment
modulates Nur77 activities through phosphorylation in the central
DNA-binding domain (Ser354), it is not clear whether
modification of the N- and C-terminal domains might also play a
significant role in regulating Nur77 activities. To assess this
possibility, we employed a Nur77-Gal4 fusion construct (designated
pSG77Gal4), in which the Nur77 DNA-binding domain was replaced by that
of the yeast transcription factor Gal4 (25). This fusion protein thus
has both the N- and C-terminal domains of Nur77 intact, but binds to
the Gal4-binding sites. Transcriptional activation mediated by this
fusion protein can be quantified using the plasmid p17MX2tkCAT, which
encodes the CAT reporter driven by the thymidine kinase promoter
containing the Gal4-binding sites (26). The activity of the endogenous
Nur77, on the other hand, can be simultaneously detected using the
plasmid p77RE2-PRL-LUC, which contains the luciferase reporter gene
driven by the Nur77 response elements linked to the PRL basal promoter.
Y1 cells were cotransfected with pSG77Gal4, p17MX2tkCAT, and
p77RE2-PRL-LUC together with the ß-galactosidase expression vector
pPGKßGal to monitor transfection efficiencies (24). Upon ACTH
treatment, the trans-activation activity of endogenous Nur77
in Y1 cells was greatly increased (Fig. 4A), as reflected by a large
increase in luciferase activity. This could be attributed to the
synthesis and modification of endogenous Nur77 in the active form. In
contrast, ACTH treatment resulted in only a slight, but not
significant, increase in CAT activity driven by the Nur77-Gal4 chimera
(Fig. 4B). These results indicate that
any modification of the N- or C-termini of Nur77 as a result of ACTH
treatment contributes to the activity of Nur77 in only a minor way, if
at all.
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We tested this hypothesis by manipulating the activities of MKP-1 (also
called 3CH134), a dual specificity phosphatase that inactivates MAP
kinase by removing both the tyrosyl and threonyl phosphates of MAP
kinase (27, 45). This hypothesis predicts that overexpression of MKP-1
would lead to an increase in Nur77 activity by down-regulation of MAP
kinase. A cotransfection assay was developed to simultaneously measure
MAP kinase and Nur77 activities, using transcription as an end point.
MAP kinase-mediated transcription was measured with the reporter
plasmid pFos-CAT, which contains the CAT gene driven by the
c-fos basal promoter containing the serum response element.
Transcription of this reporter gene is dependent on MAP kinase
activity, most likely due to the activation of an Ets family ternary
complex factor by MAP kinase (28, 46). Y1 cells were cotransfected with
pSG-MKP to constitutively express MKP-1, with pFos-CAT to measure MAP
kinase activity, with p77RE2-PRL-LUC to measure Nur77
trans-activation activity, and with pPGKßGal to measure
transfection efficiencies. Overexpression of MKP-1 nearly abolished the
expression of the CAT reporter, indicating that MAP kinase activity had
been effectively down-regulated by MKP-1 (Fig. 5B). This effect was not altered by ACTH
treatment, which alone did not diminish MAP kinase-mediated
transcription (Fig. 5B).
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). This result shows that
Nur77 activity is unchanged by decreased MAP kinase activity,
suggesting that the MAP kinase/RSK pathway is not involved in ACTH
regulation of Nur77 activity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein phosphorylation as a regulatory mechanism for transcription factor activities was first established for the cAMP response element-binding proteins (47) and is now thought to play a role in controlling the activities of a broad range of transcription factors (48). The possibility that Nur77 might be regulated by phosphorylation was first suggested by the observation that a bacterially expressed Nur77 fragment exhibited reduced DNA-binding activity when phosphorylated at Ser354 in vitro (23). The in vivo significance of phosphorylation at this site is demonstrated in this study by the finding that ACTH stimulates the synthesis of Nur77 that is unphosphorylated at Ser354, and only this population of Nur77 can bind DNA. Nur77 protein constitutively present in Y1 cells is phosphorylated at Ser354 and is thus inactive. Consequently, the DNA-binding activity of Nur77 is negatively regulated by phosphorylation, similar to some other transcription factors (e.g. c-Myb, c-Jun, Max, and Oct-1) (48). The related transcription factor SF-1 has a threonine residue in a position comparable to that of Ser354 in Nur77; this threonine can also be phosphorylated in vitro, leading to decreased DNA binding (49). Thus, the regulatory features described herein for Nur77 may be more generally applicable to other closely related transcription factors.
Mutational analyses indicates that the serine per se at residue 354 is not important for Nur77 activity, as substitution of this residue to the neutral amino acid alanine did not alter either the DNA-binding or trans-activation activity of Nur77. By contrast, substitution at this site with a negatively charged amino acid, such as aspartate or glutamate, resulted in significant decreases in DNA-binding and trans-activation activities. Thus, the effect of phosphorylation may be explained by the introduction of a negatively charged moiety at residue 354.
Ser354 is located within a region in the DNA-binding domain of Nur77 known as the A box, which lies adjacent to the two DNA-binding zinc fingers (22). The counterpart of the Nur77 A box can be found in the A helix of the thyroid hormone receptor (TR) (50). The TR A helix significantly increases the TR interface with DNA through hydrogen bonding with the phosphate backbone and the minor groove edges of base pairs (50). Thus, it is not surprising that both the TR A helix and the Nur77 A box are endowed with positively charged amino acids that may help to mediate interaction with the negatively charged DNA. Phosphorylation within the A box would disrupt this electrostatic interaction. The results of our mutational analyses are consistent with this view, underscoring the importance of charge at residue 354 to the activity of Nur77.
Nur77 is extensively phosphorylated at the amino-terminus at sites other than Ser354 (21). However, any modification induced by ACTH at either the N- or C-terminal domains of Nur77 does not appear to exert a major effect on activity. Using a fusion construct (Nur77Gal4) in which the Nur77 DNA-binding domain was replaced by the DNA-binding domain of the yeast transcription factor Gal4, we showed that the activity of the fusion protein was not affected in Y1 cells by ACTH treatment, whereas endogenous Nur77 activity was induced. Although we cannot rule out the possibility that the DNA-binding domain substitution might affect protein conformation with unpredictable consequences, it is most likely that ACTH treatment does not cause modification of the chimeric Nur77Gal4 in a manner that affects activity.
ACTH is known to work through a receptor coupled to adenylate cyclase
to increase cAMP levels (51). It is thus not surprising that Nur77
synthesized upon stimulation by ACTH and cAMP are similarly modified
(Fig. 1A and data not shown). However, although ACTH increases cAMP
levels and activates PKA, which can phosphorylate Nur77 at Ser354in vitro (23), the net result of ACTH stimulation is
hypophosphorylation at Ser354. Thus, it is unlikely that
PKA is directly involved in phosphorylation of Nur77 in
vivo.
Despite the finding that cAMP down-regulates MAP kinase in some cell types (39, 40, 41, 42, 43, 44), our results indicate that ACTH treatment by itself does not diminish MAP kinase-mediated transcription in Y1 cells. In addition, down-regulation of MAP kinase activities by ectopic expression of MKP-1 did not increase Nur77 trans-activation activity. These results argue against MAP kinase or its downstream kinases, such as pp90RSK, being involved in the phosphorylation of Nur77 in vivo. Although Nur77 is an in vitro substrate of pp90RSK (21), the specific kinase that phosphorylates Nur77 at Ser354 in vivo remains unknown. Although a kinase activity has been identified in PC12 cells that can phosphorylate Nur77 at Ser354 (52), such a kinase has not yet been found in Y1 cells.
It is likely that both kinases and phosphatases play a role in the regulation of Nur77. In Y1 cells, ACTH treatment in the presence of OA resulted in Nur77 that is hyperphosphorylated at multiple sites, including Ser354. These results suggest that Nur77 synthesized during ACTH treatment might be first phosphorylated at Ser354 and subsequently dephosphorylated to render the active form. Alternatively, ACTH-modulated phosphatases may directly or indirectly inhibit the kinases that phosphorylate Nur77, resulting in Nur77 unphosphorylated at Ser354. As ACTH-induced alteration of gene expression and consequent steroid biosynthesis may depend on its regulation of transcription factor activities, understanding the identity and function of both the kinases and the phosphatases that work to regulate transcription factors during ACTH treatment will be an important future endeavor.
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Acknowledgments |
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Footnotes |
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2 Established Investigator with the American Heart Association.
Received February 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. M. Fernandez, F. Brunel, M. A. Jimenez, J. M. Saez, S. Cereghini, and M. M. Zakin Nuclear Receptors Nor1 and NGFI-B/Nur77 Play Similar, Albeit Distinct, Roles in the Hypothalamo-Pituitary-Adrenal Axis Endocrinology, July 1, 2000; 141(7): 2392 - 2400. [Abstract] [Full Text] [PDF]
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P. C. White and P. W. Speiser Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency Endocr. Rev., June 1, 2000; 21(3): 245 - 291. [Abstract] [Full Text]
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V. Giguère Orphan Nuclear Receptors: From Gene to Function Endocr. Rev., October 1, 1999; 20(5): 689 - 725. [Abstract] [Full Text]
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