Department of Molecular Physiological Chemistry (X.L., H.T., Y.K., J.D., N.H., A.M., M.O.), Osaka University Medical School H-1, Osaka, 565-0871, Japan; and College of Nutrition (Y.N.), Koshien University, Hyogo, 665-0006, Japan
Address all correspondence and request for reprints to: Mitsuhiro Okamoto, Department of Molecular Physiological Chemistry, Osaka University Medical School H-1, 22 Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: mokamoto{at}mr-mbio.med.osaka-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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ACTH activates membrane-associated adenylate cyclase in adrenocortical cells by means of the signal transduction machinery including the ACTH-bound receptor and G protein. The activated adenylate cyclase generates cAMP, which then activates cAMP-dependent PKA (9, 10, 11). The delayed response to ACTH stimulation is thought to occur as the result of PKA activation.
We previously isolated a cDNA clone encoding salt-inducible kinase (SIK), a novel serine/threonine kinase, from adrenal glands of rats fed with a high-salt diet (12). The primary amino acid sequence of the SIK kinase domain indicated that it belonged to a sucrose-nonfermenting-1 protein kinase (SNF-1)/AMP-activated protein kinase (AMPK) family (13). SIK mRNA was expressed in Y1 mouse adrenocortical tumor cells immediately after the addition of ACTH (12). Feldman et al. (14) recently reported that it was induced in PC12 rat pheochromocytoma cells by KCl, forskolin, and A23187. Physiological roles played by SIK in adrenocortical and adrenomedullary functions, however, remain to be elucidated.
In this study we demonstrate that SIK mRNA was expressed in rat adrenal glands 1 h after ACTH administration. In Y1 cells the level of SIK mRNA rose within 1 h after ACTH addition and then gradually fell to the basal level. SIK mRNA expression was mediated by the cAMP-dependent signaling system. The increase in SIK protein with the concomitant increase in its kinase activity occurred in the early stage of ACTH stimulation. The expression of CYP11A mRNA was found to be repressed in SIK-overexpressing Y1 cells. The ACTH-dependent transcriptional activation of CYP11A and steroidogenic acute regulatory (StAR) protein genes was also repressed in the SIK-overexpressing Y1 cells. A human CYP11A gene promoter-linked reporter gene, introduced in Y1 cells, was activated by the addition of forskolin to the culture medium, but the forskolin-dependent elevated promoter activity was inhibited by transfecting a SIK gene in the cells. These results suggest that SIK played important roles in the gene expression of CYP11A and StAR during the early stage of ACTH stimulation.
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RESULTS |
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cAMP/PKA Signaling-Dependent Induction of SIK mRNA
To explore the mechanism underlying the ACTHdependent
transcription of the SIK gene in adrenocortical cells, we first
examined the effect of actinomycin D, an mRNA synthesis inhibitor, or
cycloheximide, a protein synthesis inhibitor, on the expression of SIK
mRNA in Y1 cells. Actinomycin D completely abolished, whereas
cycloheximide enhanced, the ACTH-dependent transcription of the SIK
gene; thus, prior protein biosynthesis seemed not necessary for SIK
gene transcription (data not shown).
ACTH stimulated SIK mRNA expression in Y1 cells
in a dose-dependent manner (upper panel in Fig. 2A), the distinct elevation of SIK mRNA
being detected with 10-11
M ACTH, a physiologically significant
concentration. A significant amount of cAMP accumulated in the
cells with ACTH concentrations higher than 10-11
M (lower panel in Fig. 2A
). To confirm
further the involvement of cAMP/PKA signaling in ACTH-dependent SIK
mRNA induction, ACTH, forskolin, and 8bromo-cAMP (8-Br-cAMP) were
tested as stimulants (Fig. 2B
). These activators of PKA similarly
induced SIK mRNA in Y1 cells, but not in Kin-7 cells, a Y1-derived cell
line with less PKA activity. Kin-7 cells, once transformed with an
expression vector of PKA catalytic subunit, could express SIK mRNA
without ACTH treatment (Fig. 2C
). These results suggest that PKA
signaling is necessary for SIK gene transcription.
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Expression and Enzyme Activity of SIK Protein
To explore further the mechanism underlying SIK gene
expression, it was essential to establish the assay system of SIK
enzyme activity. A cDNA fragment encoding a
glutathione-S-transferase (GST)-linked SIK kinase domain
[GST-SIK(N)] was expressed in Escherichia coli, and the
protein was purified as described in Materials and Methods.
The purity of GST-SIK(N) was more than 90%, as judged by SDS-PAGE
followed by Coomassie-brilliant blue staining and immunoblot analysis
(left and middle panels in Fig. 4A). GST-SIK(N)K56M, a SIK having Met56
instead of Lys56 in the ATP binding motif, was also expressed and
purified. These GST-fused SIK proteins were incubated with
[
-32P]ATP, and the autophosphorylated
enzymes were visualized (right panel in Fig. 4A
). As
expected, GST-SIK(N) was strongly autophosphorylated, but not
GST-SIK(N)K56M. A weak radioactive band found in the lane of
GST-SIK(N), indicated by an asterisk, was probably that of a
SIK-dependent phosphorylated peptide derived from degradation of
GST-SIK(N) or contaminated E. coli proteins.
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To confirm further the enzyme nature of SIK, a full-length SIK protein [SIK(F)] was expressed in COS-7 cells, purified by immunoprecipitation from the cell lysates, and assayed for the kinase activity. The results indicated that SIK(F) could also catalyze autophosphorylation as well as phosphorylation of GST-Syntide 2 (data not shown).
ACTH-Stimulated Induction of SIK Protein
The time course of ACTH-stimulated SIK protein expression in
Y1 cells was investigated by immunoprecipitation of SIK protein from
cell lysates, followed by immunoblot analysis as well as in
vitro kinase assays. A significant elevation in SIK protein level
was detected 1 h after the addition of ACTH (upper
panel in Fig. 5A). The level
remained high from 26 h after ACTH addition, and then it began to
decline. The kinase activity of SIK, both autophosphorylation and
phosphorylation of Syntide 2, increased concomitantly with the SIK
protein level and reached the maximum after 2 h, and then began to
decrease (lower panel in Fig. 5A
). It was noted that the
decline of the enzyme activity seemed to begin earlier than that of the
protein level. This might suggest a possibility that the enzyme
activity, or protein stability, of SIK might be changed during the
early stage of ACTH stimulation. To test whether this was the case, Y1
cells that had been transformed with an overexpression vector of
hemagglutinin (HA)-tagged SIK(F) were incubated with ACTH. At the
indicated times SIK protein was immunopurified from cell lysates by
using anti-HA antibody and examined for kinase activity. As shown in
Fig. 5B
, SIK enzyme activity, both autophosphorylation and
phosphorylation of Syntide 2, was constant during the 6-h incubation of
Y1 cells with ACTH, suggesting that the specific kinase activity of SIK
might not vary in the ACTH-stimulated Y1 cells. Therefore, the apparent
discrepancy between the protein level and the kinase activity during
the 2- to 6-h incubation with ACTH might be due to the fact that the
degree of immunostaining did not accurately reflect the amount of
protein.
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To exclude from consideration a possibility that the SIK
transformants might have defective cAMP/PKA signaling, we assayed the
cellular content of cAMP (Fig. 6E) and PKA activity in cell homogenates
(Fig. 6F
). The extent of ACTH-induced increase in cAMP in pIRES-SIK1
did not significantly differ from that in the parent Y1. The PKA
activity in pIRES-SIK1 cell homogenates, assayed in the presence
of cAMP, was also not different from that in Y1 cell homogenates. These
results suggested that SIK might negatively regulate the expression of
CYP11A mRNA in the resting Y1 cells as well as in the ACTH-stimulated
cells. The ACTH-stimulated gene expression of StAR protein also seemed
to be negatively regulated by SIK.
SIK Inhibits Forskolin-Induced CYP11A Promoter Activity
We decided to explore further the mechanism underlying the
repression by SIK of CYP11A gene transcription. As the first step of
this line of research, we constructed several expression vectors of SIK
fragments to use them for expression assays of a CYP11A
promoter-linked reporter gene. Three kinds of HA-tagged SIK
peptides, SIK(F), a full-length SIK protein, SIK(N), an N-terminal
343-amino acid fragment having the kinase domain, and SIK(C), a
C-terminal 435-amino acid fragment that did not contain the kinase
domain, were overexpressed in Y1 cells, immunoprecipitated by anti-HA
antibody, and subjected to immunoblot analysis with anti-SIK antibody
and in vitro kinase assay (Fig. 7A). The immunostaining of HA-SIK(N) was
weaker than that of HA-SIK(F) or HA-SIK(C) (upper panel). In
addition, HA-SIK(N) had no autophosphorylation activity, even though it
had a lower, but significant, phosphorylation activity of GST-Syntide2,
when compared with HA-SIK(F) (lower panel). Therefore, the
protein stability of HA-SIK(N) might be a little different from that of
HA-SIK(F).
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CYP11A gene expression in steroidogenic cells is under the regulation
by a transcription factor Ad4BP/SF-1 (5, 6). Therefore, it
might be argued that the presence of Ad4BP/SF-1 in Y1 cell nuclei might
influence the inhibitory effect of SIK on CYP11A promoter-linked
reporter gene expression. To examine this point, mouse NIH3T3
fibroblasts that did not express Ad4BP/SF-1 were used for the
promoter assays, and the results were compared with those obtained with
Y1 cells (Fig. 7D). In NIH3T3 cells, the PKA-dependent activated CYP11A
promoter activity was inhibited by coexpressing SIK(N), even though the
extent of the activation by PKA seemed somewhat weaker than in Y1
cells.
Stably Overexpressed SIK Inhibits Basal CYP11A Promoter
Activity
The CYP11A mRNA level in the SIK-expressing transformants,
pIRES-SIK1 and pIRES-SIK2, under the nonstimulated conditions
was quite low (Fig. 6, A and D), whereas the degree of inhibition
attained by transiently expressed SIK(F) on the basal CYP11A
promoter activity in Y1 cells was only 20% (Fig. 7B
and Fig. 8A
). It might be argued that in the
latter case the episomal status of the introduced CYP11A promoter, as
well as the SIK gene, might have influenced, and attenuated, the
effectiveness of SIK. To clarify this point, Y1 cells were transformed
with linearized SIK overexpression vector or control vector, CYP11A
promoter-linked reporter plasmid, and internal control
Renilla luciferase vector, and the cell population that
stably expressed the neomycin resistance gene was selected by G418 for
2 wk. The selected cells were then assayed for promoter activity (Fig. 8B
). The promoter activity of the cells stably expressing both SIK and
CYP11A promoter-linked reporter was significantly lower than that of
those expressing only CYP11A promoter-linked reporter. Consistent with
these results was the finding that the previously isolated stable SIK
transformant pIRES-SIK1, when transfected with the CYP11A
promoter-linked reporter plasmid, had a significantly lower level of
reporter activity compared with the similarly treated parent Y1 cells
(Fig. 8C
). These results suggested that the repression of the basal
level of CYP11A gene transcription could occur by the constitutively
expressed SIK gene.
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DISCUSSION |
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The induction of early response genes such as c-Fos (22, 23), JunD (24), and c-myc
(25) by ACTH is mediated by signaling pathways that
involve MAPK (26), stress-activated protein kinase
(27), PKC (28), Ca2+
(29, 30), or arachidonic acid (18). The SIK
gene, however, was induced by ACTH, forskolin, or 8-Br-cAMP in Y1
cells, but not in Kin-7 cells (Fig. 2B) (31). Therefore,
the ACTH-stimulated SIK mRNA induction is thought to occur probably by
means of the PKA-dependent signaling pathway.
Since the SIK kinase domain is similar to the kinase domain of the
SNF-1/AMPK family of enzymes (32), the knowledge of this
family may be helpful to formulate the plans for future investigation
of SIK. AMPK is composed of three subunits, , ß, and
, of which
is the catalytic subunit (33) and ß and
are
important in the regulation of
s kinase activity (34, 35). The activity of AMPK is strictly regulated by cellular
energy metabolism. On the other hand, the activity of SIK may not vary
under the various cellular metabolic conditions; thus, SIK expressed in
Y1 cells had the constant level of kinase activity, whether or not the
cells were treated with ACTH (Fig. 5B
). Similarly, SIK kinase activity
expressed in COS-7 cells was not influenced by forskolin treatment
(data not shown).
Several investigators (36, 37) have reported that
cAMP-mediated CYP11A gene expression in Y1 cells was enhanced, rather
than inhibited, by cycloheximide, suggesting that the initiation of
CYP11A gene expression may not require de novo protein
synthesis. Since CYP11A mRNA began to increase after several hours of
incubation with ACTH, it has been proposed that some negatively
regulating factors may control the CYP11A gene expression in the early
stage of ACTH stimulation. To test a possibility that SIK might be
somehow involved in this negative regulation, we established Y1 cell
lines that stably expressed high levels of SIK mRNA and its protein
(Fig. 6). The results showed that the ACTH-stimulated induction of
CYP11A mRNA was strongly repressed in the SIK-expressing Y1
transformants. Moreover, when Y1 cells, after having been transfected
with a CYP11A promoter-linked reporter gene and a SIK expression
vector, were treated with forskolin, the forskolindependent
activated reporter activity was inhibited (Fig. 7B
). This inhibition
did not occur in Y1 cells transfected with a SIK expression vector
having defective kinase (Fig. 7C
). When the SIK-expressing Y1
transformants were used in these experiments, the reporter activity was
significantly inhibited (Fig. 8C
). These results suggested that SIK
might be involved in the negative regulation of CYP11A gene expression
in the early stage of ACTH stimulation. SIK protein, as well as kinase
activity, increased in the ACTH-stimulated Y1 cells for the first
2 h, and then gradually decreased during the period of 412 h,
whereas CYP11A gene expression appeared to begin after 8 h, at
which time the level of SIK was still substantial (Figs. 3
and 5
). In
the future this observation must be further elucidated with regard to
the action of SIK on gene transcription.
In the human CYP11A gene promoter, a region responsible for
cAMP-mediated gene activation was located at -1.8 kb to -1.5 kb,
where two Ad4 elements and one cAMP response element (CRE)-like
sequence were clustered (38). To gain more insight into
the inhibitory effect of SIK on CYP11A promoter activity, Y1 cells were
transfected with a human CG gene-derived CRE-linked luciferase reporter
gene. When the transformed cells were treated with forskolin, the
reporter activity was induced via CRE. This forskolin-dependent induced
CRE-reporter activity was inhibited by transfecting a SIK expression
vector. By contrast, when a bovine CYP11B1 gene-derived Ad4-linked
reporter gene was used for the transformation of Y1 cells, SIK could
not inhibit the reporter activity (J. Doi, unpublished results).
Moreover, the inhibitory effect of SIK on CYP11A promoter was found in
nonsteroidogenic NIH3T3 fibroblasts as well as in Y1 (Fig. 7D). These
results suggested that the molecular target of SIKs action might be
the transcriptional machinery composed of ubiquitously expressed
transcription factors like CRE binding proteins rather than
steroidogenic tissue-specific transcription factors such as Ad4BP/SF-1
or dosage-sensitive sex reversal adrenal hypoplasia congenita X
chromosome gene-1.
The basal expression of both CYP11A gene and StAR protein gene was
repressed in Y1 cells that overexpressed an exogenous dosage-sensitive
sex reversal adrenal hypoplasia congenita X chromosome gene-1
(7) and the other mutant Y1 lines, 10r-6 and 10r-9
(39, 40). In these cells the action of a nuclear
transcription factor Ad4BP/SF-1 was found to be defective. In the
SIK-expressing Y1 transformants the level of CYP11A mRNA was quite low
(Fig. 6A) and the basal CYP11A promoter activity was repressed (Fig. 8C
). On the other hand, the level of StAR protein mRNA in the SIK
transformants was similar to that in the control cells (Fig. 6A
). When
the SIK transformants were incubated with ACTH, the level of StAR
protein mRNA was elevated during the first 2 h of incubation (Fig. 6D
), but 12 h later the mRNA expression was strongly repressed
(Fig. 6D
). Forskolin or 8-Br-cAMP treatment of the SIK transformants
could also induce StAR protein mRNA during the first 2 h of
incubation (data not shown). Considering that the mode of basal
expression of steroidogenic genes in SIK-overexpressing Y1 cells was
different from that in the Ad4BP/SF-1-defective cells, we could
conclude that the repression of the gene expression by SIK might occur
by the mechanism independent of the action of Ad4BP/SF-1.
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MATERIALS AND METHODS |
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The following reagents were obtained from commercial sources: plasmids pIRES1neo and pGEX6P-1 were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA) and Pharmacia Biotech (Piscataway, NJ), respectively; forskolin, 8-Br-cAMP, cycloheximide, actinomycin D, FCS, dithiothreitol (DTT), and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (St. Louis, MO); geneticin and trypsin-EDTA were from Life Technologies, Inc. (Gaithersburg, MD); and ACTH (Cortrosyn) was from Daiichi Pharmaceutical Company Ltd., Tokyo, Japan.
Experimental Animals
Sprague Dawley rats, purchased from SLC Co. Ltd. (Shizuoka,
Japan), were maintained under standard conditions of light and
temperature. All experiments were carried out in accordance with the
guidelines for animal care of Osaka University Medical School.
Cell Culture
Y1 and Kin-7 cells were maintained in DMEM (Life Technologies, Inc.) containing 10% FCS and antibiotics at 37 C
under an atmosphere of 5% CO2-95% air.
Northern Blot Analysis and Immunoblot Analysis
These analytical methods were described previously
(42).
cDNAs
SIK cDNA fragments were amplified by PCR from a rat adrenal
zona glomerulosa cDNA library (15) by using the following
sets of primer, sense: 5'-gcggccgcATGGTGATCATGTCGGAGTTC and antisense1:
5'-gaattcTCACTGTACCAGGACGAACGTCC, or sense and antisense2:
5'-gaattcCTGTACCAGGACGAACGTCCC (the lowercase letters
indicate the linker sequences). The amplified products were introduced
into pT7(R) vector (Novagen), the resultant plasmids being named
pT7-SIK and pT7-SIK(-stop), respectively. To construct pIRES-SIK, a
NotI-EcoRI fragment of pT7-SIK was ligated into
the NotI-EcoRI site of pIRES1neo vector
(CLONTECH Laboratories, Inc.). To prepare pGEX-SIK(N) and
pET-SIK(N) plasmids, a BamHI-XhoI fragment of
pT7-SIK was introduced into the BamHI-XhoI site
of pGEX6P-1 (Pharmacia Biotech) and pET28a (Novagen,
Madison, WI), respectively.
An E. coli expression plasmid of a mutant SIK having defective kinase activity was constructed by replacing an AAA (Lys56) codon with a ATG (Met) codon using a mutagenic primer, 5'-ACGCAGGTTGCAATTATGATAATTGACAAGACACGG, a template, pGEX6P-1-SIK(N), and a site-directed mutagenesis kit, GeneEditor (Promega Corp., Madison, WI).
To construct HA-tagged SIK expression vectors, a set of oligonucleotides corresponding to the HA-tag, 5'-AATTCTATCCATATGATGTTCCAGATTATGCTTAG and 5'-GATCCTAAGCATAATCTGGAACATCATATGGATAG, were annealed and ligated into the BamHI/EcoRI site of pIRES1neo vector, the resultant vector being named pIRES(HA). EcoRI/NotI linker oligonucleotides, 5'-TCGAATTCGCGGCCGCAATGG and 5'-TCGACCATTGCGGCCGCGAAT, were ligated into the XhoI site of pT7-SIK(-stop). Then, the EcoRI/NotI cDNA fragments encoding the N-terminal, C-terminal, and full-length SIK were purified and ligated into the NotI/EcoRI site of pIRES(HA), and the resultant plasmids were named pIRES(HA)-SIK(N), pIRES(HA)-SIK(C), and pIRES(HA)-SIK(F), respectively.
cDNA fragments of mouse StAR protein, Ad4BP/SF-1, and ACTH receptor were produced by PCR by using cDNA templates prepared from Y1 cells (15) and one of the following primer sets; for StAR protein cDNA, 5'-CGGGGACGAAGTGCTAAGTA and 5'-CAGGTGGTTGGCGAACTCTA, for Ad4BP cDNA, 5'-gaattcCCGCGGCATGGACTACTGG and 5'-ggatccTCAAGTCTGCTTGGCCTGCA, and for ACTH-R cDNA, 5'-gaattcATGAAGCATATTATCAAATTCGTA and 5'-ggatccTAATACCGGTTGCAGAAGAG. cDNA fragments of rat CYP11A and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) were described previously (15).
To construct pGEX6P-1-Syntide2, a set of oligonucleotides, 5'-gatcCCCGCTGGCACGTACCCTGTCCGTTGCAGG-TCTGCCGGGTAAAAAG and 5'-aattcTTTTTACCCGGCAGACCTGCAACGGACAGGGTACGTGCCAGCGGG was annealed and ligated into the BamHI/EcoRI site of pGEX6P-1.
To construct a PKA expression vector, pIRES-PKAc, a cDNA fragment of
mouse PKA catalytic subunit , amplified by RT-PCR with primers,
5'-ACGCGGCCGCGATGGGCAACGC and 5'-ATGGATCCCCTAAAACTCAGTAAACTC, was
digested with NotI/BamHI and ligated into the
NotI/BamHI site of pIRES1neo.
Stable Expression of SIK mRNA in Y1 Cells
Plasmid pIRES-SIK or pIRES1neo (0.5 µg), after having been
linearized by Bst1107I digestion, was incubated with 2.5
µl of Lipofectamine Plus reagent (Life Technologies, Inc.) in 50 µl serum-free medium for 15 min, after which the
mixture was diluted in 400 µl serum free-medium. Y1 cells (1 x
105), plated 48 h previously in a 12-well
plate, were washed twice with serum-free media and incubated with the
DNA solution for 3 h. The culture medium was then changed to 1 ml
serum-containing medium. The cells were harvested by trypsinization
after 48 h, diluted in 50 ml serum-containing medium with 1 mg/ml
Geneticin, and then plated in 98-well plates. The cell culture was
conducted for 2 wk in the presence of Geneticin (Life Technologies, Inc., Rockville, MD) and the Geneticin-resistant
cells, 50 colonies, were selected.
Purification of GST-Tagged SIK Protein
In a previous report we expressed full-length SIK protein as a
GST-fused protein in E. coli (12), but the
yield of the recombinant protein was poor. According to the recent
report by Feldman et al. (14), an N-terminal
polypeptide of SIK, composed of 343 amino acids, could be purified as a
stable GST-fused protein with better yield. We, therefore, constructed
an expression vector for GST-SIK(N), a GST-fused N-terminal kinase
domain of SIK, which was similar to that prepared by Feldman et
al. We also developed a coexpression system of GST-SIK(N) with
chaperone proteins, such as GroEL and GroES, which were expected to
produce more soluble and active proteins during biosynthesis in
E. coli (41, 43). As a result, our improved
E. coli expression system, compared with our previous
expression system, provided a 10-fold increase in the overall recovery
of active GST-SIK(N) protein in a soluble form.
Plasmid [pGEX6P-1-SIK(N) or pGEX6P-1-SIK(N)K56M] was cotransformed with pGro12 into bacterial strain BL21 (codon plus) (Stratagene, La Jolla, CA). The resultant transformant was grown in 2.5 liters of 2YT medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl per liter) containing 8% glycerol and 50 mM potassium phosphate (pH 7.4) at 28 C. When the optical density at 600 nm of the culture solution reached 1.0, recombinant proteins were induced by the addition of 0.2 mM isopropylthiogalactoside and 10 mM L-arabinose. Three hours after induction, the bacteria were harvested by centrifugation, and the pellet was suspended in 50 ml sonication buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol and 1 mM PMSF]. The E. coli cells were broken by sonication, and the lysed cell suspension was subjected to centrifugation at 1000x g for 10 min. The resultant supernatant was again centrifuged at 100,000x g for 45 min. The active GST-SIK(N) protein was distributed to both the pellet and the soluble fraction in almost equal amounts. Because GST-SIK(N) protein in the soluble fraction was tightly bound to GroEL/ES complex, the pellet fraction was used for further purification. The pellet was solubilized by 20 mM sonication buffer containing 1% Triton X-100 and 1% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate for 30 min, and the solution was subjected to centrifugation at 100,000 x g for 45 min. The GST-SIK(N) protein was recovered in the supernatant. The supernatant was applied onto a 1-ml glutathione-Sepharose (Pharmacia Biotech) column, and the column was washed with 10 ml of sonication buffer. The addition of glutathione (10 mM) to the buffer eluted an apparently single protein peak with molecular mass of 63 kDa. The peak fractions were pooled, diluted with 5 vol of buffer A [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, 10% glycerol] and applied onto a 1-ml HiTrap Q column (Pharmacia Biotech). The column was washed with 10 ml buffer A, and then bound proteins were eluted in a linear gradient of 0 to about 1 M NaCl in 15 ml buffer A at 0.5 ml/min flow rate. As a result, two protein peaks were produced; the minor peak, about one-tenth of the major in size, was eluted at the NaCl concentration 150250 mM, and contained a protein of molecular mass 62 kDa, while the major, eluted at 200300 mM, contained a protein of molecular mass 63 kDa. An immunoblot analysis performed by using an anti-GST antibody revealed that the minor peak protein was the GST-fused SIK(N) protein. Further examinations using various antibodies against several E. coli proteins suggested that the major peak protein was the GroEL protein, one of the chaperone proteins. The fractions containing GST-SIK(N), but not GroEL, were pooled, determined for protein concentration using a protein assay kit (Bio-Rad Laboratories, Inc.), and kept at -80 C until use.
Purification of GST-Syntide2
The purification of GST-Syntide2 was performed according to the
above method described for the purification of GST-SIK(N) without using
the GroEL/ES system. The GST- Syntide2 protein in the sonicated
E. coli was solubilized with 1% Triton X-100 for 30 min,
and the solution was centrifuged at 100,000 x g for 45
min. The GST-Syntide2 protein was recovered in the supernatant. The
glutathione-Sepharose and HiTrap Q column chromatographies were
performed as described above.
Immunoprecipitation
Y1 cells treated with ACTH (10-6 M) for
15 min to about 24 h were lysed with 0.4 ml of lysis buffer, and
DNA in the lysate was sheared by using a syringe with an 18-G needle.
The lysate was centrifuged at 100,000 x g for 45 min.
The supernatant was transferred into a 1.5-ml tube and mixed with 5
µl of anti-SIK IgG and 25 µl of protein G-Sepharose
(Pharmacia Biotech), and then the mixture was incubated at
4 C for 2 h. The SIK/IgG/protein G Sepharose complex was
precipitated by centrifugation at 3,000 x g for 5 sec,
washed three times with 1 ml of lysis buffer, and washed once with 1 ml
of SIK-reaction buffer [50 mM Tris-HCl (pH 7.4),
10 mM MnCl2, 1
mM DTT, 100 mM NaCl, and
10% glycerol]. The final precipitate was suspended in the
SIK-reaction buffer with a final volume of 50 µl. Two aliquots of the
SIK-IgG suspension, 20 µl and 30 µl, were used for the immunoblot
analysis and kinase assay, respectively. The HA-tagged SIK protein was
precipitated with 25 µl of anti-HA agarose (Roche Laboratories, Inc., Nutley, NJ).
In Vitro Kinase Assay
GST-SIK(N) (0.2 µ g), [-32P]ATP
(0.5 µCi or 18.5 kBq), and substrate (GST or GST-Syntide2) (5 µg)
were mixed in 10 µl SIK-reaction buffer. Reactions were performed at
30 C for 5 min for approximately 1 h, and stopped by the addition
of 8 µl 3x SDS sample buffer. The reaction mixture was boiled for 5
min, and proteins in the mixture were separated in 15% SDS-PAGE. The
gel was dried and exposed to x-ray films (at room temperature for 30
min). To examine the kinase activity of the immunopurified SIK protein
in Y1 and COS-7 cells, 30 µl of the immunoprecipitated samples were
mixed with 10 µl of SIK-reaction buffer containing 20 µg of
GST-Syntide2 and 1 µCi (37 kBq) of
[
-32P]ATP, and the mixture was incubated at
30 C for 1 h. After the addition of 15 µl of 3x SDS sample
buffer, the reaction mixture was heated at 100 C for 5 min, and an
aliquot (20 µl) was subjected to SDS-PAGE and autoradiography as
described above.
Assays of cAMP and PKA
Cells, 1 x 105, were plated in
96-well plates to determine the cellular cAMP level. After incubation
for 24 h, cells were treated with or without ACTH
(10-6 M) for 1 h, and the
levels for cAMP were determined using a cAMP enzyme immunoassay kit
(Amersham Pharmacia Biotech, Arlington Heights, IL)
according to the manufacturers recommendations. To examine PKA
activity in Y1, SIK-overexpressing Y1 cells (pIRES-SIK-1) and Kin-7
cells, cells (5 x 106) were plated in 10-cm
dishes and incubated for 24 h. Cells were washed with 5 ml of PBS,
harvested by using a scraper with 0.5 ml of cold extraction buffer [25
mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5
mM EGTA, 1 mM DTT, 1 µg/ml leupeptin, 1
µg/ml aprotinin, 1 mM PMSF], and homogenized. PKA
activity in the homogenate was measured with a SignaTECT PKA assay
system (Promega Co.) in the presence or absence of cAMP
(0.1 or 5 µM). Total protein concentration was measured
by a protein assay kit (Bio-Rad Laboratories, Inc.).
Anti-SIK Antibody
Polyclonal antibodies were raised against a full-length SIK
protein or a SIK(N) fragment (amino acid residues 1343). The antigen
was produced in E. coli as a fusion protein using T7 RNA
polymerase expression system. Expression vectors pET28-SIK(F) and
pET28-SIK(N) were transfected into an E. coli strain,
BL21(DE3) (Novagen). The expressed proteins were solubilized from the
particulate fraction of cell homogenates with SDS sample buffer
containing 1% SDS and 10% 2-mercaptoethanol and subjected to
SDS-PAGE. The fusion protein (2 mg) was electrophoretically
extracted from the gel slices as described previously
(44). Its aliquot, about 0.4 mg, was emulsified with two
volumes of complete Freunds adjuvant and used to immunize Japanese
white rabbits (females, 2.0 kg body weight). To purify specific
antibody against the SIK(N) protein, an affinity column coupled with
the GST-SIK(N) protein was prepared. GST-SIK(N), 1 mg, was dialyzed
overnight against coupling buffer [0.1 M
NaCO3 (pH 8.3), 0.5 M
NaCl] at 4 C and loaded onto a 1 ml HiTrap NHS-activated column
(Pharmacia Biotech), and the coupling reaction was done
according to the manufacturers instruction. The antiserum (15 ml)
against the SIK(N) peptide was diluted with 15 ml of 50 mM
TBST [Tris-HCl (pH 8.0), 0.9% NaCl, 0.1% Tween 20] buffer,
the solution was applied onto the GST-SIK(N) column, and the column was
washed with 15 ml of 50 mM TBST buffer. Bound IgG was
eluted with 5 ml of 100 mM glycine-HCl (pH 2.0),
and Tris-HCl (pH 9.5) was added to the eluate to adjust the solutions
pH to 7.
Reporter Assay
A CYP11A (pS2.3H Luc) promoter construct (0.25 µg), one of the
SIK expression vectors (0.25 µg), pIRES(HA)-SIK(F), pIRES(HA)-SIK(N),
or pIRES(HA)-SIK(C), and pRL-SV40 (Renilla luciferase
expressing vector; Promega Corp., 0.03 µg), an internal
standard, were mixed, and the mixture was incubated with 2.5 µl of
Plus reagent (Life Technologies, Inc.) in 50 µl
serum-free medium for 15 min. To the DNA solution 50 µl of serum-free
medium containing 2.5 µl of lipofectamine (Life Technologies, Inc.) was added, and the mixture was further incubated for 15
min and diluted with 400 µl of the serum-free medium. Y1 cells,
washed twice with serum-free media, were incubated with the DNA
solution for 3 h, and the cell suspension was transferred to 1 ml
of serum-containing medium. After the 12-h incubation, cells were
treated with or without forskolin (50 µM) for
8 h and harvested to measure luciferase activities by the
Dual-luciferase Reporter Assay System (Promega Corp.).
Transformation efficiencies were corrected by Renilla
luciferase activities. The specific promoter activity of the CYP11A
gene was expressed as fold-expression compared with the reporter
activity of empty vector, pGL3.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to this paper.
Abbreviations: AMPK, AMP-activated protein kinase; 8-Br-cAMP, 8-bromo-cAMP; CRE, cAMP responsive element; CYP11A, cholesterol side-chain cleavage cytochrome P450; DTT, dithiothreitol; G3PDH, glyceraldehyde 3-phosphate dehydrogenase GST, glutathione S-transferase; HA, hemagglutinin; PMSF, phenylmethylsulfonyl fluoride; SF-1, steroidogenic factor 1; SIK, salt-inducible kinase; SIK(C), a C-terminal 435-amino acid fragment that does not contain the kinase domain; SIK(N), an N-terminal 343-amino acid fragment containing the kinase domain; SNF-1, sucrose-nonfermenting-1; StAR, steroidogenic acute regulatory.
Received for publication December 5, 2000. Accepted for publication April 20, 2001.
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REFERENCES |
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