Salt-Inducible Kinase Is Involved in the ACTH/cAMP-Dependent Protein Kinase Signaling in Y1 Mouse Adrenocortical Tumor Cells

Xing-zi Lin1, Hiroshi Takemori1, Yoshiko Katoh, Junko Doi, Nanao Horike, Ariko Makino, Yasuki Nonaka and Mitsuhiro Okamoto

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, 2–2 Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: mokamoto{at}mr-mbio.med.osaka-u.ac.jp


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1–2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase’s inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
WHEN STIMULATED BY ACTH, adrenocortical cells biosynthesize, and secrete, steroid hormones in a few minutes. This acute response to ACTH stimulation is followed by a delayed response that occurs several hours after ACTH addition. The delayed response implicates transcriptional activation of steroidogenic genes, such as the gene of steroidogenic acute regulatory (StAR) protein that facilitates the intracellular transport of cholesterol to mitochondrial membranes (1, 2) and the gene of cholesterol side-chain cleavage P450 (CYP11A) that catalyzes the conversion of cholesterol to pregnenolone (3, 4). Although several investigations have focused on the transcription of these genes (5, 6, 7, 8), the mechanism underlying the delayed initiation of gene transcription is not well understood.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue Distribution of SIK mRNA
We previously reported that the treatment of rats with ACTH for several days induced SIK mRNA in the adrenal glands, whereas the treatment of Y1 cells with ACTH induced SIK mRNA within a few hours (12). To test whether or not the induction of SIK mRNA in the in vivo ACTH treatment occurred as one of the acute responses, SIK mRNA content in several tissues taken from rats administered with, or without, ACTH 1 h previously was examined (Fig. 1AGo). The amount of SIK mRNA in the adrenal glands of ACTH-treated rats was larger than that of controls, indicating that SIK gene transcription was indeed activated within 1 h after ACTH administration. Brain had a rather large amount of SIK mRNA, irrespective of ACTH treatment. Unexpectedly, the level of SIK mRNA was elevated in the ovary after ACTH treatment. Whether this ACTH-dependent elevation occurred in rats only during specific time of the estrous cycle was tested with 12 randomly selected 6-wk-old female rats (six rats were treated with ACTH, and the other were given the sham treatment). SIK mRNA levels in individual ovaries from the ACTH-treated rats were higher than those from the sham-treated rats and did not significantly vary from one another (data not shown). Further analysis of this phenomenon will be described elsewhere.



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Figure 1. Tissue Distribution of SIK mRNA in ACTH-Treated, and Nontreated, Rats

A, Sprague Dawley rats (6 wk old) were administered 0.1 mg ACTH (+) or vehicle alone (-) for 1 h. Total RNAs (10 µg), each prepared from the indicated organs, were electrophoresed in 1% agarose gel and subjected to Northern blot analysis. cDNA fragments of SIK (upper panel) and G3PDH (lower panel) were used as probes. Positions of 28S- or 18S-ribosomal RNAs were indicated. B, The adrenal glands of normal rats were separated into the capsular portion (zona glomerulosa, ZG) and the noncapsular portion (zona fasciculata/reticularis and medulla, ZF/ZR/M), and total RNAs were prepared from the respective portions, as described previously (15 ). Probes used for the Northern blot analysis were SIK cDNA (upper panel), preadipocyte factor-1 (Pref-1, a zona glomerulosa specific marker) cDNA (middle panel), and G3PDH cDNA (lower panel).

 
The level of SIK mRNA was elevated in the adrenal glands of rats fed with either high-K+, or high-Na+ diet (12). Whether SIK mRNA was expressed in specific zones of adrenal glands was examined by Northern blot analysis (Fig. 1BGo). The expression of SIK mRNA was seen not only in the zona glomerulosa, but also in the inner portion of adrenal gland including zonae fasciculata and reticularis and medulla. These results sharply contrasted with the zona glomerulosa-specific expression of Pref-1 mRNA (15).

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. 2AGo), 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. 2AGo). 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. 2BGo). 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. 2CGo). These results suggest that PKA signaling is necessary for SIK gene transcription.



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Figure 2. SIK mRNA Is Induced by ACTH via a cAMP/PKA Signaling Pathway

A, Y1 cells (5 x 105) that had been cultured in 10-cm dishes for 48 h were incubated for 2 h with ACTH at the concentrations indicated and then harvested to extract total RNAs or to prepare cell lysates. Total RNAs (10 µg) were separated in 1% agarose gel, and mRNA levels of SIK (upper panel) and G3PDH (middle panel) were examined. The cellular content of cAMP (lower panel) was determined by an enzyme immunoassay as described in Materials and Methods. Asterisks indicate the values under detectable levels (<100 pmol/well). Means and SEMs from triplicate experiments are shown. B, Y1 and Kin-7 cells were treated with vehicle, ACTH (10-6 M), forskolin (20 µM), or 8-Br-cAMP (1 mM) for 2 h, and total RNAs were extracted from the cells to examine the mRNA levels of SIK (upper panel) and G3PDH (lower panel). C, Kin-7 cells (1 x 106) that had been cultured in 10-cm dishes for 24 h were transformed with pIRES-PKAc plasmids (2.5 µg) or an empty plasmid (pIRES) using 20 µl Plus reagent and 20 µl Lipofectamin. The cells were harvested after a 4-h incubation and total RNAs were prepared for Northern blot analysis. A cDNA fragment of PKA catalytic subunit {alpha} was used to confirm the transformation of the cells. The autoradiograms are representative of three independent experiments.

 
The time course of ACTH-stimulated SIK mRNA expression was examined (upper panel in Fig. 3AGo). The cellular level of SIK mRNA rapidly rose after the addition of ACTH, reached the highest point in 1 h, and then gradually fell, almost reaching the basal level after 12 h. Because the half-life of plasma ACTH was reportedly short (16), it might be argued that the peaking-off in SIK mRNA level during prolonged incubation was caused by loss of ACTH in the culture medium. To determine whether or not this was the case, experiments were designed in which cells were incubated with one dose of ACTH for 2 h, with one dose for 12 h, or with one dose at the start and five doses further added at intervals of 2 h during the 12-h incubation (Fig. 3BGo). The cells harvested after the 2-h incubation with one dose of ACTH had a higher level of SIK mRNA than those harvested after the 12-h incubation in which ACTH was replaced with one dose each time for five times at every 2-h interval. These results suggest that the loss of ACTH during the prolonged incubation was not the reason for the decline of SIK mRNA level after 2 h.



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Figure 3. Induction of SIK mRNA Occurs Earlier Than That of CYP11A- and StAR Protein-mRNAs

A, Y1 cells were incubated with ACTH (10-6M) for the indicated periods. Total RNAs (10 µg), extracted from the cells, were separated in an 1% agarose gel, and the mRNA levels of SIK, CYP11A, StAR protein, and G3PDH were examined. The experiments were conducted in duplicate, and one of the results is shown. B, Three methods of ACTH treatment were designed as illustrated in the left panel: First, one dose of ACTH (10-6M) was added to Y1 cells, and the cells were incubated for 2 h. Second, one dose of ACTH was added to Y1 cells, and the incubation was conducted for 12 h. Third, one dose of ACTH was added to Y1 cells at the start, and the additional doses were added at intervals of 2 h during the 12-h incubation. mRNA levels after the incubation were examined by Northern blot analysis. The experiments were conducted in duplicate, and one of the results is shown.

 
The time course of ACTH-stimulated CYP11A- and StAR protein-mRNA expression provided an interesting contrast to that of SIK mRNA expression (Fig. 3AGo). After the addition of ACTH, a time lag of several hours was present before the distinct elevation of CYP11A- and StAR protein-mRNA levels occurred. The mRNA levels reached the highest points after 12 h and remained there after 24 h. Two StAR protein transcripts of 3.4 kb and 1.6 kb in size were detected, as was reported previously in the case of mouse Leydig MA-10 cells (17, 18).

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. 4AGo). 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 [{gamma}-32P]ATP, and the autophosphorylated enzymes were visualized (right panel in Fig. 4AGo). 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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Figure 4. In Vitro Kinase Assay of SIK

A, One microgram each of GST, GST-SIK(N), or GST-SIK(N)K56M was subjected to SDS-PAGE in a 12% acrylamide gel, and proteins were visualized by staining with Coomassie brilliant blue R-250 (CBB: left panel), or by immunoblotting with anti-SIK IgG (middle panel). The enzyme (0.2 µg each) was incubated with 0.5 µCi (18.5 kBq) of [{gamma}-32P]ATP in 10 µl of SIK-reaction buffer for 10 min, and the reaction was stopped by adding 8 µl 3xSDS sample buffer. The phosphorylated proteins were separated in a 15% acrylamide gel and exposed to x-ray films (right panel) as described in Materials and Methods. Positions of the GST and GST-SIK(N) proteins are indicated. An asterisk indicates a phosphoprotein derived from a degraded GST-SIK(N) protein. B, GST-SIK(N) or GST-SIK(N)K56M protein, 0.2 µg each, was incubated with 0.5 µCi (18.5 kBq) of [{gamma}-32P]ATP in the presence or absence of GST-Syntide2 (5 µg) in 10 µl of SIK-reaction buffer for 5–60 min (upper panel). pGST-SIK(N) and pGST-Syntide2 denote the phospho-GST-SIK(N) and phospho-GST-Syntide2 proteins, respectively. The similar experiment was performed by using cold ATP, and the proteins were visualized with Coomassie brilliant blue R-250 (lower panel). Marker proteins (M) with apparent molecular masses of 175, 83, 62, 47.5, 32.5, 25, and 16.5 kDa were used. WT: wild type of the GST-SIK(N) protein.

 
Syntide2, a synthetic peptide containing a consensus target motif of SNF-1/AMPK family kinases, is a good substrate for several protein kinases including Ca2+-dependent calmodulin kinase II, PKC, and SIK (14, 19). We incubated GST-SIK(N) with GST-Syntide2 and [{gamma}-32P]ATP, and then the reaction mixtures were subjected to SDS-PAGE. The autoradiograms (upper panel in Fig. 4BGo) indicated that GST-SIK(N) phosphorylated GST-Syntide2 as well as itself in a time-dependent manner, but GST-SIK(N)K56M did not. It should be noted that GST-SIK(N) could not phosphorylate GST protein.

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. 5AGo). The level remained high from 2–6 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. 5AGo). 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. 5BGo, 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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Figure 5. Levels of SIK Protein and SIK Kinase Activity in ACTH-Stimulated Cells

A, Y1 cells (5 x 105), cultured in 10-cm dishes for 48 h, were incubated with ACTH (106 M) for indicated periods. SIK protein was immunoprecipitated from cell lysates with anti-SIK(N) IgG and protein G-Sepharose, and the precipitate was suspended in SIK-reaction buffer with a final volume of 50 µl. An aliquot of the suspension, 20 µl, was subjected to immunoblot analysis with anti-SIK(N)-IgG (upper panel). The blotted membrane was finally visualized by incubating with peroxidase-labeled anti-rabbit IgG. Positions of SIK protein and IgG are indicated. An asterisk denotes a nonspecific signal. Marker proteins with apparent molecular masses of 83, 62, and 47.5 kDa were indicated. The remaining aliquot, 30 µl, was mixed with 10 µl of SIK-reaction buffer containing 20 µg GST-Syntide2 and 1 µCi (37 kBq) of [{gamma}-32P]ATP, and the mixture was incubated at 30 C for 1 h. After the addition of 15 µl 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 followed by autoradiography (lower panel). The results are representative of duplicate experiments. B, Y1 cells (1 x 106), cultured in 10-cm dishes for 24 h, were transformed with pIRES(HA) or pIRES(HA)-SIK(F) plasmids (2.5 µg) using 20 µl of Plus reagent and 20 µl of Lipofectamin. After the 24-h incubation, the cells were treated with ACTH (10-6 M) for 15 min to 6 h and lysed with 0.4 ml lysis buffer as described in Materials and Methods. The HA-tagged SIK protein was immunoprecipitated with HA-agarose, and the precipitate was suspended in SIK-reaction buffer with the final volume adjusted to 50 µl. Aliquots of the suspension were subjected to immunoblot analysis and in vitro kinase assay. The results are representative for experiments performed in triplicate.

 
Effect of Overexpression of SIK on Steroidogenic Gene Expression
Taking into account the findings that in the ACTH-stimulated Y1 cells, the levels of SIK mRNA and protein rose within 1 h and returned to the basal levels after 8 h, whereas the levels of CYP11A- and StAR protein-mRNAs were significantly raised after 8 h, we asked whether SIK played a regulatory role(s) in the steroidogenic gene expression. Therefore, we established Y1 cell lines that stably expressed SIK and examined their steroidogenic gene expression (Fig. 6AGo). Expression of CYP11A mRNA in two independently isolated SIK transformants, pIRES-SIK1 and pIRES-SIK2, was significantly repressed compared with that in the control cells. The expression of mRNA for StAR protein, Ad4BP/steroidogenic factor 1 (SF-1), and ACTH receptor in the SIK transformants was similar to that in the controls. Immunoblot analyses (Fig. 6BGo) and the kinase assays (Fig. 6CGo) ascertained that both SIK protein and enzyme activity were expressed in the SIK transformants.



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Figure 6. Effect of Overexpression of SIK mRNA on the Steroidogenic Gene Expression

A, Two SIK-overexpressing Y1 lines, pIRES-SIK1 and pIRES-SIK2, transformed stably with the SIK overexpression plasmid (pIRES-SIK), were analyzed. Y1 (cont) and stable transformants with a vector alone [pIRES(vector)], pIRES-SIK1, and pIRES-SIK2 (5 x 105) were cultured in 10-cm dishes for 48 h. Total RNAs (10 µg) extracted after culture were subjected to Northern blot analyses with cDNA probes for CYP11A, StAR protein, Ad4BP/SF-1, ACTH receptor, SIK, and G3PDH. B, Cells were lysed in 0.4 ml of lysis buffer, and the lysate, 20 µl, was subjected to Western blot analysis with anti-SIK IgG. C, In vitro kinase assay was performed after immunoprecipitating SIK, as described in the legend for Fig. 4Go. D, Y1 (cont), pIRES(vector), pIRES-SIK1, and pIRES-SIK2 cells were plated in 10-cm dishes and incubated for 48 h. The cells were then treated with or without ACTH (10-6 M) for 2 h or 12 h. Total RNAs (10 µg) extracted from the cells were subjected to Northern blot analyses using CYP11A-, StAR protein-, and G3PDH-cDNA fragments as probes. The autoradiogram is representative of three independent experiments. E, Parent Y1 or SIK-overexpressing Y1 (pIRES-SIK1), 1 x 105 cells each, were plated in 96-well plates. After incubation for 24 h, cells were treated with or without ACTH (10-6 M) for 1 h, and the level of the cellular cAMP was examined as described in Materials and Methods. N.D. denotes the values under the detectable level (<100 pmol/well). F, Cells, 5 x 106, were plated in 10-cm dishes. After incubation for 24 h, cells were harvested, and the PKA-activity of the cell lysate was determined by using the SignaTECT PKA assay system in the presence or absence of cAMP (0.1 or 5 µM). Means and SEMs from triplicate experiments are shown.

 
We then examined the effect of ACTH on steroidogenic gene expression in the SIK transformants. The addition of ACTH could not induce CYP11A mRNA in the SIK transformants during the 24-h incubation (only the results of 2-h and 12-h post-ACTH treatment were shown in Fig. 6DGo). By contrast, the level of StAR protein mRNA in the SIK transformants was significantly elevated within 2 h after the addition of ACTH as is the case for the control cells. However, its level in the SIK transformants was still low after 12 h, at which time its level in the control cells was markedly elevated. The levels of Ad4BP/SF-1 and ACTH receptor mRNAs in the ACTH-treated SIK transformants were not varied during this experimental period (data not shown).

To exclude from consideration a possibility that the SIK transformants might have defective cAMP/PKA signaling, we assayed the cellular content of cAMP (Fig. 6EGo) and PKA activity in cell homogenates (Fig. 6FGo). 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. 7AGo). 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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Figure 7. Effect of SIK on CYP11A Gene Promoter Activity

A, Y1 cells (1 x 106), cultured in 10-cm dishes for 24 h, were transformed with pIRES(HA)-SIK(F), -SIK(N), and -SIK(C) plasmids (2.5 µg) or an empty plasmid, pIRES(HA), using 20 µl of Plus reagent and 20 µl of Lipofectamine. After incubation for 24 h, the cells were lysed with 0.4 ml lysis buffer. The HA-tagged SIK protein was immunoprecipitated with HA-agarose, and the precipitate was suspended in the SIK-reaction buffer with the final volume adjusted to 50 µl. Aliquots of the suspension were subjected to immunoblot analysis (upper panel) and in vitro kinase assay (lower panel). An asterisk denotes a nonspecific signal. B, Y1 cells (1 x 105/well), cultured in 12-well plates for 48 h, were transformed with 0.25 µg of reporter constructs (pS2.3H Luc or pGL3), 0.25 µg of SIK expression vectors [pIRES(HA)-SIK(F), pIRES(HA)-SIK(N), pIRES(HA)-SIK(C), or pIRES(HA)] and 0.03 µg of pRL-SV40, as an internal control, using 2.5 µl of Plus reagent and 2.5 µl of Lipofectamine. After incubation for 12 h, 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. Transformation efficiencies were corrected by Renilla luciferase activities. The specific promoter activities of CYP11A were expressed as fold-expression of the reporter activity of empty vector, pGL3. KD, Kinase domain; SNH, SNF-1 homology; HA, HA-tag. C, Y1 cells were transformed with CYP11A reporter plasmid (0.25 µg) and different amounts (0, 30, 100, 300 ng: left to right) of pIRES(HA)-SIK(N) or pIRES(HA)-SIK(N)K56M, after which the cells were treated with forskolin as described above. To keep the total amount of expression plasmid constant, pIRES(HA) (300, 100, 30, 0 ng; left to right) was added to the transformation solution. Means and SEMs from triplicate experiments are shown. D, Y1 (upper panel) and NIH3T3 (lower panel) cells (1 x 105/well), cultured in 12-well plates for 48 h, were transformed with 0.25 µg of the reporter constructs, 0.15 µg of SIK expression vectors, and 0.1 µg of PKA expression vector and 0.03 µg of pRL-SV40 as an internal control, using 2.5 µl of Plus reagent and 2.5 µl of Lipofectamine. After incubation for 12 h, cells were harvested to measure luciferase activities by the Dual-Luciferace Reporter Assay System. Means and SEMs from triplicate experiments are shown.

 
Y1 cells were transfected with plasmids containing a 2.3-kb human CYP11A promoter-linked luciferase reporter gene together with the expression vectors of SIK fragments. The transformed cells were incubated with or without forskolin, and then the cell lysates were prepared and assayed for luciferase. The forskolin-dependent elevated CYP11A promoter activity, shown by luciferase activity, was significantly inhibited by coexpressing SIK(F) or SIK(N), but not SIK(C) (Fig. 7BGo). The inhibition by SIK(N) was more extensive than that by SIK(F). The coexpression of SIK(N)K56M, a SIK(N) with defective kinase activity, could not inhibit the forskolin-induced CYP11A promoter activity (Fig. 7CGo).

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. 7DGo). 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. 6Go, 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. 7BGo and Fig. 8AGo). 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. 8BGo). 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. 8CGo). 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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Figure 8. Stable Expression of SIK on CYP11A Gene Promoter Activity

A, Y1 cells (1 x 105/well), cultured in 12-well plates for 48 h, were transformed with 0.25 µg of reporter constructs (pS2.3H Luc or pGL3), 0.25 µg of SIK expression vectors [pIRES(HA)-SIK(F) or pIRES(HA)], and 0.03 µg of pRL-SV40 as an internal control, using 2.5 µl of Plus reagent and 2.5 µl of Lipofectamine. After 48 h of incubation, cells were harvested to measure luciferase activities by the Dual-Luciferase Reporter Assay System. B, Y1 cells were transformed with linearized plasmids, 0.25 µg of pS2.3H Luc/pGL3, 0.25 µg of pIRES(HA)-SIK(F)/pIRES(HA), and 0.03 µg of pRL-SV40. After 48 h of incubation, transformants were selected by G418 (1 mg/ml) for 2 wk, and then cell populations resistant against G418 were harvested to measure luciferase activities. C, Parent Y1 and SIK-overexpressing Y1 (pIRES-SIK1) cells were transformed with 0.5 µg of reporter constructs (pS2.3H Luc or pGL3) and 0.03 µg of pRL-SV40. After 48 h of incubation, cells were harvested to measure luciferase activities. Means and SEM from triplicate experiments are shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It might be expected that SIK mRNA, first cloned from the adrenal glands of rats fed with a high-salt diet (12), was present in the specific zone of the cortex. However, its adrenocortical distribution was not zone specific (Fig. 1BGo). It was induced in the adrenal glands by ACTH administration (Fig. 1AGo, and Ref. 12). In addition, the level of SIK mRNA in Y1 cells was elevated during the incubation with ACTH (Fig. 2AGo), but not influenced by high salt concentration in the culture medium (data not shown). Feldman et al. (14) recently reported that the mRNA level of SIK in PC12 cells was elevated by K+-mediated depolarization of the membrane. The KCl concentration used for depolarizing the membrane was nonphysiological (35–50 mM). These findings led us to conclude that the increase in SIK mRNA detected in the adrenal glands of high-salt-fed rats had been caused by the elevation of plasma ACTH, which possibly occurred as one of the stress responses in rats being fed the special diet (20, 21).

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. 2BGo) (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, {alpha}, ß, and {gamma}, of which {alpha} is the catalytic subunit (33) and ß and {gamma} are important in the regulation of {alpha} ’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. 5BGo). 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. 6Go). 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. 7BGo). This inhibition did not occur in Y1 cells transfected with a SIK expression vector having defective kinase (Fig. 7CGo). When the SIK-expressing Y1 transformants were used in these experiments, the reporter activity was significantly inhibited (Fig. 8CGo). 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 4–12 h, whereas CYP11A gene expression appeared to begin after 8 h, at which time the level of SIK was still substantial (Figs. 3Go and 5Go). 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. 7DGo). These results suggested that the molecular target of SIK’s 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. 6AGo) and the basal CYP11A promoter activity was repressed (Fig. 8CGo). On the other hand, the level of StAR protein mRNA in the SIK transformants was similar to that in the control cells (Fig. 6AGo). When the SIK transformants were incubated with ACTH, the level of StAR protein mRNA was elevated during the first 2 h of incubation (Fig. 6DGo), but 12 h later the mRNA expression was strongly repressed (Fig. 6DGo). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cells and plasmids were generously given by several scientists as follows: Y1 cells and pS2.H Luc were given by Dr. K. Morohashi at National Institute for Basic Biology, Okazaki, Japan; Kin-7 cells (31) were provided by Dr. B. P. Schimmer at University of Toronto, Toronto, Ontario, Canada; and an expression vector for GroEL/ES proteins, pGro12 (41), was supplied by Dr. K. Nishihara at HSP Research Institute, Kyoto, Japan.

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 {alpha}, 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 150–250 mM, and contained a protein of molecular mass 62 kDa, while the major, eluted at 200–300 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), [{gamma}-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 [{gamma}-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 manufacturer’s 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 1–343). 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 Freund’s 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 manufacturer’s 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 solution’s 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.


    ACKNOWLEDGMENTS
 
We thank Dr. Ken-ichirou Morohashi (National Institute for Basic Biology, Okazaki, Japan), Dr. Bernard P. Schimmer (University of Toronto, Toronto, Ontario, Canada) and Dr. K. Nishihara (HSP Research Institute, Kyoto, Japan) for providing us with pS2.3H Luc plasmid, Kin-7 cells, and pGro12 plasmid, respectively. We are also grateful to Dr. Ronald W. Estabrook and Dr. Sergey A. Usanov (University of Texas Southwestern Medical Center, Dallas, TX) for their advice about the E. coli expression system.


    FOOTNOTES
 
A part of this work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

1 These authors contributed equally to this paper. Back

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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