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Biochemistry and Biophysics |
Manitoba Institute of Cell Biology, Winnipeg, Manitoba, R3E 0V9 Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Ciras-3 (H-ras-transformed 10T) mouse fibroblasts. These studies demonstrated that MSK1, but not Rsk-2, was the H3 kinase activated in these cells. Furthermore, assays with Rsk-2 showed that this kinase phosphorylates H2B but not H3 in vitro. H89, a potent MSK1 inhibitor, prevented TPA induction of H3 phosphorylation and diminished the TPA-induced expression of the c-fos and urokinase plasminogen activator genes. We propose that persistent activation of the Ras-MAPK pathway and MSK1 resulting in the elevation of phosphorylated H3 levels may contribute to the aberrant gene expression observed in the oncogene-transformed cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H3 phosphorylation at Ser-10 occurs concurrently with the transcriptional activation of the immediate early response genes (e.g., c-fos; Ref. 4 ). We were the first to provide direct evidence that after stimulation of the Ras-MAPK signaling pathway, the newly phosphorylated H3 is associated with the c-fos gene (1) . Furthermore, indirect immunolocalization studies demonstrated that multiple nuclear sites become associated with phosphorylated H3 after stimulation of the Ras-MAPK signaling pathway (1) . It is conceivable that several of these sites correspond to sites where phosphorylated H3 is associated with genes that are rapidly transcriptionally activated by the Ras-MAPK signaling pathway (5) . One such gene may be the uPA gene, which is activated on stimulation of the Ras-MAPK pathway (6 , 7) . Expression of uPA, which is involved in tumor invasion and metastasis, is increased in several malignancies (7) .
Rsk-2 and MSK1 have been identified as H3 kinases that are activated on stimulation of the Ras-MAPK pathway (8 , 9) . Fibroblasts from Coffin-Lowry syndrome patients, in whom the Rsk-2 gene is mutated, fail to exhibit an EGF-stimulated phosphorylation of H3. MSK1 is activated by the Ras-MAPK and the p38 stress kinase pathways, both of which, when stimulated, result in the phosphorylation of H3 (9 , 10) . Rsk-2 is activated by extracellular signal-regulated kinases, but not by p38.
In this study, we investigated the contribution of Rsk-2 and MSK1 to TPA- and EGF-stimulated phosphorylation of H3 in 10T and Ciras-3 mouse fibroblasts. Our studies show that inhibition of MSK1, but not Rsk-2, prevented phosphorylation of H3. Furthermore, we show that inhibition of MSK1 suppressed the induction of c-fos and uPA genes in the parental and oncogene-transformed cells. Our results provide evidence that MSK1 is the kinase persistently activated in Ciras-3 cells, resulting in the elevated levels of phosphorylated H3.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
10T and Ciras-3 mouse fibroblasts were cultured in 
-MEM supplemented with 10% (v/v) fetal bovine serum. Cells were grown in complete medium until 100% confluence. They were then washed twice with PBS and cultured in -MEM supplemented with 0.1% fetal bovine serum for 24 or 48 h, respectively, to synchronize cells in the G0 phase of the cell cycle. Quiescent cells were pretreated with various inhibitors [H89 (10 µM), PD 98059 (100 µM), or Ro 318220 (5 nM)] in DMSO for 30 min and then stimulated with EGF (50 ng/ml) or with TPA (100 nM) for various times. After treatment, cells were rinsed twice with PBS and harvested with 0.2% trypsin-EDTA, pelleted, and kept at -70°C until further analysis.
Preparation of Cell Extracts.
After treatment, cells were harvested and lysed in 400 µl of ice-cold SB-250 Buffer [250 mM NaCl, 25 mM Tris-HCl (pH 7.5), 5 mM Na-EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 25 mM ß-glycerophosphate]. Cell extracts were spun at 10,000 x g for 10 min at 4°C, and the supernatant was retained. Protein in the supernatant was quantified using the Bio-Rad Protein Assay as per the manufacturer’s instructions.
Histone Preparation.
Isolation of histones was carried out as described previously (1)
. Briefly, frozen pellets of approximately 1 x 108 cells were lysed with NP40 Buffer [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1.5 mM MgCl2, 0.1% NP40, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 25 mM ß-glycerophosphate]. The nuclei were collected by centrifugation and resuspended in Reticulocyte Standard Buffer [10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 10 mM NaCl, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 25 mM ß-glycerophosphate]. Histones were isolated by extraction with 0.4 N H2SO4. Quantification of histone content was performed by the trichloroacetic acid protein precipitation assay.
Isolation of Histone H3-H4 Fraction.
A histone H3-H4 fraction was isolated from chicken mature erythrocytes as described previously (11)
. Nuclei were digested with micrococcal nuclease, and the chromatin was fractionated as described previously (11)
, except that the duration of incubations with nuclease was 12 min. Digested nuclei were resuspended in 10 mM EDTA, and solubilized chromatin fragments (fraction SE) were obtained. Total chromatin fragments were applied to a hydroxylapatite (Bio-Rad) column at a ratio of 1 mg of DNA:0.25 g of hydroxylapatite. The column was washed with 0.63 M NaCl in 0.1 M potassium phosphate (pH 6.7) to remove H1 histones and nonhistone chromosomal proteins. A linear NaCl gradient from 0.63 to 2 M in 0.1 M potassium phosphate (pH 6.7) containing 1 mM DTT was run through the column at a rate of 24 ml/h, and the absorbance at 230 nm of each fraction was measured. Fractions containing the H3-H4 fraction were pooled, dialyzed against deionized distilled water, and lyophilized.
Kinase Assays.
Purified rabbit Rsk-2 was preincubated in the absence or presence of inhibitors Ro 318220 (5 nM in DMSO) or GF 109203X (2 µM in DMSO), total histones or H3-H4 purified by chromatographic methods, and 10 µCi of [-32P]ATP (ICN Biomedicals, Costa Mesa, CA) for 10 min at 4°C and then for 10 min at 30°C. Histones were then acid-extracted as described above, resolved by SDS-15% PAGE (or acetic acid-urea-Triton X-100 15% PAGE; Ref. 12
), and analyzed by Coomassie Blue staining and autoradiography. The radiolabeled histone bands were identified by aligning the bands on the autoradiogram with the stained bands on the SDS and acetic acid-urea-Triton X-100 polyacrylamide gels.
Electrophoresis and Immunoblotting.
Proteins were analyzed by SDS-15% PAGE. The proteins were visualized by Coomassie Blue staining or transferred to nitrocellulose membranes as described previously (12)
. Membranes containing the histones were stained immunochemically with anti-pH3 and horseradish peroxidase-conjugated goat antirabbit antibody (Bio-Rad) using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) as described previously (1)
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Antibodies.
Anti-phosphorylated-histone H3 rabbit polyclonal antibodies were purchased from Upstate Biotechnology.
RNA Purification and Northern Blotting.
Total RNA was isolated from frozen cell pellets using a Qiagen RNeasy Midi Kit according to the manufacturer’s instructions. Seven µg of RNA were resolved on 1% formaldehyde/agarose gels and transferred onto nylon membranes (Zeta probe; Bio-Rad). Hybridizations were carried out using a 32P-labeled probe of c-fos, uPA, or GAPDH.
Probes.
A 1.2-kb insert of mouse uPA cDNA was obtained from Dr. Mike Ostrowski. A 1.0-kb mouse GAPDH insert was supplied by Dr. Robert Shiu. The mouse c-fos insert was purchased from American Type Culture Collection (Manassas, VA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Ciras-3 cells with TPA elevates the steady-state levels of H3 phosphorylated at Ser-10 (Fig. 1)
. To determine whether the kinase phosphorylating H3 was being activated through the Ras-MAPK pathway in the parental and oncogene-transformed cells, the cells were incubated with PD 98059, a specific inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase. The mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor blocked TPA-induced phosphorylation of H3 in 10T and Ciras-3 cells. Thus, TPA induces phosphorylation of H3 through activation of the Ras-MAPK signaling pathway.
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and Ciras-3 cells did not affect TPA-induced phosphorylation of H3. This observation suggested that neither Rsk-2 nor MSK1 was the H3 kinase.
To test directly whether Rsk-2 was an H3 kinase, purified Rsk-2 was incubated with mouse fibroblast histones and a histone preparation enriched in avian erythrocyte H3 and H4. As controls, the Rsk-2 preparation was incubated with GF 109203X, an inhibitor of Rsk-2 (13)
, and the histones were incubated with [32P]ATP in the absence of enzyme. The autoradiogram shown in Lane 1 of Fig. 2
shows that two bands were excellent substrates for Rsk-2. One band was identified as H2B, which has two perfect Rsk-2 consensus phosphorylation sites (RXXS). The other band was not known. The labeling of these proteins was reduced when the Rsk-2 preparation was incubated with GF 109203X (Lane 3). Weak labeling of the H3 band was observed when the enriched H3 and H4 preparation was incubated with Rsk-2 (Lane 2), with GF 109203X-treated Rsk-2 (Lane 4), and without enzyme (Lanes 6 and 7). Noncovalent binding of radiolabeled ATP to the histones may explain the weak labeling of the acid-extracted histones and H3/H4. We conclude that H3 is not a substrate for Rsk-2.
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and Ciras-3 cells were incubated with H89 before the addition of TPA. H89 is a potent inhibitor of MSK1 and inhibits the enzyme’s ability to phosphorylate H3. However, H89 does not inhibit MSK1 phosphorylation activity toward CREB or inhibit the phosphorylation of transcription factors involved in EGF- and TPA-induced expression of immediate early genes (9)
. Fig. 3
shows that H89 inhibited TPA-induced phosphorylation of H3 in mouse 10T and Ciras-3 cells. The inhibition was reproducibly most pronounced with Ciras-3 cells. Similar results were obtained with EGF-simulated cells (data not shown). In contrast to our results with Ro 318220, these observations provide evidence that MSK1 is the TPA-induced H3 kinase.
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and Ciras-3 cells. Fig. 4
shows that c-fos mRNA levels were greatest at 15 min in 10T and 30 min Ciras-3 after the addition of TPA. In other Northern blot experiments, the time of maximal c-fos mRNA levels was 30 min for both 10T and Ciras-3. Pretreatment of the 10T cells before the addition of TPA with H89 did not prevent the TPA-induced increase in the c-fos mRNA level at 15 min. However, H89 did suppress the extent of c-fos gene expression and the duration of c-fos mRNA detection. Thomson et al. (9)
obtained similar results. With H89-treated Ciras-3 cells, the TPA-induced c-fos gene expression was severely repressed.
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cells 30 min after the addition of TPA (Fig. 5)
. At 90 min, uPA mRNA levels declined. Similar results were obtained with Ciras-3 cells, in which uPA message levels peaked at 60 min and then declined. In 10T and Ciras-3 cells, the addition of H89 was equally effective in preventing a TPA-induced increase in uPA mRNA (Fig. 5)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fibroblasts and H-ras-transformed fibroblasts. H3 phosphorylation in association with histone acetylation destabilizes higher-order chromatin structures and chromatin fiber oligomerization (1)
. These chromatin remodeling events aid in transcription of the gene.
Rsk-2 has been reported to phosphorylate H3 at Ser-10 (8)
. Our results, however, demonstrate that in vitro, Rsk-2 phosphorylates H2B but not H3. An identical result was obtained with immunoprecipitated Rsk-2 isolated from TPA-stimulated 10T cells. The two Rsk-2 phosphorylation sites in H2B are not typically accessible to Rsk-2 because this region of H2B is buried within the nucleosome (14)
. Furthermore, Rsk-2 inhibitors Ro 318220 and GF 109203X failed to inhibit the phosphorylation of H3 in vitro and in situ.
Ro 318220 and H89 are inhibitors of MSK1. H89, but not Ro 318220, inhibits phosphorylation of H3. In contrast, Ro 318220, but not H89, inhibits CREB phosphorylation (9 , 10) . Studies with mouse embryonic stem cells homozygous for disruption of the MSK1 gene failed to phosphorylate CREB or ATF1 after stimulation of the Ras-MAPK pathway with TPA or EGF (15) . To explain these observations, it is conceivable that the two kinase domains of MSK1 phosphorylate different substrates. The NH2-terminal kinase domain of MSK1 may phosphorylate transcription factors such as CREB and ATF1, whereas the COOH-terminal kinase domain may phosphorylate H3. Studies with MSK1 mutants will be necessary to decide whether the two kinase domains have different substrate preferences.
We observed that the maximal level of TPA-induced c-fos mRNA expression was lower in Ciras-3 cells than in 10T cells, consistent with the findings of others (16, 17, 18)
. Previous studies have shown that activation of fra-1 by oncogenic ras and the greater abundance of c-Jun and Fra-1 (AP1) dimers repress c-fos expression (17
, 18)
.
TPA-induced c-fos and uPA expression was reduced in H89-treated 10T and Ciras-3 cells. We have shown that on stimulation of the Ras-MAPK pathway, H3 associated with the c-fos coding region becomes phosphorylated. The phosphorylation of H3 may aid in releasing the block in elongation (1)
. Inhibiting MSK1 kinase activity prevents phosphorylation of H3, which may obstruct the complete removal of the elongation block. Similarly, our results suggest that H3 bound to the coding region of the uPA gene also becomes phosphorylated when the Ras-MAPK pathway is activated and that preventing the phosphorylation of H3 hinders the transcription of the gene.
H89 was very effective in preventing TPA-induced H3 phosphorylation in Ciras-3 cells (H-ras-transformed 10T cells). This observation suggests that persistent activation of the Ras-MAPK pathway results in a prolonged activation state of MSK1, leading to the elevated levels of phosphorylated H3 that we observe in these cells (1)
. Considering the effect of H89 in repressing the expression of c-fos and uPA, the converse (that is, the persistent activation of the Ras-MAPK pathway and MSK1) may contribute to the decondensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells (1)
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FOOTNOTES |
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1 Supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society. J. R. D. is a Canadian Institutes of Health Research Senior Scientist. 
2 To whom requests for reprints should be addressed, at Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, R3E OV9 Canada. Phone: (204) 787-2391; Fax: (204) 787-2190; E-mail: Davie{at}cc.umanitoba.ca
3 The abbreviations used are: MAPK, mitogen-activated protein kinase; uPA, urokinase plasminogen activator; TPA, 12-O-tetradecanoylphorbol-13-acetate; EGF, epidermal growth factor; PMSF, phenylmethylsulfonyl fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CREB, cAMP-responsive element-binding protein.
Received 7/17/01. Accepted 10/30/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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