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[Cancer Research 65, 3596-3603, May 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

The p53 Protein Is a Novel Substrate of Ribosomal S6 Kinase 2 and a Critical Intermediary for Ribosomal S6 Kinase 2 and Histone H3 Interaction

Yong-Yeon Cho, Zhiwei He, Yiguo Zhang, Hong Seok Choi, Feng Zhu, Bu Young Choi, Bong Seok Kang, Wei-Ya Ma, Ann M. Bode and Zigang Dong

Hormel Institute, University of Minnesota, Austin, Minnesota

Requests for reprints: Zigang Dong, Hormel Institute, University of Minnesota, 801 16th Avenue Northeast, Austin, MN 55912. Phone: 507-437-9600; Fax: 507-437-9606; E-mail: zgdong{at}hi.umn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tumor suppressor p53 protein is one of the most highly connected nodes in cellular signal transduction pathways and acts as a central regulatory switch in networks controlling cell proliferation and apoptosis. It is involved in the activation of genes that maintain control over cellular responses to DNA errors such as DNA repair, chromosomal recombination, and chromosome segregation. Here we show that ribosomal S6 kinase 2 (RSK2) activates and phosphorylates p53 (Ser15) in vitro and in vivo and colocalizes with p53 in the nucleus. Deficiency of p53 diminishes RSK2-mediated phosphorylation of histone H3 (Ser10) and adding back p53 to p53–/– embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. These results show that the p53 protein is an important substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. The RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mitogen-activated protein kinase (MAPK) cascades regulate many cellular responses induced by various environmental stimuli, resulting in the transcriptional activation of immediate-early-response genes (IEG; ref. 1). The rapid induction of IEG by external stimuli is associated with the delivery of intracellular signals to transcription factors and cofactors at regulatory elements as well as nucleosomes present both at the promoter and within the transcribed region of the genes (2). Especially, the Ras/extracellular signal–regulated kinase (ERK) pathway regulates cell proliferation, survival, growth and motility (3, 4), and tumorigenesis (5).

The 90-kDa ribosomal S6 kinase (p90 RSK) comprises a family of broadly expressed serine/threonine kinases in response to many growth factors, peptide hormones, and neurotransmitters via ERK1 and ERK2 (6, 7). The RSK2 is a member of the p90 RSK family that is activated by ERK1/ERK2 and PDK1 (8, 9). When a cell is stimulated by a hormone such as epidermal growth factor (EGF), RSK2 phosphorylates Ser133 of cyclic AMP–responsive element-binding protein (CREB; ref. 10), which is a critical regulator of IEG transcription. RSK2 also phosphorylates histone H3 at Ser10 during mitosis (11). Moreover, RSK2 is required for growth factor–stimulated expression of c-fos and transcriptional activation of Elk-1 and the serum response factor but not for activation of CREB or the MAPK pathway in response to platelet-derived growth factor or insulin-like growth factor stimulation (12).

Homozygous deletion and mutations in RSK2 are genetically linked to Coffin-Lowry Syndrome (CLS) in human, an X-linked disease marked by cognitive disabilities, short stature, skeletal abnormalities, and abnormal characteristics of the face, trunk, and limbs (13). However, although RSK2 knockout (KO) mice show impaired glycogen metabolism and altered extracellular signaling (14), insulin resistance, and lipodystrophy (15), no characteristics of CLS observed in humans have been specifically observed in the KO mice. In addition, the mechanisms underlying the many complicated phenotypes induced by one gene deletion or mutation are unclear. When activated, RSK2 translocates to the nucleus, where it may phosphorylate various nuclear proteins such as c-Fos, Elk-1, histones, CREB (10, 11, 16–18), and ATF4 (19). Based on these findings, RSK2 proteins are likely to regulate a variety of cellular processes. Despite extensive studies, the physiologic roles of the RSK2 protein remain elusive. Moreover, because RSK enzymes have broad substrate specificity, RSK2 may be able to bind and/or phosphorylate a number of diverse substrates that regulate cell cycle, depending on the specific situation.

Here, we show that the tumor suppressor p53 protein is a novel substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. In addition, the RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. Chemical reagents, including Tris, NaCl, and SDS, for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). Restriction enzymes and some modifying enzymes were purchased from New England BioLabs, Inc. (Beverly, MA). Superscript II RNase H– reverse transcriptase was from Life Technologies, Inc. (Rockville, MD) and Taq DNA polymerase was from Qiagen, Inc. (Valencia, CA). The DNA ligation kit (version 2.0) was purchased from TAKARA Bio, Inc. (Otsu, Shiga, Japan). The checkmate mammalian two-hybrid system, including expression vectors and the reporter luciferase vector was from Promega Co. (Madison, WI). Cell culture medium and other supplements were purchased from Life Technologies (Rockville, MD). Antibodies for immunoblotting, immunoprecipitation, and immunocytochemical analysis were purchased from Cell Signaling Technology, Inc. (Beverly, MA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Charlottesville, VA).

Cell culture and transfections. 293T and p53+/+ and p53–/– mouse embryonic fibroblast cells were cultured with DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a 5% CO2 incubator. The cells were maintained by splitting at 90% confluence and media were changed every 3 days. For transfection experiments, cells were split and the expression vectors introduced when cells were 50% to 60% confluent using jetPEI (Qbiogen, Inc., Montreal, Quebec, Canada) following the manufacturer's suggested protocol.

PCR amplification of human p53 and ribosomal S6 kinase 2. To clone p53, human mRNAs were reverse transcribed with an oligo-dT primer and SuperScript II RNase H– Reverse Transcriptase (Life Technologies, Grand Island, NY). Primers were synthesized as follows: p53 sense 5'-GCAGGATCCTGGAGGAGCCGCAGTCAGA-3' (BamHI underlined) and p53 antisense 5'-TCGACGCGTGAATGTCAGTCTGAGTCAGG-3' (MluI site underlined); RSK2 sense 5'-CGGGATCCGTATGCCGCTGGCGCAGCTGGCGGAC-3' (BamHI site underlined) and RSK2 antisense 5'-GCTCTAGATCACAGGGCTGTTGAGGTGATTTT-3' (XbaI site underlined). Using these primers, p53 and RSK2 cDNAs were amplified by PCR and introduced into the BamHI and MluI sites of pACT and BamHI and XbaI sites of pBIND, which are the mammalian two-hybrid vectors. The pACT-p53WT and pBIND-RSK2WT constructs were confirmed by restriction mapping and DNA sequencing.

Construction of ribosomal S6 kinase 2 deletion mutants. Deletion mutants of RSK2 were constructed by a PCR-based method as for p53. To amplify the DNA fragments deleted from NH2-terminal, four primers were made: (a) RSK2-3 5'-CGGGATCCGTTTTGAACTTTTAAAAGTATTAGGG-3', (b) RSK2-4 5'-CGGGATCCGTCATTCATTTTTCTCAACAATAGACTGG-3', (c) RSK2-5 5'-CGGGATCCGTATTTCAGTTTACTGATGGATATGAAGTA-3', (d) RSK2-10 5'-CGGGATCCGTCATCCTTGGATTGTCCACTGGTAC-3'. The underlined GGATCC indicates the BamHI sites. These primers were each used with the RSK2 antisense primer. To construct the NH2-terminal kinase (NTK) deletion mutant, RSK2-6 5'-GAAAAGGCAGATCCTTCCCAGCATTCATTTTTCTCAACAATAGAC-3' and RSK2-7 5'-CTGGGAAGGATCTGCCTTTTCATGTCCTTCCTTCACATGATGTGT-3' were combined with RSK2 sense and RSK2 antisense primers and amplified by PCR. DNA fragments corresponding to the deleted size were eluted, digested with BamHI and XbaI, and introduced into the BamHI and XbaI sites of pBIND. To delete the COOH-terminal kinase (CTK) of RSK2, RSK2-8 5'-CATCCTTGGATTGTCCACTGGGACCAACTACCACAATACCAACTAAAC-3' and RSK2-9 5'-GTCCCAGTGGACAATCCAAGGATGTTCATATCCATCAGTAAACTGAAT-3' were combined with RSK2 sense and RSK2 antisense primers, amplified by PCR and introduced into pBIND as described above. All vectors designated pBIND-RSK2D68, pBIND-RSK2D323, pBIND-RSK2D415, pBIND-RSKD674, pBIND-RSK2D69-323, and pBIND-RSK2D416-674 were confirmed by restriction mapping and DNA sequencing.

Mammalian two-hybrid assay. 293T cells (2.0 x 104) were seeded into 48-well plates and incubated with 10% FBS-DMEM for 18 hours before transfection. The DNAs, pACT-p53WT, pBIND-RSK2, and pG5-luciferase, were combined in the same molar ratio and the total amount of DNA was not more than 100 ng per well. The transfection was done using jetPEI following the manufacturer's recommended protocols. The cells were disrupted by addition of cell lysis buffer directly into each well of the 48-well plate and aliquots of 20 µL were added to each well of a 96-well luminescence plate. The luminescence activity was measured automatically by computer program (MTX Lab, Inc., Vienna, VA). The relative luciferase activity was calculated and normalized based on the pG5-luciferase basal control. For assessment of transfection efficiency and protein amount, the luciferase assay, Renilla luciferase activity assay, or Lowry protein assay were used.

Cell cycle analysis. 293T cells (4 x 105) were seeded into 60-mm culture dishes and cultured for 16 hours at 37°C in a 5% CO2 incubator. Cells were transfected with a single or various concentrations of pKH3-RSK2 and cell cycle was analyzed at various time points with 10% FBS, 0.1% FBS, or after stimulation with 50 ng/mL of EGF.

Immunocytofluorescence assay. 293T cells (7.5 x 104) were seeded in two-chamber slides and transfected with mock, pACT-p53WT, pBIND-RSK2WT, or pACT-p53WT/pBIND-RSK2WT. The transfection was followed by a 12-hour incubation, 36-hour starvation, and 10% FBS restimulation for 12 hours. The cells were washed at each time point, fixed in 4% formalin, and hybridized with an anti-VP16 mouse monoclonal antibody to detect p53 and RSK2 rabbit antibody (1:500) at room temperature for 2 hours. The cells were washed and hybridized at room temperature for 1 hour with an antimouse goat antibody conjugated with Texas Red for detection of p53 and/or an antirabbit mouse monoclonal antibody conjugated with FITC for RSK2 detection. Cells were washed again and visualized by fluorescence microscope (x250).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The tumor suppressor p53 protein is a direct substrate of ribosomal S6 kinase 2 in vitro and in vivo. Although RSK2 deficiency is directly linked to CLS, the physiologic roles of the RSK2 protein remain highly elusive. RSK2 KO mice show several metabolic disorders that could be associated with CLS, suggesting that the substrates of RSK2 may be linked to development of this syndrome. To identify novel protein substrates that interact with RSK2, we cloned 32 transcription factors and each candidate transcription factor was introduced into the pACT mammalian two-hybrid (M2H) system vector (pACT-TFs). The RSK2 wild-type cDNA (RSK2WT) was introduced as bait into the corresponding M2H pBIND vector (pBIND-RSK2WT). One of the pACT-TFs and the G5-luciferase reporter plasmid was cotransfected into 293T cells. Results indicate that pBIND-RSK2WT/pG5- luciferase activity was induced 17-fold compared with pG5-luciferase only (Fig. 1A). However, when pACT-p53WT and pBIND-RSK2WT were combined, pG5-luciferase activity was increased to 175-fold, indicating a strong interaction between p53 and RSK2 (Fig. 1A). In addition, cotransfection of various known RSK substrates, including ATF4 (19) and CREBs (10), with RSK2, showed significant increases in pG5-luciferase activity (Fig. 1A) indicating their interaction. These results confirmed that a specific interaction occurred between p53 and RSK2.



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  		Figure 1. p53 protein is a direct substrate of RSK2 in vitro and in vivo. A, protein binding determined by the mammalian two-hybrid (M2H) system. Either one or both of the M2H vectors, pACT-p53WT and pBIND-RSK2WT, were cotransfected with the pG5-luciferase reporter plasmid into 293T cells, and cells were analyzed for firefly luciferase activity. Relative luciferase activity (fold change) was normalized against pG5-luciferase activity. pACT-ATF4, pACT-CREB1, and pACT-CREB3 transfectants were used positive controls. Columns, mean of values obtained from triplicate experiments; bars, ±SD. Significant differences were evaluated using the Student's t test (*, P < 0.001). B, in vitro kinase assay to determine the ability of various kinases to phosphorylate p53. Different kinases were tested for their ability to phosphorylate a GST-p53 fusion protein in vitro. C, Western blotting analysis to determine phosphorylation of p53 at Ser15 using a phospho-specific antibody. Total p53 protein is used as a loading control in this case. D, effect of RSK2 deficiency on p53 phosphorylation. RSK2+ (GM09621) and RSK2– (GM03321) cells were cultured, starved, and stimulated with 4 kJ/m2 UVB. Phosphorylation of p53 (Ser15) was visualized by Western blotting using a phospho-specific antibody. Total RSK2 and ß-actin were used as internal controls for confirmation of RSK2 deficiency and equal protein loading.

 
To determine whether this interaction involved p53 phosphorylation, we did an in vitro kinase assay using various kinases and a glutathione S-transferase (GST)-p53 fusion protein in the presence of [{gamma}-32P] ATP (Fig. 1B). Results showed that RSK2, ERK2, and RSK1 each phosphorylated p53, whereas S6K1, MSK1, MAPKAPK2, MEK1, or Akt1 did not (Fig. 1B). The specific phosphorylation site was Ser15 (Fig. 1C). Importantly, in cells from RSK2-deficient CLS patients (RSK2–), p53 phosphorylation at Ser15 was markedly diminished after UVB stimulation, indicating a dependence on RSK2 (Fig. 1D). These results strongly support our hypothesis that the tumor suppressor p53 protein is an important RSK2 substrate that is phosphorylated directly by RSK2 in vitro and in vivo and the phosphorylation response is deficient in CLS patients.

Binding domain analysis of ribosomal S6 kinase 2 for the tumor suppressor p53 protein. To identify the domain of RSK2 that specifically interacts with p53, six deletion mutants (Fig. 2A; RSK2D68, 1-68 deleted; RSK2D323, 1-323 deleted; RSK2D415, 1-415 deleted; RSK2D674, 1- 674 deleted; RSK2D69-323, 69-323 deleted; RSK2D416-674, 416-674 deleted) were amplified. Each deletion mutant fragment was introduced into the pBIND mammalian two-hybrid system vector and the binding domain of RSK2 interacting with p53 was analyzed by luciferase activity (Fig. 2B). The wild-type RSK2 interacted strongly with wild-type p53; however, none of the NH2-terminal deletion mutants were able to interact with p53 (Fig. 2B), suggesting that the NH2-terminal domain of RSK2 plays an important role in the binding with p53. In addition, the RSK2D69-323 (NTK) deletion mutant still exhibited binding activity with wild-type p53, suggesting that binding did not occur here. Furthermore, the RSK2D416-674 (CTK) deletion mutant did not show any binding activity with wild-type p53 (Fig. 2B), indicating that the CTK domain may also be important in the RSK2 interaction with p53. Our data clearly showed that the p53 protein interacted with NH2-terminal residues 1-68 and CTK domain of RSK2.




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  		Figure 2. RSK2 and p53 colocalize in the nucleus. A, schematic diagrams for construction of RSK2 deletion mutants. B, binding domain analysis of the RSK2 protein interaction with the p53 wild-type protein. pBIND-RSK2 or individual deletion mutants were cotransfected with pACT-p53WT and the pG5-luciferase reporter gene, and their respective interaction was visualized by firefly luciferase activity in the mammalian two-hybrid system. Luciferase activity was adjusted for protein concentration. Columns, mean of values obtained from triplicate experiments; bars, ±SD. Significant differences were evaluated using the Student's t test (*, P < 0.001) normalized against pG5-luciferase reporter activity. C, nuclear translocation of the p53 protein and RSK2. 293T cells were transfected with mock vector, pACT-p53WT, pBIND-RSK2WT, or pACT-p53WT/pBIND-RSK2WT. Ectopically expressed proteins were visualized at 12 hours after transfection under a fluorescence microscope using Texas Red specific for the p53 protein or FITC for RSK2. Pictures of Texas Red, FITC, and light microscopy represent exactly the same region of cells for each (x250). D, effects of RSK2 expression on the nuclear translocation of p53 protein under various conditions. 293T cells were transfected with pACT-p53WT and/or pBIND-RSK2WT and nuclear localization of the p53 protein was visualized at the indicated time point using a Texas Red–conjugated antibody.

 
Ribosomal S6 kinase 2 induces nuclear accumulation of the tumor suppressor p53 protein. In response to DNA damage and other types of cellular stress, p53 normally translocates to the nucleus where it activates a variety of target genes, most of which are involved in cell cycle arrest or apoptosis (20). To examine whether the interaction of RSK2 and p53 induced nuclear localization, we used immunocytofluorescence analysis. 293T cells were transfected with mock vector, pACT-p53WT, pBIND-RSK2WT, or pACT-p53WT/pBIND-RSK2WT. The cells were fixed and hybridized with an anti-VP16 monoclonal and an RSK2 rabbit antibody. Secondary antibodies conjugated with Texas Red were also used for p53 detection and FITC for RSK2 detection (Fig. 2C). Cells showed similar morphology and extensive nuclear accumulation of RSK2 and p53 was detected in cells transfected with pACT-p53WT and pBIND-RSK2WT together compared with cells transfected with either alone (Fig. 2C). Nuclear localization of p53 with and without RSK2 was also examined under normal and stress conditions. In these experiments, pACT-P53WT or pACT-P53WT/pBIND-RSK2WT transfected cells were starved for 36 hours and restimulated with 10% FBS. Cells were fixed and hybridized as for Fig. 2C for detection of p53. Results indicated that in cells transfected with pACT-p53WT alone, p53 was mainly found in the cytosol (Fig. 2D). On the other hand, nuclear accumulation of the tumor suppressor p53 protein was detected in cells cotransfected with pBIND-RSK2WT and pACT-p53WT and the accumulation was enhanced by either 36-hour starvation or 36-hour starvation followed by restimulation with FBS (Fig. 2D). These results provide strong evidence that RSK2 colocalizes in the nucleus with p53 and suggest a novel and important role for the RSK2-p53 interaction.

Activation of p53 by ribosomal S6 kinase 2 is not required for apoptosis. To begin to determine a specific role of endogenous p53 in the RSK2-p53 interaction, we analyzed epidermal growth factor (EGF)–induced p53 activation in cells transfected with a luciferase linked p53 (pG-13) reporter plasmid with or without transfection of RSK2 (pKH3-RSK2; Fig. 3A). Cells transfected with pKH3-RSK2 and treated with EGF showed a dramatic induction of pG-13 luciferase activity compared with mock-transfected control cells (Fig. 3A). Increasing concentrations of EGF had no additional effect suggesting that p53 phosphorylation (Ser15) in cells transfected with mock or pKH3-RSK2 occurred independently of EGF (Fig. 3B) further indicating that phosphorylation of p53 at Ser15 was associated with RSK2 expression rather than stimulation by EGF (Fig. 3A and B). The Ser15 phosphorylation is a critical event in the up-regulation and functional activation of p53 during cellular stress (21), indicating that RSK2 activates p53 by phosphorylation at Ser15.



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  		Figure 3. Activation of p53 by RSK2 under various conditions. A, activation of p53 by RSK2 following exposure to EGF. 293T cells were transfected with the pG-13 luciferase reporter plasmid with or without pKH3-RSK2. Cells were cultured 12 hours, starved in 0.1% FBS-DMEM, and stimulated with the indicated EGF concentration. Cells were tested for firefly luciferase activity. Luciferase activity was adjusted for protein concentration. Columns, mean of values obtained from triplicate experiments; bars, ±SD. Significant differences were evaluated using the Student's t test (*, P < 0.01 and **, P < 0.001). B, 293T cells were or were not transfected with pKH3-RSK2, cultured 12 hours, starved, and stimulated with the indicated concentration of EGF. Overexpression of RSK2 and phosphorylation of p53 (Ser15) were analyzed with an RSK2 and a phospho-specific antibody, respectively. Equal protein loading was confirmed using the ß-actin antibody on the same membrane. C, 293T cells were transfected with the mock vector or pKH3-RSK2, cultured for 12 hours, starved for 36 hours, and stimulated with EGF for 12 hours. From each time point, cells were harvested, fixed, stained with propidium iodide and analyzed for cell cycle phase. Columns, mean of values obtained from triplicate experiments; bars, ±SD. Significant differences were evaluated using the Student's t test (*, P < 0.05 and **, P < 0.01). D, 293T cells were transfected identically as in (C). Cells were harvested, lysed, and treated with proteinase K, and total genomic DNAs were isolated by phenol/chloroform extraction. DNA fragments were visualized with ethidium bromide staining after 2% agarose gel electrophoresis.

 
To further study the role of endogenous p53 protein activation induced by RSK2, we examined cell cycle distribution in cells transfected with mock vector or pKH3-RSK2WT at time points following transfection, starvation, or restimulation with EGF (Fig. 3C). Under standard culture conditions (10% FBS, no starvation), cells overexpressing RSK2 exhibited cell cycle distribution as follows: 40% S, 45% G1, and 15% G2-M compared with 20% S, 50% G1, and 30% G2-M in mock vector control cells (*, P < 0.05 and **, P < 0.01; Fig. 3C). Starvation in 0.1% FBS followed by restimulation with 10% FBS in cells ectopically expressing RSK2 induced an S-phase accumulation of about 86%, G1 to 2%, and G2-M to 12%. This was markedly different from mock vector control cells, which accumulated to only about 27% in the S phase, 23% G1, and 47% G2-M (**, P < 0.01; Fig. 3C). In addition, stimulation of RSK2 overexpressing cells with EGF following starvation resulted in an S phase of about 50%, G1 16%, and G2-M 36% compared with S phase of about 60%, G1 21%, and G2-M 17% for mock control cells (*, P < 0.05; Fig. 3C). Moreover, although RSK2 expression was increased, DNA fragmentation was not detected but instead disappeared, suggesting that overexpression of RSK2 protected cells from apoptosis (Fig. 3D). Cells were confirmed by microscope to be viable. These results strongly suggested that interaction and phosphorylation of p53 protein involving RSK2 could play a novel function in S-phase cell cycle checkpoint regulation, or genomic or chromatin stability, but not apoptosis. This hypothesis is supported by evidence that p53 and ATM are required in the regulation of G1-S and S-phase checkpoint (22) and p53 targets chromatin structure alteration (23).

Tumor suppressor p53 protein is required for phosphorylation of histone H3 by ribosomal S6 kinase 2 in vitro. The p53 protein plays a pivotal role in activating and integrating adaptive cellular responses to a wide range of environmental stresses (24). It acts as a central node in a highly complex signal transduction network that functions primarily to minimize errors that can lead to cancer or other pathologies (25). RSK2 was reported to phosphorylate histone H3 in vivo (11); however, the exact mechanism for phosphorylation of histone H3 by RSK2 is not yet clearly understood and a role for p53 has not been reported. To explore the possibility of a p53, histone H3, and RSK2 interaction, we first analyzed the interaction of p53 and histone H3. Cells were transfected with pACT-p53, pBIND-histone H3, and pG5-luciferase. Under normal cell culture conditions, the interaction between p53 and histone H3 was about 8-fold compared with cells transfected with only pG5-luciferase (Fig. 4A). Stimulation with UVB increased the interaction to about 115-fold in cells transfected with both histone H3 and p53 compared with pG5-luciferase–only cells (Fig. 4A). Moreover, cells transfected with pACT-p53 and pBIND-RSK2 exhibited an interaction between RSK2 and p53 that was increased from 150-fold to about 221-fold after UVB stimulation (Fig. 4A). Furthermore, when cells were transfected with pACT-histone H3 and pBIND-RSK2, pG5-luciferase activities were increased by UVB stimulation from 23- to 79-fold compared with pG5-luciferase only (Fig. 4A). These results suggested that RSK2, p53, and histone H3 could together form a protein complex. We also considered the possibility that p53 could act as a possible cofactor or intermediate between the RSK2 and histone H3 signal transduction network. To investigate this idea, we conducted an in vitro phosphorylation and Western blot analysis using RSK2 and histone H3 in the presence or absence of p53. Results indicated that when combined with p53, RSK2 strongly phosphorylated histone H3, compared with a weak H3 phosphorylation in the absence of p53 (Fig. 4B, lane 2). In addition, the presence of the MDM-2 binding peptide of p53 (residues 17-26 against human p53) did not interfere with the interaction of RSK2 and histone H3 (Fig. 4C, lane 1 versus lane3). These results clearly showed that the p53 protein is required for optimal phosphorylation of histone H3 by RSK2 in vitro.



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  		Figure 4. p53 protein is required for phosphorylation of histone H3 by RSK2 in vitro. A, protein-protein interactions of p53 with histone H3, p53 with RSK2, and RSK2 with histone H3. 293T cells were transfected with pACT-p53WT and pBIND-histone H3, pACT-p53WT, and pBIND-RSK2WT or pACT-histone H3 and pBIND-RSK2WT each combined with the pG5- luciferase reporter plasmid. They were analyzed for luciferase activity with or without stimulation with UVB. Relative luciferase activities were obtained by normalizing against pG5-lucifease activity (value of 1). B, p53 protein mediates histone H3 phosphorylation by RSK2. Active RSK2 and histone H3 were or were not combined with the p53 protein (GST-p53) and supplemented with a final concentration of 200 µmol/L ATP. Reactions were carried out at 30°C for 30 minutes and proteins were visualized by Western blotting using the indicated antibody. C, MDM-2 binding site of p53 protein is not involved in the interaction of RSK2 and histone H3. RSK2 and histone H3 proteins were combined with the p53 protein or the MDM-2 binding peptide of the p53 protein and supplemented with a final concentration of 200 µmol/L ATP. Reactions were carried out at 30°C for 30 minutes and phosphorylated (Ser10) and levels of total histone H3 protein were visualized by Western blotting with specific antibodies.

 
Adding back p53 to p53–/– embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. To examine whether p53 is essential for phosphorylation of histone H3 in vivo, we used p53 knockout embryonic fibroblasts (p53–/–) alone or after adding p53 back. The p53–/– cells were treated with various doses of EGF or UVB, harvested at indicated times and phosphorylation of histone H3 was determined by Western blot using a phospho-histone H3 Ser10 antibody. Phosphorylation of histone H3 at Ser10 was markedly diminished in p53–/– cells regardless of stimulation type (Fig. 5A). On the other hand, transfection of p53 into p53–/– cells restored phosphorylation of histone H3 at Ser10 after stimulation with EGF or UVB in a time- and dose-dependent manner (Fig. 5B). These results strongly suggest the presence of a protein triad complex in which p53 is a required cofactor or intermediate for RSK2 phosphorylation of histone H3 (Fig. 5C).



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  		Figure 5. p53 protein is essential for phosphorylation of histone H3 by RSK2 in vivo. A, wild-type or knockout p53 embryonic fibroblasts were stimulated with EGF or UVB for the indicated time and dose courses, and histone H3 was isolated from the acid-soluble fraction of the nucleus. Phosphorylated (Ser10) and total histone H3 proteins were visualized by Western blotting using specific antibodies. B, wild-type p53 was ectopically expressed in p53–/– cells. The dose-dependent expression of p53 protein was analyzed by Western blotting using a p53 antibody. p53–/– cells expressing wild-type p53 were stimulated with EGF or UVB according to the indicated dose. Histone H3 proteins were isolated from the acid-soluble fraction of the nucleus. Phosphorylation (Ser10) and total histone H3 proteins were visualized by Western blotting using specific antibodies. C, novel function of p53 for chromatin remodeling. RSK2 and p53 interact with high affinity in cytosol. When cells are stimulated, RSK2 is activated through the MAPK cascade and phosphorylated p53 protein at Ser15. RSK2/p53 complex translocates to the nucleus where RSK2 then phosphorylates histone H3 at Ser10 and induces expression of target genes. p53 protein is cofactor or intermediary for RSK and histone H3 interaction.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the p53 protein is a transcriptional regulator, its ability to inhibit cell growth is dependent upon its transactivation function. For example, the transcription factor CBP, which is implicated in cell proliferation and differentiation, functions as a coactivator of p53 and potentiates p53's transcriptional activity (26). The ability of p53 to physically and functionally interact with CBP, which has histone acetyltransferase activity (27), and with components of the general transcription machinery indicates that p53 may have different functions in a multistep activation pathway (26).

The p53 protein forms complex with p300/CREB and is necessary for histone H3 acetylation at the proximal p21 promoter (28). Our data clearly showed that p53 mediates histone H3 phosphorylation by RSK2. Taken together, these data suggest that p53 mediates histone H3 modification by acetylation as well as phosphorylation. Furthermore, our data suggest that the p53 protein is involved in human chromatin diseases such as CLS as well as functioning as a pivotal tumor suppressor. Chromatin structure is important for regulating gene expression and for the proper condensation and segregation of chromosomes during cell division. CLS is a human disease with underlying defects in chromatin structure and modification (29). Over 75 RSK2 mutations have been observed in CLS patients resulting in phenotypes that are severe in males and milder and more variable in females (30). The most common defects include abnormal bone and cognitive development and short stature. However, no consistent relationship has been established between specific mutations and severity or expression of a distinct phenotype, suggesting that various substrates of RSK2 could be involved. For example, RSK2-mediated phosphorylation of CREB is also defective in CLS and was suggested to be associated with deficits in neurologic function in CLS patients (31). Because of p53's role in normal embryonic development, the loss of RSK2-mediated p53 phosphorylation and activation could very likely contribute to various phenotypes of CLS.

Several protein kinases such as ATM, ATR, DNAPK, ERKs, and p38 kinase phosphorylate p53 at Ser15 upon stimulation with UV, DNA damage, or {gamma}-radiation (32) and also RSK2 phosphorylates p53 at Ser15 (Fig. 1C and D). In our study, a slight phosphorylation of p53 at Ser15 was still observed in RSK2 CLS patient cells (Fig. 1D), suggesting that one or more other kinases also may be involved. Recently, phosphorylation of p53 on Thr55 was shown to be induced by treatment with doxorubicin treatment, a DNA intercalating agent, leading to cell death via ERK2 (33, 34). This finding suggested that ERKs and the RSK2 pathway are involved at different site(s) of p53 phosphorylation. RSK2 has been shown to phosphorylate histone H3 at Ser10 when induced by EGF (11). UVB induces phosphorylation of histone H3 through the p38 kinase and ERK pathways (35). However, the exact mechanism is unknown. Here, we provide evidence that the p53 protein is a novel substrate of RSK2 and a critical intermediary for RSK2 and histone H3 interaction. This is the first evidence for a novel function of p53 as an intermediate or cofactor molecule between a serine/threonine kinase and its substrate.


   Acknowledgments
 
Grant support: Hormel Foundation and NIH grants CA77646 and CA81064.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. M.E. Greenberg (Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA) and Dr. J.A. Smith (Department of Pathology, Center for Cell Signaling, University of Virginia, Charlottesville, VA) for the generous gift of the pMT2-HA-RSK2 and pKH3-RSK2, respectively and Andria Hansen for secretarial assistance.


   Footnotes
 
Note: Y-Y. Cho and Z. He contributed equally to this work.

The University of Minnesota is an equal opportunity educator and employer.

Received 11/ 2/04. Revised 1/25/05. Accepted 2/24/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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