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J. Biol. Chem., Vol. 278, Issue 34, 32367-32372, August 22, 2003
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From the Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854
Received for publication, April 16, 2003 , and in revised form, June 9, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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One set of targets of ERK is the RSK family of proteins including RSK2 (8). RSK2 is a serine/threonine kinase activated by ERK1/2 MAP kinase and PDK1. Like other RSK family members, RSK2 consists of two kinase domains, an NH2-terminal catalytic domain (NTD) that is similar to protein kinases B and C and a COOH-terminal catalytic domain (CTD) that is similar to calcium/calmodulin-dependent protein kinases I and II (9). Activation of RSK2 is complex. In response to growth factors like EGF, inactive RSK2 is phosphorylated by ERK at the catalytic loop of the CTD (10, 11). The CTD autophosphorylates RSK2 at serine 386 in the connecting domain (12). This enables further phosphorylation by the constitutively active kinase PDK1 at serine 227 in the NTD (10). The NTD is the kinase domain responsible for phosphorylation of substrate molecules such as histone H3, CREB, and Akt (13). Mutations in RSK2 are linked to Coffin Lowry syndrome in humans, an X-linked disease marked by cognitive disabilities and skeletal malformations (14). In cells derived from Coffin Lowry syndrome patients, EGF-induced CREB phosphorylation and c-fos transcription did not occur (15). Hence RSK2 appears to be required specifically for these functions. Finally, RSK2 is also required for EGF-induced phosphorylation of histone H3 (16) implicating RSK2 in the regulation of chromatin remodeling. The regulation of RSK activity presents a complex problem that is just beginning to be solved.
We have previously reported that the small death effector domain protein PEA-15 regulates the ERK MAP kinase pathway. PEA-15 is a 15-kDa protein that was originally identified as a major astrocytic phosphoprotein (17, 18). The first 80 amino acids of PEA-15 correspond to the canonical death effector domain (DED) sequence found in proteins that regulate apoptotic signaling pathways (19, 20). The remaining 51 amino acids contain a serine (Ser-104) that is phosphorylated by protein kinase C (17) and a serine (Ser-116) phosphorylated by calcium calmodulin kinase II (21). PEA-15 blocks an H-Ras to integrin signal (22). More recently we have shown that PEA-15 blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their accumulation in the nucleus (23). PEA-15 contains a nuclear export sequence required for its capacity to maintain ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with a consequent increase in c-fos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase (23).
We now show that PEA-15 binds RSK2 and alters its activity. PEA-15 does not bind to RSK1 and therefore appears to exhibit some binding specificity. PEA-15 expression impairs RSK2 kinase activity and prevents RSK2 accumulation in the nucleus. Moreover, PEA-15 blocks the ability of RSK2 to activate CREB mediated transcription and to phosphorylate histone H3. Hence we have identified a potentially crucial RSK2 binding partner that can regulate the outcome of RSK2 signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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[35S]Methionine Labeling and GST Pull-downs—CHO-K1 cells were cultured to 90% confluency in a 100-mm tissue culture dish. Cells were then cultured for at least 4 h in the presence of 35S-labeled methionine. Cells were lysed in MLB buffer (25 mM HEPES, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl2, 1mM EDTA, and protease inhibitors) and then subjected to a pull-down assay using equal amounts of purified GST or GST-PEA-15 immobilized on agarose beads. The cell lysates were incubated with the purified proteins for at least 1 h at 4 °C. The beads were washed extensively with MLB buffer and then once with buffer containing 50 mM Tris-Cl (pH 7.5) and 150 mM NaCl. The proteins were then cleaved with 1 unit of thrombin in a buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 2.5 mM of CaCl2. The beads were discarded, and the supernatant was subjected to SDS-PAGE. The gels were dried and autoradiographed.
Immunoprecipitations and Kinase Assays—COS-7 cells were
transfected with pMT2 HA-RSK2 in 100-mm dishes. The cells were serum-starved
for 24 h prior to stimulation with 10 ng/ml EGF for 10 min. Cells were washed
in phosphate-buffered saline, and lysates were obtained by scraping the cells
in cold lysis buffer containing 20 mM Tris (pH 8), 137
mM NaCl, 10% glycerol and 1% Nonidet P-40. Cell debris was
discarded after centrifuging at 14,000 rpm for 20 min at 4 °C. The
supernatant was used to immunoprecipitate HA-RSK2 using the monoclonal
HA-antibody 12CA5. Lysates were incubated with the antibody for 2–3 h on
ice. Immune complexes were precipitated with 50 µl of protein A-Sepharose
(Amersham Biosciences) beads for 1 h at 4 °C. The precipitates were washed
extensively in lysis buffer supplemented with 1 M NaCl. For kinase
assays, the precipitates were resuspended in 40 µl of buffer containing 20
mM MOPS (pH 7.2), 25 mM 
-glycerophosphate, 5
mM EGTA, 1 mM dithiothreitol and split into two equal
aliquots. One aliquot was used in an S6 peptide kinase assay (Upstate
Biotechnology), while the other aliquot was subjected to immunoblotting to
verify equal RSK2 precipitation. The kinase assay was carried out according to
the manufacturer's protocol using an S6 peptide substrate to determine
radiolabeled phosphate incorporation by scintillation counting. Alternatively,
the S6 peptide was substituted with 10 µg of myelin basic protein,
GST-PEA-15, or GST, and the reaction was arrested with sample buffer after 10
min. Proteins were separated by SDS-PAGE, dried, and visualized by
autoradiography.
His-tagged Protein Precipitations—His6-tagged constructs of PEA-15 (wt and mutants) were transfected into HeLa cells using Polyfect (Qiagen) according to the manufacturer's protocol. The cells were cultured for 48 h prior to lysing with 1 ml of Nonidet P-40 lysis buffer/100-mm dish. Lysates were obtained after centrifugation at 14,000 rpm for 20 min at 4 °C to remove cell debris. For each sample, 50 µl of Probond Ni+2 column (Invitrogen) was used after washing the column once with distilled water and lysis buffer. Lysates were incubated with the columns for 45–60 min at 4 °C. The pellets with the bound protein were washed extensively with lysis buffer and finally resuspended in 100 µl of sample buffer. Proteins were subjected to SDS-PAGE and immunoblotting for endogenous RSK2.
Immunofluorescence Assays—COS-7 cells were co-transfected with HA-RSK2 and GFP or PEA-15 fused to GFP. Cells were grown for 24 h and then serum-starved for 24 h. Cells were then treated with 50 ng/ml EGF for 0, 20, or 50 min before fixing with 4% paraformaldehyde for 10 min. Cells were then stained overnight at 4 °C with anti-RSK2 antibody (Upstate Biotechnology) in 3% bovine serum albumin and 0.2% Triton X-100 in phosphate-buffered saline. The antibody was detected using anti-rabbit Texas Red-conjugated antibody from Molecular Probes. The cells were imaged in a Zeiss Axiovert 100M fluorescent microscope using a 100X oil-immersion objective.
Transcription Assays—Transcription assays for RSK2 activity were carried out using the Stratagene path detect kit specific for CREB-mediated transcription. The strategy involves the use of a Gal4DBD-tagged CREB construct (pFA2-CREB) and a luciferase construct carrying the Gal4 promoter region (pFR-Luc). Using this approach, only the effect of transfected CREB on luciferase expression is measured. 250 ng of pcDNA3 or PEA-15 (wt and mutants) was co-transfected with 500 ng of pFR-Luc and 50 ng of pFA2-CREB constructs. Cells were left serum-starved for 16–20 h prior to stimulation with 20% serum for 1 h. Luciferase readings were obtained using the Promega assay kit according to the manufacturer's protocol. To verify the specificity of the effect for RSK2, activated RSK2 (Y707A) was co-transfected along with the other plasmids, and cells were left serum-starved prior to lysis. Luciferase readings were obtained in a luminometer for 5 µg of total cellular protein according to the manufacturer's protocol.
In Vitro Transcription/Translation—pcDNA3-RSK2 plasmid was used to transcribe RNA with a linked protein transcription/translation system (Ambion). The proteins were labeled using [35S]methionine according to the manufacturer's protocol. One-tenth of the reaction was retained to load on the gel as input. The remaining 40 µl of the reaction was diluted to 500 µl in buffer containing 25 mM HEPES, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, and protease inhibitors. Purified GST-PEA-15 or GST immobilized on agarose beads was used as bait to carry out a protein pull-down. The translation mixture containing full-length proteins was first precleared with GST for one-half hour at 4 °C. Equal amounts of GSTPEA-15 or GST were used for a pull-down experiment with the precleared reaction mixtures. The proteins were incubated for 2–3 h at 4 °C before washing the pellet extensively with MLB buffer. The samples were resuspended in sample buffer and subjected to SDS-PAGE, drying, and autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To determine whether the interaction is direct, we used a linked in vitro transcription and translation system to produce [35S]methionine-labeled RSK2 and RSK1 proteins. We then mixed the labeled RSKs with agarose beads coated with purified GST-PEA-15. As controls we included GST-coated agarose beads. PEA-15 binds RSK2 but not RSK1 or GST (Fig. 1C). Hence PEA-15 and RSK2 can interact directly as purified proteins.
To confirm the interaction we used an additional in vivo assay to isolate the protein complex. We co-transfected His tagged PEA-15 with HA-tagged RSK2 in COS-7 cells and allowed expression for 48 h. We then immunoprecipitated RSK2 using an anti-HA antibody (Cell Signaling Technology) and immunoblotted for PEA-15. We found a significant amount of PEA-15 co-immunoprecipitated with RSK2 (Fig. 1D). Additionally, we were able to observe endogenous PEA-15 that had also been precipitated in a complex with RSK2 (data not shown). Thus we have shown that PEA-15 and RSK2 interact with each other and that the interaction is direct.
To determine the regions of PEA-15 that may be necessary for this interaction, we used point mutants of PEA-15 in the DED as well as the COOH-terminal regions of the protein. These two regions have been shown to be important for the interaction of PEA-15 with ERK, and mutations in these regions affect the ability of PEA-15 to alter ERK function (24). The D74A mutation in the DED and the L123R mutation in the COOH-terminal tail abolish ERK binding to PEA-15 in vitro, and these mutants were therefore used as representatives. We also used a previously characterized point mutant in the nuclear export sequence of PEA-15 (I15A), which allows PEA-15 accumulation in the nucleus. We observed that whereas the D74A mutant and the I15A mutant bind RSK2 in vivo, the L123R tail mutant does not (Fig. 1E). Hence the interaction of PEA-15 with RSK2 involves the COOH-terminal domain of the protein. Furthermore, although the D74A mutation completely prevents ERK binding it has no effect on RSK2 binding. This supports a model in which PEA-15 can interact with ERK1/2 and RSK2 independently.
Because PEA-15 binds RSK2, we determined whether the two proteins co-localize in the cell. We found that whereas PEA-15 appeared to be strongly co-localized with RSK2, control cells transfected with GFP did not show similar co-localization patterns (Fig. 2). Neither PEA-15 nor RSK2 appeared to be confined to any particular compartment of the cytoplasm but instead, under these conditions, were diffusely distributed throughout. These co-localization experiments provided further in vivo evidence for an interaction between PEA-15 and RSK2. Thus we have shown that PEA-15 interacts directly with RSK2, co-localizing with it in the cell, and that the interaction involves the COOH-terminal domain of PEA-15.
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PEA-15 Affects RSK2-dependent Transcription—Having found that PEA-15 binds to RSK2, we sought to determine whether PEA-15 expression affects RSK2 function. One known function of RSK2 is the activation of the transcription factor CREB (15, 25). To examine the functional consequences of PEA-15 binding to RSK2 we carried out in vivo transcription assays using a CREB-driven luciferase construct in the Path-detect system. Cells were treated with serum or not and then lysed and assayed for luciferase activity as a measure of CREB-mediated activation of transcription. Cells transfected with PEA-15 showed impaired CREB activity (Fig. 3). For comparison, PEA-15 had little impact on the activation of the c-Jun transcription factor.
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We then sought to exclude the possibility that PEA-15 blocks CREB activation by an RSK2-independent mechanism. We therefore examined whether PEA-15 expression could alter activation of CREB by an activated mutant of RSK2 (Fig. 4, Y707A). PEA-15 was co-transfected with the activated RSK2 construct. Cells transfected with wt PEA-15 strongly inhibited RSK2-dependent CREB-mediated transcription (Fig. 4). Furthermore a non-ERK-binding tail mutant (L123R) and the nuclear export sequence mutant, I15A, did not inhibit transcription (Fig. 4). Activated RSK2 expression was verified by blotting for RSK2 in the lysates (Fig. 4). Hence, PEA-15 is able to impair RSK2 activation of CREB.
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Over-expression of PEA-15 Decreases Phosphorylation of Histone H3—RSK2 is reportedly required for the phosphorylation of Histone H3 (16). To further determine the functional consequences of PEA-15 expression on RSK2 kinase, we examined the effect of PEA-15 on the RSK2-specific nuclear substrate histone H3. We co-transfected COS-7 cells with RSK2 and PEA-15 or vector control. The MEK inhibitor was used to remove the high background phosphorylation of histone H3 at Ser-10, which was observable even upon serum starvation (data not shown and previous reports). Cells were then stimulated with EGF. We observed that PEA-15 expression blocked H3 phosphorylation compared with the vector control (Fig. 5) in response to EGF stimulation.
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PEA-15 Affects RSK2 Kinase Activity—One mechanism by which PEA-15 may block RSK2 activation of CREB and histone H3 phosphorylation is by blocking the kinase activity of RSK2. To address this hypothesis, we looked at the effects of PEA-15 on RSK2 kinase activity using an S6 peptide-based kinase assay. In this assay PEA-15 or control vector alone were cotransfected with HA-tagged RSK2 into cells followed by immunoprecipitation of the RSK2 and determination of its ability to phosphorylate S6 peptide. We found that PEA-15 inhibits RSK2 kinase activity by 50% compared with a vector control (Fig. 6A). Surprisingly, the nuclear localized PEA-15 mutant (I15A) did not appear to inhibit RSK2 kinase activity (Fig. 6A) suggesting that the inhibition of RSK2 kinase activity by PEA-15 occurs in the cytoplasm and is not simply a function of PEA-15 binding itself.
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Alternatively, PEA-15 may impair RSK2 phosphorylation of S6 by acting as a competitive substrate for RSK2. To determine whether PEA-15 is a substrate of RSK2, we immunoprecipitated RSK2 after stimulation with EGF and carried out a kinase assay using purified GST-PEA-15, myelin basic protein (a good substrate for RSK2 activity), or GST as substrate. After the kinase reaction, proteins were separated by SDS-PAGE and subjected to autoradiography. Although myelin basic protein was efficiently phosphorylated by RSK2, neither PEA-15 nor GST was phosphorylated (Fig. 6B). Hence, PEA-15 is not a substrate of RSK2 and does not act as a competitive substrate in blocking S6 kinase activity.
PEA-15 may inhibit RSK2 kinase activity by preventing full activation of RSK2 in cells. Complete activation of RSK2 requires phosphorylation of Ser-386 by the COOH-terminal kinase domain. To determine whether PEA-15 expression alters phosphorylation at this site, cells stimulated with EGF in the presence or absence of over-expressed PEA-15 were immunoblotted with a Ser-386 phospho-epitope antibody. PEA-15 did not affect phosphorylation at Ser-386 (Fig. 6C). Therefore PEA-15 does not affect RSK2 activity by altering its activation state.
PEA-15 Alters RSK2 Localization—A mutant of PEA-15 that accumulates in the nucleus (I15A) did not impair the kinase activity of RSK2 (Fig. 6A). In addition, PEA-15 has been shown to bind ERK and sequester it in the cytoplasm (23). This suggests that PEA-15 might regulate RSK2 similarly by altering its subcellular localization. To test this hypothesis, we investigated whether PEA-15 regulates the translocation of RSK2 after EGF stimulation. Serum-starved cells co-transfected with RSK2 and either GFP or wt PEA-15 fused to GFP showed RSK2 predominantly localized to the cytoplasm (Fig. 7). On stimulation with EGF for 20 min, nuclear translocation of RSK2 was observed in control cells transfected with GFP alone. However, in cells transfected with the PEA-15 construct, RSK2 fails to undergo nuclear translocation. At 50 min post-EGF stimulation RSK2 re-entered the cytoplasm in the control cells, whereas no change was observed in cells transfected with PEA-15. RSK1 has been suggested to undergo transient membrane localization as an immediate response to EGF stimulation (26). However, we were unable to detect this in our system for RSK2 even at very early time points post-EGF stimulation (data not shown). Our results therefore suggest that PEA-15 changes the subcellular localization of RSK2. This may contribute to the effect of PEA-15 on CREB-mediated transcription and appears to be a requirement for PEA-15 inhibition of RSK2 kinase activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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PEA-15 is reported to affect ERK MAP kinase signaling and apoptosis (23, 28–30). Perhaps some of these effects are due to the interaction of PEA-15 with RSK2. For example, c-fos activation in PEA-15-null mice was shown to be twice that of wild type mice. RSK2 has been shown to be required for activation of c-fos (31). Hence, PEA-15 binding to RSK2 may normally play a role in limiting the ability of RSK2 to access nuclear targets such as c-fos, CREB, and histone H3. Cells lacking PEA-15 may then have abnormally high levels of these activities of RSK2 leading to effects on the cell cycle. Interestingly PEA-15-null cells are also reported to sustain an increased rate of proliferation (23).
We have also shown that PEA-15 binds ERK1/2 (23, 24). Here we show that RSK2 binds PEA-15 in a manner that is distinct from that of ERK. Specifically, RSK2 binds to a mutant PEA-15 that cannot bind ERK (D74A). This suggests that the two binding sites are different and that PEA-15 may be able to bind to both ERK and RSK2 at the same time, perhaps preventing or promoting their interaction. However the fact that neither RSK2 nor ERK could bind the PEA-15 tail mutant (L123R) indicates that the binding sites on PEA-15, although distinct, may overlap. The precise nature of these interactions remains to be determined.
RSK2 phosphorylates histone H3 in response to EGF stimulation (16). The phosphorylation of histone H3 by RSK2 was hypothesized by Sassone-Corsi and colleagues (16) to be important in MAP kinase regulation of transcription. Here we show that expression of PEA-15 inhibits RSK2 phosphorylation of histone H3. This is likely because of the perturbation of RSK2 translocation to the nucleus. Rapid phosphorylation of histone H3 correlates with EGF-induced expression of immediate-early response genes such as c-fos (32). We previously reported that cells lacking PEA-15 have a significant increase in c-fos expression (23). This is in agreement with the hypothesis that PEA-15 negatively regulates RSK2 activity.
It is interesting that PEA-15 alters both the subcellular localization of RSK2 and its activity. We have found that a mutant of PEA-15 that binds RSK2 but is localized to the nucleus does not block RSK2-mediated activation of transcription (Fig. 4, I15A). Moreover, the same mutant does not block RSK2 kinase activity (Fig. 6A). We propose that this is evidence that effects on transcription and RSK2 activity in the cell are predominantly due to the altered localization of RSK2. However it remains possible that binding of the I15A mutant of PEA-15 is physically different from that of wild type and therefore is incapable of blocking RSK2 kinase activity. Alternatively, retention of RSK2 in the cytoplasm may decrease RSK2 activity by an as yet undetermined mechanism.
PEA-15 is one of the first RSK-binding proteins suggested to regulate RSK
function negatively. It is the first that binds specifically to RSK2. Recently
the adapter protein 14-3-3 was reported to bind specifically to RSK1 and
to regulate it negatively by interfering with RSK1 phosphorylation
(33). Together these data
suggest that there may be several RSK binding partners that can maintain
signal specificity and modulate the growth factor response by different
mechanisms. As such, this work provides important new insight for unraveling
the complex regulation of RSKs.
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FOOTNOTES |
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To whom correspondence should be addressed: Nelson Biological Laboratories,
Rutgers, The State University of New Jersey, 604 Allison Rd., Piscataway, NJ
08854. Tel.: 732-445-8084; Fax: 732-445-5870; E-mail:
ramos{at}biology.rutgers.edu.
1 The abbreviations used are: ERK, extracellular signal-regulated kinase;
CREB, cAMP response element-binding protein; CTD, COOH-terminal catalytic
domain; DED, death effector domain; GFP, green fluorescent protein; GST,
glutathione S-transferase; HA, hemagglutinin; EGF, epidermal growth
factor; MBP, myelin basic protein; MEK, MAP/ERK kinase; NTD,
NH2-terminal domain; PDK1, phosphoinositide-dependent protein
kinase 1; PEA-15, phosphoprotein enriched in astrocytes, 15 kDa; RSK,
ribosomal S6 kinase; wt, wild type; MOPS, 4-morpholinepropanesulfonic
acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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