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J. Biol. Chem., Vol. 280, Issue 14, 13304-13314, April 8, 2005
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From the
Department of Clinical Biochemistry, Glostrup Hospital, 2600 Glostrup, Denmark, the **Department of Human Genetics, University Medical Centre St. Raboud, P. O. Box 9101, Nijmegen 6500 HB, the Netherlands, the Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4558 Basel, Switzerland, Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, Medical Sciences Institute/Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom, and the ||Kinase Signalling Laboratory, Biotech Research and Innovation Centre, Fruebjergvej 3, 2100 Copenhagen Ø, Denmark
Received for publication, July 20, 2004 , and in revised form, December 17, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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50% of the constitutive RSK4 activity. Finally experiments in mouse embryonic stem cells with targeted deletion of the PDK1 gene suggested that PDK1 was not required for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent findings suggesting that RSK4 can participate in non-growth factor signaling as for instance p53-induced growth arrest. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The substrates of RSK are phosphorylated by the N-terminal kinase (NTK) domain (24–27) whose activity is regulated by the C-terminal kinase domain and a linker region between the two kinase domains (Fig. 1). The activation mechanism of RSK is complex and involves sequential phosphorylation of four regulatory sites (28), which are Ser232, Ser372, Ser389, and Thr581, using RSK4 numbering. First ERK, bound to a C-terminal MAP kinase docking site (29, 30), phosphorylates Ser372 in the linker and Thr581 in the activation loop of C-terminal kinase (28, 31). Phosphorylation of Thr581 activates C-terminal kinase, which thereafter autophosphorylates RSK at Ser389, located in a so-called hydrophobic motif in the linker (32). Phosphorylation of Ser389 in the hydrophobic motif generates a transient docking site that recruits as well as stimulates the activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1) (25), which then phosphorylates Ser232 in the activation loop of the NTK of RSK (33, 34). After dissociation of PDK1 from RSK, the Ser386-phosphorylated hydrophobic motif interacts with a binding pocket within the NTK domain and thereby activates the NTK in synergy with phospho-Ser232 (35). The phosphorylation of Ser372 enhances the activity of the NTK by a yet unknown mechanism (28, 33). Apart from these four activating sites, Thr368 is phosphorylated by ERK, but the site has not been found to regulate kinase activity (28). Moreover Ser742 appears to be phosphorylated by the activated NTK (28, 36), which results in decreased affinity of RSK for ERK, serving as an intramolecular feedback inhibitory mechanism that operates in RSK1 and RSK2 but not in RSK3 (36). The activation mechanism of MSK is thought to be very similar to that of RSK except for two features (22, 23, 37–39). First, the MAP kinase docking site can interact with both ERK and p38 MAP kinase, explaining why MSK is activated by two MAP kinase pathways. Second, the activation loop of the NTK is not phosphorylated by PDK1 but probably via autophosphorylation.
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In this study, we characterized RSK4 and demonstrated that RSK4 is distinct from other RSKs by being constitutively activated in serum-starved cells in the absence of growth factor. Our data suggested that constitutive activity was due to constitutive phosphorylation of Ser232, Ser372, and Ser389 induced in part by very low basal levels of ERK activity and in part by less well defined mechanisms that may include enhanced autophosphorylation ability of RSK4 compared with other RSKs. The constitutive, growth factor-independent activity of RSK4 may help explain how RSK4 can participate in non-growth factor signaling, such as p53-induced growth arrest.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-tubulin was from the YL1/2 mouse hybridoma cell line. Epidermal growth factor (EGF) was from PreproTech Inc. (Rocky Hill, NJ). S6 peptide (RRLSSLRA) and cross-tide (GRPRTSSFAEG) were synthesized by K. J. Ross-Petersen AS (Copenhagen, Denmark). U0126 was from Promega (Madison, WI). SB203580 was from Calbiochem. [
-32P]ATP was from Amersham Biosciences. Other chemicals were from Sigma.
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2, the JNK22 (48) coding sequence was amplified by PCR with primers introducing a 5' BamHI and a 3' NotI site, and the digested PCR product was cloned into the mammalian expression vector pEBG in-frame with GST. The expression construct for HA-c-Jun is described in Ref. 49.
Transfection, Stimulation, and Immunoprecipitation—All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in atmospheric air with 5% CO2. Medium for wild-type and PDK1-deficient mouse embryonic stem (ES) cells (37) also contained 10 ng/ml leukemia inhibitory factor and 0.1 mM 2-mercaptoethanol. For transfection, 90% confluent cells in 9.6-cm2 wells were incubated with 4 µg of DNA and 11 µl of Lipofectamine 2000 reagent (Invitrogen) as described in the manufacturer's protocol. Transfection of ES cells was scaled up to 56-cm2 culture dishes. Serum starvation was performed by two washes with serum-free Dulbecco's modified Eagle's medium, aspiration, and incubation in serum-free Dulbecco's modified Eagle's medium. Phorbol 12-myristate 13-acetate (PMA), anisomycin, U0126, and SB203580 were added from 1,000x stock solutions in Me2SO. Cells were harvested by washing with phosphate-buffered saline (PBS) and solubilization for 15 min in 500 µl of lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, 5 mM EDTA, 25 mM sodium fluoride, 10 nM calyculin A, 1mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, and 200 kallikrein inhibitor units/ml aprotinin) on ice. Subsequent manipulations were performed at 0–4 °C. Cell extracts were clarified by centrifugation for 5 min at 14,000 x g, and the supernatant was incubated for 90 min with antibody with the addition of 20 µl of 50% protein G-agarose beads (Santa Cruz Biotechnology, Inc.) during the final 30 min. For GST fusion protein pull-down assays, glutathione-Sepharose 4B (Amersham Biosciences) was used. Agarose bead-antibody complexes were precipitated by centrifugation, washed five times with lysis buffer, drained, and dissolved in SDS-PAGE sample buffer (2% sodium dodecyl sulfate, 62 mM Tris (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 0.1% bromphenol blue). For kinase assays, the final two washes were with 1.5x kinase assay buffer (30 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol).
Kinase Assays—Agarose beads with immunoprecipitated kinase were drained with a syringe and resuspended in 20 µl of 1.5x kinase assay buffer. The kinase reaction was initiated by the addition of 10 µl (final concentrations) of ATP (100 µM, 0.5 µCi of [-32P]ATP) and 800 µM S6 peptide (RSK assays) or 166 µM cross-tide (MSK assays). After 10 min at 30 °C (the reaction was linear with time), 20 µl of the supernatant was removed with a Hamilton syringe (leaving behind precipitated kinase) and spotted onto phosphocellulose paper (Whatman p81) that was washed four to five times with 150 mM orthophosphoric acid. [32P]phosphate incorporated into protein substrate was quantified on a STORMTM PhosphorImager using ImageQuantTM software (Amersham Biosciences). Due to the low activity, a reaction blank (an assay performed without antibody during immunoprecipitation) was performed for all the conditions in endogenous RSK4 and MSK1 assays and subtracted from the respective kinase assay values.
In Vitro Activation of RSK—Endogenous RSK4 or RSK2, immunopurified from 3 x 106 serum-starved embryonic kidney (HEK) 293 cells per assay point, were incubated for 30 min at 30 °C with 50 µM MgATP in the absence or presence of 50 ng of active ERK2 (Upstate Biotechnology) and 50 ng of active PDK1 (35) in 20 µl of 1.5x kinase assay buffer. Thereafter the kinase activity of RSK4 and RSK2 was determined as described under "Kinase Assays" except that in the present assay the blank reaction contained ERK2 and PDK1.
Immunocytochemistry—Subconfluent cells cultured on gelatin-coated glass coverslips were transfected with pMT2-HA-RSK4. One day after transfection, cells were fixed in 4% (w/v) paraformaldehyde for 15 min followed by permeabilization with 0.2% (v/v) Triton X-100 for 5 min and blocking in 10% horse serum for 20 min. Cells were then incubated for 60 min at 37 °C with rabbit anti-HA antibody diluted 1:500 in PBS. After washing with PBS, cells were incubated for 45 min at 37 °C with fluorescein isothiocyanate-conjugated anti-rabbit antibody (F0511, Sigma) diluted 1:300 in PBS. Coverslips were then washed with PBS, mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA), and analyzed in a laser scanning confocal microscope (Leica).
Cell Fractionation—HEK293F cells were harvested in cold PBS, pelleted, and resuspended in hypotonic RSB buffer (10 mM HEPES (pH 6.2), 10 mM NaCl, 1.5 mM MgCl2, inhibitors as for lysis buffer above). The cell suspension was incubated for 30 min at 4 °C and then homogenized by 20 strokes in a tight fitting homogenizer. The homogenate was centrifuged at 375 x g (2 min), and the pellet was washed twice with RSB buffer and then resuspended in lysis buffer (50 mM Tris (pH 8.0), 250 mM NaCl, 1% Triton X-100, inhibitors as for lysis buffer above) to generate the nuclear fraction. The supernatant was centrifuged for 60 min at 150,000 x g. The resulting pellet was washed once in RSB buffer and then resuspended in lysis buffer to generate the membrane fraction, whereas the supernatant of this centrifugation represented the cytosolic fraction. All procedures were carried out at 4 °C.
Immunoblotting—Immunoblotting was performed as described previously (33) except that the membrane was blocked in Bailey's Irish Cream liquor in immunoblots using phosphospecific Abs or anti-RSK4 Ab.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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RSK4 immunoblotting on the nuclear, cytosolic, or membrane fraction of HEK293 cells suggested that RSK4 is localized to the cytoplasm (Fig. 2D). Control blots showed the expected distribution of the marker proteins lamin A/C, -tubulin, and p63 in the various cell fractions. A predominantly cytosolic localization of RSK4 was confirmed by immunocytochemistry and confocal microscopy to HA-RSK4 transiently expressed in various cell types (Fig. 2E). However, a weak nuclear RSK4 signal was also observed, suggesting that a minor fraction of cellular RSK4 may be localized to the nucleus. This fraction might have leaked to the cytoplasmic fraction during the cell fractionation experiment depicted in Fig. 2D. The subcellular distribution of transiently expressed RSK4 was not affected by exposure of the cells to a variety of stimuli that activate MAP kinase pathways. These stimuli included 20 nM EGF or 150 nM PMA for 10, 20, 30, or 60 min; 120 J of UV irradiation and analysis after 20, 40, or 60 min or 2, 4, 7, or 10 h; and 10 µg/ml anisomycin for 30 or 60 min (data not shown). We noted an optimal bipartite nuclear localization signal in a loop within the N-terminal kinase domain of RSK4: 325KRHLFFANIDWDKLYKR341. Although this nuclear localization signal is conserved in all RSKs, it does not appear to be functional since the low nuclear RSK4 signal in transfected cells was not affected by its mutation as in the triple mutant RSK4-K325Q/R326A/K337A (data not shown).
To investigate how the kinase activity of RSK4 is regulated, endogenous RSK4 was immunoprecipitated and assayed from serum-starved HEK293 cells after exposure, or not, to EGF and/or U0126, a specific inhibitor of MEK1 (50). The anti-RSK4 Ab precipitated RSK4 to the same extent under all the conditions (Fig. 3A, lower left panel). Surprisingly the kinase activity of RSK4 was only slightly stimulated by EGF (Fig. 3A, lower left panel). In contrast, the activity of RSK2 precipitated from the same cell lysates was stimulated 10-fold by EGF as expected (Fig. 3A, lower right panel) and as previously demonstrated with RSK1, RSK2, and RSK3 in HEK293 cells (36). Moreover ERK, assayed on the same cell lysates, was robustly activated by EGF (Fig. 3A, upper panel). Incubation with U0126 for 2 h reduced the basal and EGF-stimulated kinase activity of RSK4 by 40–50%. This demonstrates the involvement of the MEK-ERK pathway in activation of RSK4 and also suggests that the very low level of ERK activity in unstimulated cells is sufficient to induce significant activation of RSK4 but not of RSK2. Serum starvation for 18 h, instead of 4 h as in Fig. 3, did not increase the responsiveness of RSK4 to growth factor stimulation nor did incubation for 18 h with U0126 or PD98059, another specific MEK1 inhibitor (51), decrease the activity of RSK4 by more than 50% (data not shown). In agreement with the activity measurements, EGF induced a profound electrophoretic mobility shift in RSK2 (Fig. 3A, lower right panel) most likely due to profound EGF-induced phosphorylation of RSK2. In contrast, EGF induced no significant mobility shift in RSK4 (Fig. 3A, lower left panel), suggesting that no significant phosphorylation was induced. Results identical to those shown in Fig. 3A were obtained by exposure of HEK293 cells to PMA, which is another strong activator of the ERK pathway in this and other cell types (data not shown). Intriguingly also in other cell types analyzed under serum-starved conditions, the activity of endogenous RSK4 was not significantly increased by EGF or PMA but was 30–50% inhibited by U0126. In contrast, RSK2 precipitated from the same cell lysates was robustly stimulated by EGF or PMA. Fig. 3, B–D, shows the results of such experiments with U2OS osteosarcoma cells, BJ fibroblasts, and COS7 cells. These experiments indicate that RSK4 is constitutively activated in cells.
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To investigate the activity and regulation of RSK4 expressed from the RSK4 cDNA clone, HEK293 cells were transiently transfected with plasmid expressing HA-RSK4 or HA-RSK2 for comparison. Exogenous RSK4 and RSK2 responded to EGF and U0126 in basically the same way as the endogenous kinases except that a 2-fold stimulation of exogenous RSK4 was observed in response to EGF (Fig. 4A). To determine the specific kinase activity of RSK4 compared with that of RSK2, immunoprecipitations for kinase assays were titrated to precipitate similar amounts of HA-RSK4 and HA-RSK2 from transiently transfected COS7 cells. As shown in Fig. 4B, RSK4 has in fact higher specific activity than RSK2 when measured against S6 peptide, the most frequently used in vitro RSK substrate, or against cross-tide, derived from glycogen synthase kinase-3
and containing Ser9, which is a physiological RSK phosphorylation site (37). Using full-length glycogen synthase kinase-3 as a substrate, similar results were obtained (data not shown). When correlating for RSK protein levels, the specific activity of RSK4 was 10–20-fold higher than that of RSK2 in serum-starved COS7 cells and 2–3-fold higher than that of RSK2 in EGF-treated COS7 cells. As observed with HEK293 cells, exogenous RSK4 could be stimulated 2–3-fold by EGF in COS7 cells in contrast to the endogenous RSK4, which was not stimulated by EGF in these cell types, as shown in Fig. 3. It should be noted that the above in vitro results do not imply that S6 protein or glycogen synthase kinase-3 are physiological substrates of RSK4 but only reflect that the proteins contain the minimal consensus sequence (Arg-X-X-Ser), which allows in vitro phosphorylation by RSK family kinases as well as by several other kinases related to RSKs.
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and - MAPKs (52), had no effect on the kinase activity of RSK4. Serving as a control for the various treatments, the kinase activity of MSK1 was strongly stimulated by anisomycin or UV irradiation and was profoundly inhibited by SB203580. Note that treatment with anisomycin reduced precipitable RSK4 kinase activity apparently by decreasing the RSK4 protein level, which may be due to the ability of anisomycin to inhibit protein synthesis. Also in U2OS cells, the activity of RSK4 was not affected by UV irradiation or SB203580 (data not shown).
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or JNK22 (Fig. 7, B and C). As controls, p38 and JNK22 co-immunoprecipitated with MSK1 and c-Jun, respectively. Thus, these results provide further evidence that RSK4 is regulated by ERK-type MAP kinases but not by stress-activated MAP kinases.
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Phosphoblot analysis was also performed on exogenous RSK4 and RSK2 transiently expressed in COS7 cells. RSK4 was phosphorylated at Ser232 to the same extent in starved and EGF-stimulated cells, whereas RSK2 showed a 3–4-fold induction of this site in response to EGF (Fig. 8C, upper panel). RSK4 also showed high basal phosphorylation of Ser389 and Thr581 as compared with RSK2. By contrast, RSK4 showed no basal phosphorylation of Ser372. In exogenous RSK4, the phosphorylation of Ser372, Ser389, and Thr581 was induced or further increased by EGF, probably explaining why the kinase activity of exogenous RSK4 can be stimulated to a modest degree by EGF in COS7 cells in contrast to the endogenous RSK4. However, exogenous RSK4 still showed high basal kinase activity in COS7 cells that appears to result from high basal phosphorylation of Ser232 and Ser389.
Analysis of RSK4 point mutants surprisingly revealed that phosphorylation of the predicted PDK1 site, Ser232, was not affected by mutation of Ser389 in the predicted PDK1 docking site (Fig. 8C, upper panel). By contrast in RSK2, mutation of the corresponding serine in the PDK1 docking site (Ser386) completely abolished phosphorylation of the PDK1 phosphorylation site (Fig. 8C, upper panel). Also unexpectedly, mutation of Thr581 only slightly decreased the ability of EGF to induce phosphorylation of Ser389. To investigate whether this is due to an inability of the C-terminal kinase of RSK4 to be activated via phosphorylation of Thr581, we analyzed EGF-induced activation of the isolated C-terminal kinase domain with the linker including the Ser389 site (RSK4-(371–745)). As shown in Fig. 8C (lower panel), EGF promoted wild-type RSK4-(371–745), but not RSK4-(371–745)-T581A, to autophosphorylate at Ser389. This shows that the C-terminal kinase of RSK4 is functional. Kinase assays showed that mutation of Ser389 profoundly decreased the kinase activity of RSK4, suggesting that this phosphorylation site has a major role in the basal as well as EGF-stimulated catalytic activity of RSK4 (Fig. 8C). By contrast, kinase assays on mutants of Thr581 and Ser372 suggested that these sites have no role in the basal activity of exogenous RSK4 in COS7 cells but contribute to the EGF-stimulated activity.
Only in one of eight cell lines tested did RSK4 show a low basal activity compared with stimulated kinase activity. Thus, in CHO cells stably transfected with the insulin receptor (IR) (53), the activity of exogenous RSK4 was stimulated about 5-fold by insulin. No endogenous RSK4 protein could be detected in CHO-IR cells (data not shown). Since endogenous RSK4 was not stimulated in any cell type tested, the stimulation in CHO-IR cells may not be physiological. However, this cell line was considered suitable for further analysis of the role of the individual phosphorylation sites in regulation of the catalytic activity of RSK4 due to the low basal versus stimulated activity. Wild-type RSK4 and mutants of the six phosphorylation sites identified in RSKs were expressed and assayed in CHO-IR cells. Stimulation of RSK4 activity by insulin was mediated by ERK since it was completely abolished by U0126 (Fig. 9A, left panel) and insensitive to SB203580 (data not shown). Mutation of Thr368 did not affect RSK4 kinase activity as shown for the corresponding Thr in RSK1 (28) and RSK2.2 Individual mutation of Ser372 and Thr581 decreased the activity of RSK4 by 30%, whereas the double mutant S372A/T581A showed 80% reduced activity. Mutation of Ser389 most profoundly reduced RSK4 activity, whereas mutation of Ser742 had no effect. The RSK4-S232A mutant was not included in Fig. 9A due to low expression, but the mutant was completely devoid of kinase activity, indicating that Ser232 phosphorylation is strictly required for activation of RSK4. Since individual mutation of the sites corresponding to Ser372 and Thr581 were previously shown to abolish PMA-induced activity of RSK1 in COS1 cells (28), we analyzed these RSK1 mutants in CHO-IR cells. Compared with RSK4, these two sites individually contributed considerably more to activation of RSK1, although their mutation did not abolish kinase activity (Fig. 9A, right panel). Phosphoimmunoblot analysis of RSK4 from CHO-IR cells showed that, as in COS7 cells, mutation of Ser389 did not abolish phosphorylation of Ser232, although a 2-fold reduction was observed (Fig. 9B). Also as in COS7 cells, mutation of Thr581 had only a small effect on insulin-stimulated phosphorylation of Ser389, explaining why the T581A mutation had only a small effect on kinase activity.
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The finding that mutation of the PDK1 docking site in RSK4 did not significantly affect phosphorylation of Ser232 could indicate that, unlike RSK1–3 (33, 34, 37), RSK4 does not require PDK1 for activation. To address this question, RSK4 was transiently expressed in wild-type mouse ES cells or in mouse ES cells with targeted disruption of both PDK1 alleles (37) and thereafter analyzed with respect to kinase activity and phosphorylation of Ser232. As shown in Fig 10A, exogenous RSK4 had similar activity in wild-type and PDK1-deficient ES cells and showed similar phosphorylation of Ser232. In contrast, endogenous RSK2 showed greatly decreased kinase activity and phosphorylation at the PDK1 site in PDK1-deficient ES cells (Fig. 10A). To obtain maximal RSK2 activity, the ES cells were treated with PMA in these experiments, but RSK4 had similar activity in PMA-treated and untreated cells (data not shown). To investigate whether RSK4 may autophosphorylate at Ser232, PDK1-deficient ES cells were transfected with RSK4 mutants (RSK4-K105A or RSK4-P242A/E243A) in which the NTK had been inactivated by mutation of critical residues conserved in virtually all kinases. These mutants had minimal kinase activity and showed profoundly decreased phosphorylation at Ser232 compared with wild-type RSK4 (Fig 10B), suggesting that RSK4 can autophosphorylate at Ser232. Finally, like exogenous RSK4, endogenous RSK4 showed equal phosphorylation of Ser232 in wild-type and PDK1-deficient ES cells (Fig. 10C). In conclusion, RSK4 is markedly distinct from other RSKs in that it does not require PDK1 for phosphorylation of Ser232 and activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The constitutive activity of RSK4 appears to result from constitutive phosphorylation of key activating sites. Thus, endogenous RSK4 showed the same level of phosphorylation of Ser232, Ser372, and Ser389 in starved and growth factor-stimulated cells, and mutational analysis demonstrated that these sites are important in stimulation of kinase activity. In a simple explanation for the constitutive activation, the expression level (and/or the rate of dephosphorylation) of RSK4 is so low that the basal activity of a highly expressed activating kinase is sufficient to induce maximal phosphorylation of RSK4. This is likely to be partly the case since the basal activity of ERK contributed to at least 30–50% of the activity of endogenous RSK4 as evidenced by U0126 sensitivity, although basal ERK activity was very low, amounting to less than 5% of the EGF-stimulated ERK activity (Fig. 3A and data not shown). In addition, ERK is a very abundant kinase with an estimated concentration of 1–3 µM in mammalian cells (54), whereas RSK4 has very low expression as discussed below. The above explanation is also indirectly supported by the finding that, in the case of overexpressed RSK4 in COS7 cells, the basal activity of endogenous ERK was not sufficient to induce maximal phosphorylation of the ERK sites in RSK4 conceivably due to high amounts of RSK4 relative to ERK.
The experiments with exogenous RSK4 in COS7 cells also suggested that the U0126-insensitive (i.e. apparently ERK-independent) RSK4 activity derives from phosphorylation of Ser232 and Ser389 since the RSK4-S372A/T581A mutant with all regulatory ERK sites mutated showed considerable basal phosphorylation of Ser232 and Ser389 as well as considerable basal kinase activity. Mutational analysis furthermore confirmed that Ser232 and Ser389 are the most important phosphorylation sites in stimulation of RSK4 catalytic activity. This is not surprising since the two sites are conserved in many kinases of the so-called AGC kinase superfamily, which includes RSK, MSK, protein kinase B (PKB), p70 S6 kinase, and serum- and glucocorticoid-inducible kinase, where the two sites have an essential and synergistic effect on kinase activation (35). An absolute requirement for PDK1 in phosphorylation of the activation loop in the NTK of RSK1–3 has been demonstrated biochemically (33, 34) and genetically (37, 55). It is therefore surprising that PDK1 is not required for Ser232 phosphorylation and activation of RSK4 as demonstrated here genetically in PDK1-deficient ES cells. Instead RSK4 may autophosphorylate at Ser232 as indicated by the reduced Ser232 phosphorylation observed in the catalytically inactive RSK4 mutants in PDK1-deficient ES cells. Our results, however, do not exclude that PDK1 may contribute to phosphorylation of Ser232 in PDK1 wild-type cells.
Mutational analysis also suggested that phosphorylation of Ser372 and Thr581 plays a more modest role in stimulation of RSK4 kinase activity than does phosphorylation of Ser232 and Ser389. In insulin-stimulated CHO-IR cells, double mutation of Ser372 and Thr581 had a much greater effect on RSK4 kinase activity than did the single mutations, suggesting that the sites can compensate for each other. It is possible that the unexpected phospho-Thr581-independent phosphorylation of Ser389 induced by insulin in the T581A mutant (Fig. 9B) could contribute to the apparent compensating function of the two sites, but the mechanism behind this observation is unknown.
Based on this and previous studies on the RSK activation mechanism, a model for constitutive activation of endogenous RSK4 in serum-starved cells is proposed. Constitutive activation is due to maximal phosphorylation of Ser232, Ser372, and Ser389 of which Ser232 and Ser389 synergize to stabilize the NTK in the active conformation thereby inducing about 80% of the RSK4 activity. The low basal ERK activity is responsible for induction of at least 50% of the RSK4 activity via maximal phosphorylation of Ser372 and Thr581. Phosphorylation of Ser372 directly stimulates the activity of the NTK, whereas phosphorylation of Thr581 stimulates NTK by promoting partial phosphorylation of Ser389, and this event may possibly enhance autophosphorylation of Ser232. The remaining 50%, apparently ERK-independent, RSK4 activity derives from phosphorylation of Ser232 and Ser386, which may be catalyzed by autophosphorylation by the NTK and the C-terminal kinase, respectively, although a contribution from additional kinases cannot be excluded.
Two recent studies suggest that RSK4 may have unique cellular functions compared with RSK1–3. In the first study, a short interfering RNA screen was carried out to identify mediators of p53-induced growth arrest, and it was discovered that knock-down of RSK4 abolishes p53-dependent G1 cell cycle arrest induced either by conditional activation of p53 or by DNA damage via ionizing irradiation (42). It was also demonstrated that RSK4 knock-down strongly suppressed expression of mRNA for the cyclin-dependent kinase inhibitor p21cip1, a major component of the antiproliferative response to p53. However, the regulation and mechanism of action of RSK4 in the p53 response was not addressed. In the present study, we found no regulation of RSK4 by UV irradiation, which also induces DNA damage and p53 activation, or by p53 transfection in U2OS or BJ cells, the two cell types analyzed in the study mentioned above. Thus, no change in RSK4 activity or subcellular distribution was detected within 8 h after UV irradiation nor did UV irradiation or transfection with p53 plasmid increase the RSK4 protein level within 24 h, although a robust growth arrest was observed (data not shown). It is therefore possible that RSK4 may function as an indirect mediator of p53 in induction of downstream effectors for instance by phosphorylating a protein in the p53 transcriptional activation complex on the p21cip1 promotor or by stimulating p21cip1 mRNA stability in the cytoplasm. The constitutive activity of RSK4 demonstrated here would enable RSK4 to function as such an indirect mediator of p53 signaling.
In the second study, a functional screen of mouse mRNAs showed that injection of RSK4, but not RSK1–3, transcripts into X. laevis one-cell embryos disrupted the subsequent formation of mesoderm, which is induced by the fibroblast growth factor-Ras-ERK pathway (43). The mechanism of inhibition was not resolved, but the inhibitory action appeared to take place at a level downstream of Ras, resulting in reduced phosphorylation of MEK and ERK at the activating sites. In early mouse development, RSK4 shows particularly high mRNA expression in extraembryonic tissue, and RSK4 expression is inversely correlated with the presence of active ERK as detected by anti-active ERK immunostaining. The authors proposed that RSK4 is an inhibitor, rather than a mediator, of growth factor signal transduction via the Ras-ERK pathway in selected cellular contexts. This hypothesis would require that activation of RSK4 can occur in a growth factor-independent manner. In the present study, we found evidence for growth factor-independent activation of RSK4 that may occur via low basal levels of ERK activity and/or autophosphorylation.
Although a quantitative analysis was not performed, the low signal in kinase assays and immunoblots of endogenous RSK4 suggests that RSK4 may be expressed at 10–20 times lower levels than other RSKs in most cell types. This conclusion is supported by the extraordinarily low number of human RSK4 expressed sequence tags (ESTs) in the public data bases. Thus, as of January 2005, Unigene listed 16 RSK4 ESTs, 289 RSK1 ESTs, 282 RSK2 ESTs, and 351 RSK3 ESTs (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene), indicating that low RSK4 protein expression is due to low RSK4 mRNA levels. In agreement with this conclusion, Kohn et al. (41) noted that RSK4 shows relatively broad but low expression compared with RSK2 in fetal mouse tissues. Since RSK4 appears to be constitutively activated in cells and may function to suppress Ras-ERK signal transduction and cell proliferation, the expression level of RSK4 may be low in most cell types to allow cell growth. Conversely up-regulation of RSK4 expression may be one mechanism to restrict cell growth. It can further be speculated that the biological activity of RSK4 is regulated primarily at the level of expression rather than at the level of catalytic activity, the major level of regulation of RSK1–3.
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FOOTNOTES |
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Both authors contributed equally to this study.
¶ Present address: The Friedrich Miescher Inst., Maulbeerstrasse 66, CH-4558 Basel, Switzerland.
¶¶ To whom correspondence should be addressed: Biotech Research and Innovation Centre, Fruebjergvej 3, DK-2100 Copenhagen Ø, Denmark. E-mail: morten.frodin{at}bric.dk.
1 The abbreviations used are: RSK, 90-kDa ribosomal S6 kinase; MSK, mitogen- and stress-activated kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAP, mitogen-activated protein; NTK, N-terminal kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1; HA, hemagglutinin; HEK, human embryonic kidney; CHO, Chinese hamster ovary; EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ES, embryonic stem; IR, insulin receptor; Ab, antibody; GST, glutathione S-transferase; PBS, phosphate-buffered saline; EST, expressed sequence tag.
2 C. J. Jensen and M. Frödin, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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107 HEK293 cells (lane 1) or on crude lysates from 
