©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Divergent Functional Roles for p90 Kinase Domains (*)

(Received for publication, February 27, 1995; and in revised form, May 23, 1995)

Christian Bjørbæk Yi Zhao David E. Moller (§)

From theDepartment of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A unique and highly conserved structural feature of approx90-kDa ribosomal S6 kinase (p90 or RSK) is the presence of two non-identical kinase domains. To explore the mechanism of RSK activation, a cloned human RSK cDNA (RSK3) was used to generate and characterize several site-directed RSK mutants: K91A (N-Lys, NH(2)-terminal ATP-binding mutant), K444A (C-Lys, COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant), T570A (C-Thr, mutant of the putative MAPK phosphorylation site in subdomain VIII of the C-domain), S218A (N-Ser, mutant of the corresponding NH(2)-terminal residue). Epitope-tagged RSKs were expressed in transfected COS cells followed by immunoprecipitation with or without prior in vivo epidermal growth factor stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was ablated (and partially impaired with N-Ser). In contrast, both C-Lys and C-Thr retained high levels of kinase activity and were capable of responding to stimulation. C-Lys also retained partial kinase activity toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1 G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)) whereas NLys did not. The isolated NH(2)- and COOH-terminal domains were also expressed; the C-domain was inactive, whereas the N-domain retained partial activity. Relative to wild-type, both N-Lys and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein tyrosine phosphatase. We conclude that 1) the NH(2)-terminal RSK kinase domain mediates substrate phosphorylation; 2) both domains contribute to autophosphorylation; 3) the putative MAPK phosphorylation site is not required for growth factor-stimulated autophosphorylation or kinase activation.


INTRODUCTION

Protein serine/threonine kinases mediate a broad array of cellular functions including the stimulation of proliferation and differentiation by cytokines and growth factors(1, 2, 3) , cellular oncogenesis(4) , regulation of the cytoskeleton(5) , responses to stress (e.g. changes in osmolarity, UV light, ionizing radiation, 6-9), the regulation of metabolism by insulin and other hormones(10, 11, 12) , and possibly apoptosis(13) .

The p90 ribosomal S6 kinase (p90 or RSK) (^1)has been implicated as an important participant in several of these critical cellular events. Thus, activation of RSK coincides with oncogenic transformation(14) , stimulation of G(0)/G(1) transition(1, 15, 16) , T cell activation(17) , stimulated differentiation of PC12 cells(18) , platelet activation(19) , and cellular responses to heat shock (20) or ionizing radiation(9) . RSK has also been demonstrated within the cell nucleus and is thought to phosphorylate nuclear target substrates (21, 22, 23) . An additional important role for RSK involves its phosphorylation of the G-subunit of protein phosphatase 1 and glycogen synthase kinase-3, since these events contribute to the regulation of glycogen metabolism by insulin(10, 24, 25, 26, 27, 28) .

Molecular cloning of a RSK cDNA from Xenopus ovarian tissue revealed a predicted protein with a strikingly different structure from other Ser/Thr kinases(29) . Thus, the RSK polypeptide contained two non-identical complete kinase domains. The NH(2)-terminal kinase domain is related to protein kinases C and the catalytic subunit of cAMP- and cGMP-dependent kinases (40-45%); whereas the COOH-terminal domain bears 30-35% homology to phosphorylase b kinase and calcium/calmodulin kinases(29, 30) . Molecular cloning of RSK isoforms from the chicken and mouse(31) , rat(32) , and human (33, 34) have demonstrated precise conservation of this unique feature along with strong overall homology (75-85%) between different 724-752 amino acid isoforms and between species. Strong evolutionary pressure to conserve this structure was further demonstrated by the report of a highly related Drosophila RSK homolog(35) . In spite of intensive recent interest in RSK, the functional importance of dual ATP-binding sites and distinct kinase domains remains obscure. Given that RSK exhibits broader substrate specificity than the other class of S6 kinase, p70/85(12, 36) , it is possible that individual RSK kinase domains mediate phosphorylation of different substrates or have other roles in substrate recognition or activation.

RSK is regulated by phosphorylation on Ser (mostly) and Thr residues which is mediated by upstream Ser/Thr kinases and by additional autophosphorylation(12, 32) . Furthermore, the enzyme does not contain phosphotyrosine and can be dephosphorylated and deactivated by protein phosphatase 2A(12, 25, 32, 37) . Several lines of evidence suggest that isoforms of mitogen-activated protein kinase (MAPK), in particular p44 (ERK1) and p42 (ERK2), are upstream activators of RSK(25, 32, 37, 38, 39, 40) . Thus, in vitro phosphorylation of RSK by ERK1,2 results in enzyme activation(25, 32, 37) . In addition, ERK1,2 MAPKs are detected in fractions from mitogen-stimulated cell extracts which mediate RSK phosphorylation(38, 41, 42, 43) . By sequencing of a chymotryptic phosphopeptide after in vitro phosphorylation of a RSK isoform (RSK2) by MAPK, Sutherland et al.(43) identified two predominant Thr phosphorylation sites within the sequence TPCYTA located upstream of the APE motif in subdomain VIII of the second kinase domain. These residues are conserved in all known RSK isoforms(25, 31, 33) . Since the first Thr residue is followed by Pro, they suggested that this was the major site of transphosphorylation by MAPKs which are known to be proline-directed(5, 43) . Furthermore, MAPK phosphorylated only the first Thr in a peptide containing both residues(43) .

In order to explore the mechanism of RSK activation and the functions of the two-domain structure, we expressed epitope-tagged wild-type and mutated versions of a cloned human RSK cDNA (44) in COS cells and characterized their function. Here we report: (i) phosphorylation of several known RSK substrates is mediated exclusively by the NH(2)-terminal kinase domain, (ii) in vitro RSK autophosphorylation is partially impaired by mutation of either ATP-binding site, (iii) mutation of the putative MAPK-phosphorylated Thr is associated with normal kinase activity and a preserved ability to respond to growth factor stimulation, (iv) an NH(2)-terminal Ser residue that is important for maximal kinase activity is identified.


MATERIALS AND METHODS

Proteins and Peptides

S6 kinase substrate peptide (RRRLSSLRA) and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) were purchased from UBI (Lake Placid, NY) and Sigma, respectively. 40 S ribosomal subunits isolated from rat liver were a generous gift from Dr. Joseph Avruch (Massachusetts General Hospital, Boston, MA); recombinant rabbit protein phosphatase 1 (PP1G, amino acids 1-442) was kindly provided by Dr. Anna A. DePaoli-Roach (Indiana University School of Medicine, Indianapolis, IN); recombinant c-Fos was a gift from Dr. Tom Curran (Roche Institute of Molecular Biology, Nutley, NJ). Histones H1, H2B, H3 were purchased from Boehringer Mannheim; Yersinia protein tyrosine phosphatase was obtained from Calbiochem. Human epidermal growth factor (EGF) was from Sigma.

Antibodies

Monoclonal antibody 12CA5 against the influenza hemagglutinin (HA) epitope tag was from Babco (Emeryville, CA). N67, a rabbit polyclonal antibody, was raised against the synthetic peptide KFAVRRFFSVYLRR (Research Genetics, Huntsville, AL) which corresponds to amino acids 7-20 encoded by a human RSK cDNA designated as RSK3(44) .

Plasmids, Mutagenesis, in Vitro Transcription and Translation

The coding region (2199 base pairs) of the human RSK3 cDNA (on EcoRI linkers) was subcloned into pGEM-11Zf(-) (Promega, Madison, WI) to generate pGEM-RSK3. Site-directed RSK mutants were generated in pGEM-RSK3 using the method of Deng and Nickoloff (45) . Mutagenic oligonucleotide primers and a selection primer designed to ablate a unique XbaI site in the pGEM polylinker were used with the Transformer Kit (Clontech, Palo Alto, CA) according to recommendations provided by the manufacturer. The following five mutants were generated, where N and C denote the amino- and carboxyl-terminal halves of the protein, respectively: N-Lys (K91A, Lys to Ala), N-Ser (S218A), C-Lys (K444A), C-Thr (T570A) and N/C-Lys (K91A/K444A). Two additional mutants of Ser were also generated: S218E and S218D. The sites of mutagenesis in each of the RSK mutant plasmids were sequenced (Sequenase 2.0, U.S. Biochemical, Cleveland, OH) to confirm that the appropriate nucleotide substitutions were present. In order to assure that the site-directed mutant RSK coding regions could generate full-length polypeptide products with equal efficiency, we performed in vitro transcription of wild-type and mutant pGEM-RSK3 plasmids using T7 RNA polymerase (Promega) followed by in vitro translation using rabbit reticulocyte lysate (Promega) in the presence of [S]methionine (3000 Ci/mmol, DuPont NEN). Labeled polypeptides were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. Wild-type and each mutant plasmid generated a predominent 85 kDa protein band of similar intensity (data not shown). Subsequently, wild-type or mutagenized RSK3 cDNAs were excised from pGEM-RSK3 and transferred in-frame to the EcoRI site of pMT2-HA (gift of Joseph Avruch), a modified version of the pMT2 expression vector (46) which encodes the 9-amino-acid HA epitope tag (YPYDVPDYA) in a position NH(2)-terminal to the EcoRI site.

Two additional constructs designed for expression of the isolated NH(2)-terminal (RSKDeltaC) and COOH-terminal kinase (RSKDeltaN) domains were prepared as follows: for RSKDeltaC the wild-type RSK expression vector (pMT2-HA-RSK3) was digested with BsgI and DraIII followed by religation using a short double-stranded oligonucleotide adapter. This resulted in the in-frame removal of the entire COOH-terminal kinase domain via deletion of residues 384-689. For RSKDeltaN, a XhoI-MluI fragment encoding residues 33-219 was removed from pMT2-HA-RSK3/N-Ser (which had been previously modified to include a unique MluI site), followed by in-frame blunt-ended religation.

Cell Culture, Transient Transfection, and Immunoprecipitation

For transient expression of mutant and wild-type HA-tagged RSK proteins in COS-7 cells, subconfluent cells in 35-mm dishes were transfected with 1.8 µg of plasmid DNA using lipofectamine (15 µl) as recommended by the manufacturer (Life Technologies, Inc.). The pMT2-HA empty vector was included in all experiments as a control for subsequent assays. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 mg/ml streptomycin. Seventy-two h after transfection, cells were incubated for 6 h in Dulbecco's modified Eagle's medium supplemented with 0.1% fetal calf serum followed by 15 min of stimulation with or without EGF (100 ng/ml) or tetradecanoyl phorbol acetate (200 nM, Sigma). Cells were then washed with cold phosphate-buffered saline and solubilized in 500 µl of cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 5 mg/ml leupeptin, 5 mg/ml aprotinin, 1 mM sodium orthovanadate, and 0.5 mM phenylmethylsulfonyl fluoride). Cell lysates were briefly sonicated and clarified by centrifugation at 23,000 g for 15 min at 4 °C, and subsequent steps were also performed at this temperature. HA-tagged proteins were immunoprecipitated with 12CA5 (1:250) and protein A-Sepharose beads (Boerhinger Mannheim) as described previously(47) . Immunoprecipitates were washed three times with lysis buffer and once with kinase assay buffer (KA buffer, 30 mM Tris-HCl, pH 7.4, 10 mM MgCl(2), 0.1 mM EGTA and 1 mM dithiothreitol).

Immunoblot Analysis

For immunoblotting of transfected mutant and wild-type RSK polypeptides, equal aliquots of 12CA5 immunoprecipitates were resuspended in 1 Laemmli sample buffer (48) and subjected to SDS 8% PAGE. Resolved proteins were transferred to nitrocellulose and blocked with 10% non-fat dried milk as described previously(47) . Membranes were then incubated with the N67 antiserum (1:1000 dilution) for 12-15 h. After removal of unbound antibodies, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin for 1 h and washed three times in buffered saline. Rabbit antibodies were detected using enhanced chemiluminescence (Amersham, Buckinghamshire, United Kingdom) and autoluminography.

Protein Kinase Assays

S6 peptide and Kemptide were used at final concentrations of 0.3 mg/ml. Protein substrates were used at concentrations of 0.07 mg/ml. Kinase assays were initiated by the addition of 2.5 µl of 150 µM [-P]ATP (5 µCi) to the immune-complex pellet resuspended in 12.5 µl of 1.2 KA buffer, including appropriate substrates, followed by incubation at 30 °C for 10 min. In vitro RSK autophosphorylation was performed under conditions identical to kinase assays except that no substrates were added to the reaction mixture. For kinase assays including peptide substrates, the reaction was terminated by spotting the mixture onto phosphocellulose paper (Whatman p81). After thrice washing in 150 mM phosphoric acid, filters were subjected to Cerenkov counting. For kinase assays including protein substrates or for determination of autophosphorylation, the reactions were terminated by adding 7.5 µl of 3 Laemmli sample buffer(48) . After heating at 95 °C for 5 min, proteins were resolved by 15% (for protein substrates) or 8% (for RSK autophosphorylation) SDS-PAGE. Gels were dried and subjected to autoradiography and PhosphorImager (Molecular Dynamics) analysis.

For protein tyrosine phosphatase treatment, aliquots of immunoprecipitated proteins were incubated in 15 µl of 30 mM HEPES, 150 mM NaCl, 50 mM beta-glycerophosphate, and 5 mM dithiothreitol, with or without 0.8 milliunits protein tyrosine phosphatase/ml for 20 min at 30 °C. Reactions were terminated by washing once with 1 ml of ice-cold KA buffer and resuspending in ice-cold KA buffer followed by determination of kinase activity and autophosphorylation as described above.


RESULTS AND DISCUSSION

To investigate the role of the two kinase domains of the 90-kDa RSK in substrate phosphorylation and in growth factor-mediated activation of the enzyme, oligonucleotide-directed mutants of a human RSK cDNA were generated and characterized. For this purpose, we used the cDNA encoding a 733-amino-acid isoform designated as RSK3(44) . RSK3 is homologous (75-84% overall identity) to the two other known mammalian RSK isoforms (31, 33, 34) with a high degree (86-92%) of amino acid identity between the corresponding kinase domains in each isoform.

In each of the two kinase domains, the invariant lysine (Lys and Lys) in subdomain II (30) was mutated to alanine to create N-Lys and C-Lys ATP-binding site mutants (Fig.1A). Based on earlier studies(30, 49) , each of these two mutations would be expected to markedly impair phosphotransferase activity mediated by the respective kinase domain. Also, a double kinase mutant (N/C-Lys) where both lysine residues were changed to alanine was created. Furthermore, to specifically address the possible role of Thr in subdomain VIII of the COOH-terminal domain in growth factor-mediated kinase activation, this residue was also mutated (C-Thr, Fig.1A). This residue in RSK2 (also known as ISPK-1 or MAPKAP-1) Thr(34) has been implicated as the predominant site of MAPK (ERK2) phosphorylation(43) . The corresponding residue in the NH(2)-terminal kinase domain, Ser, was also mutated (N-Ser, Fig.1A). Both Ser and Thr are located within 20 residues upstream of the highly conserved A(S)PE motif present in all protein kinases, a region known to often contain phosphorylation sites that are critical for catalytic function(30) .


Figure 1: Construction and expression of site-directed RSK mutants. A, schematic drawing of a 733-amino acid human RSK polypeptide (RSK3). Conserved regions within protein kinase subdomains I-II and VIII, according to Hanks et al.(30) , are shown as filled and hatchedboxes, respectively. Four individual residues which were changed into alanine (Lys, Ser, Lys, and Thr) are shown in bold. The five mutant RSK polypeptides that were generated by site-directed mutagenesis are designated as follows: N-Lys (K91A), N-Ser (S218A), C-Lys (K444A), C-Thr (T570A), and N/C-Lys (K91A and K444A double mutant), where N and C denote the amino- and carboxyl-terminal kinase domains, respectively. B, immunoblotting of immunoprecipitated HA-tagged mutant and wild-type RSK proteins. In this example, HA-epitope-tagged cDNAs encoding wild-type (WT) or mutant RSK polypeptides or vector alone (Vect.) were transfected into COS cells. Cell lysates were immunoprecipitated with 12CA5 antibody. Equal aliquots of 12CA5 immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-RSK(N67) antibody. A single 85 kDa protein band of expected size and similar intensity was detected in each case. Densitometry was performed using autoradiograms of immunoblots from 9 experiments. Mean (± S.E.) protein levels (as percent of wild-type level) for each of the mutants were as follows: 120 ± 34 (N-Lys), 115 ± 27 (N-Ser), 85 ± 19 (C-Lys), 121 ± 23 (C-Thr), 117 ± 33 (N/C-Lys).



After using in vitro transcription/translation to verify that these five mutant and wild-type RSK polypeptides were synthesized with equal efficiency (not shown), the cDNA coding regions were cloned into pMT2-HA(32) , an expression vector containing the 9-residue influenza virus HA epitope tag. This allowed for the recombinant proteins to be isolated by immunoprecipitation with a monoclonal antibody (12CA5) directed against the HA epitope after transient expression in transfected COS-7 cells. In each of several experiments that were performed, we verified that similar amounts of wild-type or mutated RSK proteins were present in 12CA5 immunoprecipitates by immunoblotting with a polyclonal antibody raised against the NH(2) terminus of the human RSK3 protein (Fig.1B). Analysis of immunoblotting data from nine independent experiments showed that mean levels of immunoprecipitated RSK protein for each of the five mutants did not differ from wild-type (see legend to Fig.1B).

We first compared the activities of HA-tagged mutant and wild-type proteins using an in vitro immune-complex kinase assay with immunoprecipitates from transfected cells. The cells were serum starved or serum starved followed by stimulation with EGF which has been reported to activate RSK(16, 28) . A peptide (RRRLSSLRA) corresponding to amino acids 231-239 of ribosomal S6 protein was tested since it is known to be avidly phosphorylated by RSK(12) . For each experiment, a vector-only transfected control was included. Levels of background substrate phosphorylation were consistently less than 1% of kinase activity achieved using wild-type RSK transfected cells. As shown in Fig.2, wild-type RSK activity increased by 3.5-fold after in vivo stimulation. As predicted, the double ATP-binding site mutant was devoid of kinase activity. Of the two single ATP-binding site mutants, the N-Lys mutant showed no measurable activity, while the C-Lys mutant retained substantial kinase activity (approximately 50% of wild-type both in the serum-deprived and stimulated conditions). Thus, EGF stimulation of C-Lys kinase activity was also preserved. Examination of the C-Thr mutant revealed that it also retained high levels of kinase function and was still capable of responding to growth factor stimulation (2.4-fold). Mutation of the corresponding NH(2)-terminal Ser residue resulted in modest impairment of kinase activity from both serum-deprived and stimulated cells (approx90% reduced).


Figure 2: Kinase activity of mutant and wild-type RSK polypeptides derived from serum-deprived or EGF-stimulated COS cells. Following transfection of COS-7 cells with epitope-tagged mutant or wild-type RSK cDNAs, cells were serum-starved for 6 h and then stimulated (hatched bars) or not (filled bars) with 100 ng/ml EGF for 15 min. Cell lysates were immunoprecipitated with HA-epitope antibody 12CA5. Equal aliquots of immunoprecipitated protein were assayed for protein kinase activity using S6 peptide (RRRLSSLRA) as a substrate. Separate aliquots of 12CA5 immunoprecipitates were analyzed by immunoblotting (Fig.1B) to verify that similar amounts of RSK proteins were present. The results are expressed as a percentage of EGF-stimulated kinase activity achieved using wild-type (WT) RSK protein. Data are means ± S.E. of eight experiments; mean levels of stimulated WT RSK kinase activity were 2.8 ± 0.5 pmol of ATP transferred/min/reaction (6.2 10^5 counts/min/10 min reaction); background enzyme activity obtained with immunoprecipitates from vector-only transfected cells was consistently <1.0%. Similar results were obtained in a more limited number of experiments where cells were stimulated with tetradecanoyl phorbol acetate.



Although the N-Lys mutant was unable to phosphorylate S6 peptide, we sought to test whether the two kinase domains might each phosphorylate different substrates. Therefore, we isolated N-Lys, C-Lys, and wild-type RSK from stimulated cells and compared their ability to phosphorylate eight different polypeptides that were previously shown to be in vitro substrates for RSK(15, 21, 25, 32, 44, 50) . As shown in Table1, kinase activity of the N-Lys mutant was very low or unmeasurable. In contrast, the C-Lys mutant retained kinase activity toward each of the eight tested substrates. Interestingly, in all cases C-Lys kinase activity was diminished relative to wild-type RSK. This, suggests that both kinase domains may be required for maximal activity toward various substrates. Although it remains possible that the COOH-terminal kinase domain might be able to trans-phosphorylate (unknown) in vivo RSK substrate(s), these data strongly suggest that substrate phosphorylation is mediated exclusively by the NH(2)-terminal kinase domain. An additional, but less likely, possibility is that activation of the COOH-terminal kinase domain requires a unique upstream element or pathway which was not stimulated by EGF, serum, or tetradecanoyl phorbol acetate or was absent from COS cells.



In order to assess whether either RSK kinase domain was capable of functioning independently, we generated truncated proteins where either the COOH-terminal (RSKDeltaC) or NH(2)-terminal (RSKDeltaN) domain was deleted. After immunoprecipitation of lysates derived from transfected cells, epitope-tagged RSKDeltaC or RSKDeltaN proteins were readily detected by immunoblotting as 53- or 60-kDa proteins, respectively (not shown). In subsequent immune-complex kinase assays, we found that the isolated NH(2)-terminal domain (RSKDeltaC) retained low level kinase activity toward S6 peptide (5-6% relative to wild-type RSK). In contrast, the RSKDeltaN mutant displayed no detectable kinase activity (above background values obtained with vector-only transfected cells). Since the full-length molecule with mutated COOH-terminal Lys was apparently more active than the isolated NH(2)-terminal domain, this suggests that the presence of the COOH-terminal domain, even when inactive, acts to facilitate the function of the NH(2)-terminal domain.

Available evidence suggests that RSK undergoes autophosphorylation coincident with kinase activation(12, 32) . To further address the potential functional role of each domain, and in particular to assess whether the COOH-terminal kinase contributes to autophosphorylation, we performed in vitro RSK autokinase assays with or without prior in vivo stimulation with EGF. As shown in Fig.3A, incubation of wild-type RSK with [-P]ATP resulted in substantial in vitro phosphorylation that was further augmented (3.5-fold) by EGF stimulation. We were surprised to find a small amount (approx10% compared to wild-type) of RSK phosphorylation in the case of the double ATP-binding mutant (N/C-Lys, Fig.3A). This result suggested contamination with a coimmunoprecipitated kinase. Since MAPK and RSK have been reported to be coimmunoprecipitated(50, 51) , we tested the effect of pretreatment with protein tyrosine phosphatase (PTPase) which is known to fully inactivate MAPK. Using this approach, we found that prior PTPase treatment abrogated phosphorylation of N/C-Lys, whereas wild-type RSK phosphorylation was reduced by approximately 25% (Fig.3A). Importantly, PTPase treatment did not affect the subsequent determination of kinase activity toward S6 peptide (not shown). The fact that both N-Lys and N/C-Lys mutants lacked detectable kinase activity toward S6 peptide (Fig.2) also argues strongly that potential coimmunoprecipitated kinase(s) did not contribute to the enzyme activities that were measured. In order to eliminate the effects of the coimmunoprecipitated MAPK-like activity on in vitro RSK autophosphorylation, subsequent experiments were conducted after pretreatment with PTPase.


Figure 3: In vitro RSK autophosphorylation. A, RSK phosphorylation and effect of PTPase treatment. In this example, COS cells transfected with the double-lysine mutant (N/C-Lys), wild-type RSK (WT), or the expression vector alone (Vect.) were serum starved(-) or starved followed by EGF stimulation (+). Cell lysates were immunoprecipitated with antibody 12CA5 followed by in vitro treatment with (+) or without(-) PTPase and subsequent incubation with [P]ATP in the absence of substrate as described under ``Materials and Methods.'' Proteins were resolved by SDS-PAGE followed by autoradiography. Phosphorylated 85 kDa bands corresponding to RSK3 proteins are depicted. B, phosphorylation of wild-type (WT) and mutant RSK proteins. COS cells transfected with wild-type or mutant RSK cDNAs were serum-starved followed by stimulation with (hatched bars) or without (filled bars) EGF. Immunoprecipitated RSK proteins were pretreated with PTPase followed by determination of in vitro autophosphorylation as described above. Radiolabeled RSK proteins were quantitated by PhosphorImager analysis. Results are expressed as a percentage of EGF-stimulated wild-type RSK phosphorylation. Data are means ± S.E. of five independent experiments.



As shown in Fig.3B, both serum-deprived and stimulated values of in vitro RSK autophosphorylation were modestly reduced with the C-Lys mutant. This result supports the hypothesis that RSK autophosphorylation (and subsequent NH(2)-terminal kinase activation) could be mediated, at least in part, by the COOH-terminal domain. Furthermore, mutation of Thr resulted in modest effects on autophosphorylation with protein derived from both serum-deprived and -stimulated cells, exactly mirroring the effect of this mutation on kinase activity ( Fig.2and Fig. 3B). It therefore appears that Thr is not required for activation by EGF of either autophosphorylation or kinase activity toward substrates. The fact that the C-Thr mutant retained the ability to respond to stimulation suggests that MAPK(s) might phosphorylate alternative sites (on either the NH(2)- or COOH-terminal domains). Alternatively, the in vivo regulation of RSKs may involve additional upstream kinases, apart from ERK1/2 as was suggested by others(1) . Furthermore, it is possible that the RSK3 isoform may not be exactly regulated as RSK2 (or RSK1). Indeed, we recently showed that although RSK3 could be phosphorylated in vitro by ERK2, its phosphotransferase activity was unaffected(44) . In contrast, parallel in vitro incubation of ERK2 with recombinant RSK1 augmented the activity of this isoform(44) .

Despite the absence of protein kinase activity with mutation of the NH(2)-terminal ATP-binding site (Fig.2), EGF stimulated phosphorylation of this mutant (N-Lys) to levels that were 15% of the wild-type (3-fold versus protein from serum-deprived cells, Fig.3B). These results suggest that the residual capacity for RSK autophosphorylation in this case is likely to have been mediated by the COOH-terminal domain, although maximal in vitro RSK autophosphorylation appears to require that both kinase domains are intact. Additional studies may help to define whether RSK autophosphorylation occurs via an inter- or intramolecular mechanism and whether phosphorylation of the NH(2)-terminal domain might be mediated, in part, by the COOH-terminal kinase.

Data regarding the N-Ser mutant suggested that Ser might be important for autophosphorylation and kinase activity. Given that the analogous residue in the catalytic domains of several other protein Ser/Thr kinases including MAPK (Thr in the TEY motif, 52) and MAPK kinase (Ser in MEK1, 54, 55) are important sites of phosphorylation by upstream activators, this residue could represent a site of regulated phosphorylation. Therefore, we also generated mutants where Ser was replaced by Asp or Glu in an attempt to mimic a potential negative phosphate charge at this site. Both S218E and S218D epitope-tagged mutants were present at levels similar to wild-type RSK in immunoprecipitates derived from transfected cells (not shown). In subsequent kinase assays, when both mutants were isolated from serum-starved cells they exhibited activity that was approx17% of wild-type levels; stimulated values were approx40% compared to wild-type RSK. Thus, neither mutant displayed features suggestive of constitutive activation. Since all three mutants of Ser were readily stimulated by EGF (5-7-fold), this residue is clearly not required for growth factor-stimulated RSK activation. However, given that values of kinase activity with the Ala substituted mutant were approx4-fold lower relative to both Asp or Glu substitutions, Ser may represent a site of autophosphorylation that contributes to maximal enzyme activity.

Taken together, data presented in this study are consistent with a model whereby maximal growth factor-stimulated RSK activation requires the participation of both kinase domains. The COOH-terminal domain appears to participate in regulation of RSK autophosphorylation. The NH(2)-terminal domain also contributes to autophosphorylation and mediates substrate phosphorylation. Therefore, the structure of RSK provides a unique signal transducing paradigm where what might otherwise exist as two distinct enzymes in a kinase cascade are combined into one interdependent unit.


FOOTNOTES

*
This work was supported by grants from the American Diabetes Association and National Institutes of Health-NIDDK Grant RO1 45874-02 (to D. E. M.) and grants from the Juvenile Diabetes Foundation International and the Danish Natural Science Research Council (to C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Endocrinology, R-80T-100, MERCK Research Laboratories, P. O. Box 2000, Rahway, NJ 07065.

^1
The abbreviations used are: RSK, ribosomal S6 kinase; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are grateful for advice and valuable reagents provided by Joe Avruch and John Kyriakis (Massachusetts General Hospital, Boston, MA).


REFERENCES

  1. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,5889-5892 [Abstract]
  2. Marshall, C. J. (1995) Cell 80,179-185 [Medline] [Order article via Infotrieve]
  3. Hunter, T. (1995) Cell 80,225-236 [Medline] [Order article via Infotrieve]
  4. Hartwell, L. H., and Kastan, M. B. (1994) Science 266,1821-1828 [Medline] [Order article via Infotrieve]
  5. Davis, R. J. (1993) J. Biol. Chem. 268,14553-14556 [Free Full Text]
  6. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dal, T., Rubie, E., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369,156-160 [CrossRef][Medline] [Order article via Infotrieve]
  7. Galcheva-Gargova, Z., Derijard, B., Wu, I.-H., and Davis, R. J. (1994) Science 265,806-811 [Medline] [Order article via Infotrieve]
  8. Davis, R. J. (1994) Trends Biochem. Sci. 19,470-473 [CrossRef][Medline] [Order article via Infotrieve]
  9. Kharbanda, S., Saleem, A., Shafman, T., Emoto, Y., Weichselbaum, R., and Kufe, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,5416-5420 [Abstract]
  10. Cohen, P. (1992) Trends Biochem. Sci. 17,408-413 [CrossRef][Medline] [Order article via Infotrieve]
  11. Czech, M. P., Klarlund, J. K., Yagaloff, K. A., Bradford, A. P., and Lewis, R. E. (1988) J. Biol. Chem. 263,11017-11020 [Free Full Text]
  12. Kyriakis, J. M., and Avruch, J. (1993) in Protein Kinases: Frontiers in Molecular Biology (Woodgett, J. R., ed) Oxford University Press, Oxford
  13. Lahti, J. M., Xiang, J., Heath, L. S., Campana, D., and Kidd, V. J. (1995) Mol. Cell. Biol. 15,1-9 [Abstract]
  14. Erikson, E., Stefanovic, D., Blenis, J., Erikson, R. L., and Maller, J. L. (1987) Mol. Cell. Biol. 7,3147-3155 [Medline] [Order article via Infotrieve]
  15. Chen, R. H., and Blenis, J. (1990) Mol. Cell. Biol. 10,3204-3215 [Medline] [Order article via Infotrieve]
  16. Chen, R. H., Chung, J., and Blenis, J. (1991) Mol. Cell. Biol. 11,1861-1867 [Medline] [Order article via Infotrieve]
  17. Calvo, V., Bierer, B. E., and Vik, T. A. (1992) Eur. J. Immunol. 22,457-462 [Medline] [Order article via Infotrieve]
  18. Nguyen, T. T., Scimeca, J.-C., Filloux, C., Peraldi, P., Carpentier, J.-L., and Van Obberghen, E. (1993) J. Biol. Chem. 268,9803-9810 [Abstract/Free Full Text]
  19. Papkoff, J., Chen, R.-H., Blenis, J., and Forsman, J. (1994) Mol. Cell. Biol. 14,463-472 [Abstract]
  20. Jurivich, D. A., Chung, J., and Blenis, J. (1991) J. Cell. Physiol. 148,252-259 [Medline] [Order article via Infotrieve]
  21. Chen, R.-H., Sarnecki, C., and Blenis, J. (1992) Mol. Cell. Biol. 12,915-927 [Abstract]
  22. Chen, R.-H., Abate, C., and Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,10952-10956 [Abstract]
  23. Chen, R.-H., Tung, R., Abate, C., and Blenis, J. (1993) Biochem. Soc. Trans. 21,895-900 [Medline] [Order article via Infotrieve]
  24. Dent, P., Lavoinne, A., Nakielny, S., Caudwell, F. B., Watt, P., and Cohen, P. (1990) Nature 348,302-308 [CrossRef][Medline] [Order article via Infotrieve]
  25. Lavoinne, A., Erikson, E., Maller, J. L., Price, D. J., Avruch, J., and Cohen, P. (1991) Eur. J. Biochem. 199,723-728 [Abstract]
  26. Sutherland, C., and Cohen, P. (1994) FEBS Lett. 338,37-42 [CrossRef][Medline] [Order article via Infotrieve]
  27. Sutherland, C., Leighton, I. A., and Cohen, P. (1993) Biochem. J. 296,15-19 [Medline] [Order article via Infotrieve]
  28. Eldar-Finkelman, H., Seger, R., Vanderheede, R., and Krebs, E. G. (1995) J. Biol. Chem. 270,987-990 [Abstract/Free Full Text]
  29. Jones, S. W., Erikson, E., Blenis, J., Maller, J., and Erikson, R. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,3377-3381 [Abstract]
  30. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241,42-52 [Medline] [Order article via Infotrieve]
  31. Alcorta, D. A., Crews, C. M., Sweet, L. J., Bankston, L., Jones, S. W., and Erikson, R. L. (1989) Mol. Cell. Biol. 9,3850-3859 [Medline] [Order article via Infotrieve]
  32. Grove, J. R., Price, D. J., Banerjee, P., Balasubramanyam, A., Ahmad, M. F., and Avruch, J. (1993) Biochemistry 32,7727-7738 [Medline] [Order article via Infotrieve]
  33. Moller, D. E., Xia, C.-H., Tang, W., Zhu, A., and Jakubowski, M. (1994) Am. J. Physiol. 266,C351-C359
  34. Bjørbæk, C., Vik, T. A., Echwald, S. M., Yang, P. Y., Vestergaard, H., Wang, J. P., Webb, G. C., Richmond, K., Hansen, T., Erikson, R. L., Gabor-Miklos, G. L., Cohen, P. T. W., and Pedersen, O. (1995) Diabetes 44,90-97 [Abstract]
  35. Erikson, R. L., Alcorta, D. A., Sweet, L. J., Jones, S. W., Vik, T., Erikson, E., Simmons, D. L., Liu, M. A., Bedard, P. A., and Martins, T. J. (1989) Proceedings of First International Conference on Gene Regulation, Oncogenesis, and AIDS , p. 13
  36. Thomas, G. (1993) Biochem. Soc. Trans. 21,901-904 [Medline] [Order article via Infotrieve]
  37. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334,715-718 [CrossRef][Medline] [Order article via Infotrieve]
  38. Chung, J., Pelech, S. L., and Blenis, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4981-4985 [Abstract]
  39. Ahn, N. G., Seger, R., Bratien, R. L., Ditz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266,4220-4227 [Abstract/Free Full Text]
  40. Gregory, J. S., Boulton, T. G., Sang, B-C., and Cobb, M. H. (1989) J. Biol. Chem. 264,18397-18401 [Abstract/Free Full Text]
  41. Barrett, C. B., Erikson, E., and Maller, J. L. (1992) J. Biol. Chem. 267,4408-4415 [Abstract/Free Full Text]
  42. Ahn, N. G., and Krebs, E. G. (1990) J. Biol. Chem. 265,11495-11501 [Abstract/Free Full Text]
  43. Sutherland, C., Campbell, D. G., and Cohen, P. (1993) Eur. J. Biochem. 212,581-588 [Abstract]
  44. Zhao, Y., Bjørbæk, C., Weremowicz, S., Morton, C. C., and Moller, D. E. (1995) Mol. Cell. Biol. , in press
  45. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200,81-86 [Medline] [Order article via Infotrieve]
  46. Kaufmann, R. J., Davies, M. V., Pathak, V. K., and Hershey, J. W. B. (1989) Mol. Cell. Biol. 9,946-958 [Medline] [Order article via Infotrieve]
  47. Zhu, A. X., Zhao, Y., Moller, D. E., and Flier, J. S. (1994) Mol. Cell. Biol. 14,8202-8211 [Abstract]
  48. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  49. Hunter, T., and Cooper, J. A. (1986) in The Enzymes (Boyer, P. D., ed) pp. 191-246, Academic Press, Orlando
  50. Erikson, E., and Maller, J. L. (1988) Sec. Mess. Phosphoprot. 12,135-143
  51. Scimeca, J.-C., Nguyen, T. T., Filloux, C., and Van Obberghen, E. (1992) J. Biol. Chem. 267,17369-17374 [Abstract/Free Full Text]
  52. Hsiao, K.-M., Chou, S., Shih, S.-J., and Ferrell, J. E., Jr. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,5480-5484 [Abstract]
  53. Her, J. H., Lakhani, S., Zu, K., Vila, J., Dent, P., Sturgill, T. W., and Weber, M. J. (1993) Biochem. J. 296,25-31 [Medline] [Order article via Infotrieve]
  54. Pages, G., Brunet, A., L'Allemain, G., and Pouyssegur, J. (1994) EMBO J. 13,3003-3010 [Abstract]
  55. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77,841-852 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.