Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M005892200 on August 1, 2000

J. Biol. Chem., Vol. 275, Issue 41, 31588-31593, October 13, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/41/31588    most recent
M005892200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, J. A.
Right arrow Articles by Sturgill, T. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, J. A.
Right arrow Articles by Sturgill, T. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Creation of a Stress-activated p90 Ribosomal S6 Kinase

THE CARBOXYL-TERMINAL TAIL OF THE MAPK-ACTIVATED PROTEIN KINASES DICTATES THE SIGNAL TRANSDUCTION PATHWAY IN WHICH THEY FUNCTION*

Jeffrey A. SmithDagger §||Dagger Dagger , Celeste E. Poteet-SmithDagger §Dagger Dagger , Deborah A. Lannigan§**, Tiffany A. FreedDagger §Dagger Dagger , Anna J. ZoltoskiDagger §Dagger Dagger , and Thomas W. SturgillDagger §**Dagger Dagger

From the Dagger  Howard Hughes Medical Institute and the § Markey Center for Cell Signaling, Departments of Dagger Dagger  Medicine and ** Pharmacology, University of Virginia, Charlottesville, Virginia 22908

Received for publication, July 5, 2000, and in revised form, July 31, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The growing family of mitogen-activated protein kinase-activated protein kinases (MAPKAPKs)1 includes a subfamily of enzymes that contain two distinct kinase catalytic domains in one polypeptide chain and another that contains a single kinase domain (see Fig. 1). The three isozymes of the classic RSKs (RSK1/2/3) and the two isozymes of mitogen- and stress-activated protein kinase (MSK1/2) are in the former subfamily. The single kinase domain subfamily includes MAPKAPK2/3/5 and the two isozymes of MAPK-interacting kinase (MNK1/2). RSK, the initial MAPKAPK to be identified, has been well studied. The amino-terminal kinase domain of RSK phosphorylates exogenous substrates (1-3), whereas the carboxyl-terminal kinase domain regulates amino-terminal kinase domain activity (1, 2, 4, 5). With regard to primary structure, the catalytic core of the single kinase domain MAPKAPKs is related to the carboxyl-terminal kinase domain of RSK (Fig. 1).


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Schematic representation of MAPKAPK family members. A depiction of the dual kinase domain and single kinase domain MAPKAPKs is shown. The catalytic cores of the single kinase domain MAPKAPKs are related to the carboxyl-terminal kinase domain (CTD) of the RSK isozymes. GenBank accession numbers are: rat RSK1, A53300; mouse RSK2, P18654; human MSK1, AAC31171; human MSK2, RSKb, CCA09009; mouse MNK1, CAA71965; mouse MNK2, CAA71966; human MAPKAPK2, P49137. NTD, amino-terminal kinase domain.

In vivo, the MAPKAPKs demonstrate specificity for the upstream activator. RSK1/2/3 are specifically activated by ERK, whereas MAPKAPK2 is activated by p38 MAPK. MNK1 and MSK1/2 are activated by both ERK and p38 MAPK (6-9) although MSK2 (also known as RSKb) was reported to be predominantly regulated by p38 MAPK (9). In vivo activation studies with MNK2 have not been published; however, in vitro studies and yeast two-hybrid assays indicate that full-length MNK2 interacts with both ERK and p38 MAPK (6).

Examination of the amino acid sequence of the MAPKAPK family members revealed a putative MAPK docking site in each MAPKAPK similar to the ERK docking site identified in RSK (10, 11). The following study demonstrates that each of the MAPKAPKs contains a short sequence of amino acids that confer specificity for the upstream activator. The docking site determines the signaling cascade in which the MAPKAPK functions.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials

Antibodies were obtained as follows: polyclonal anti-ERK antibody (06-182), Upstate Biotechnology; monoclonal FLAG M2 antibody (IBI3025), Kodak; polyclonal anti-p38 MAPK antibody (SC-535-G), monoclonal anti-HA antibody (12CA5), and monoclonal anti-MYC antibody (9E10), the University of Virginia Lymphocyte Culture Center; polyclonal phospho-Ser-380 RSK1 antibody (06-826) and phospho-Ser-167 estrogen receptor (ER) polyclonal antibody, Upstate Biotechnology; ER (EVG alpha 78) polyclonal antibody (12); ER (EVG F9) monoclonal antibody (13); anti-mouse IgG:horseradish peroxidase antibody (NA931) and anti-rabbit IgG:horseradish peroxidase antibody (NA934), Amersham Pharmacia Biotech.

Plasmid Construction

pGEX2T bacterial expression constructs encoding the carboxyl-terminal tail residues of the wild type MAPKAPKs were created either by annealing complementary oligonucleotides corresponding to the 3' end of the MAPKAPKs or by polymerase chain reaction (PCR) amplification of the 3' end of each MAPKAPK. The annealed oligonucleotides and PCR products were subsequently subcloned into pGEX2T at the BamHI and EcoRI sites. pGEX2T bacterial expression constructs encoding GST-MAPKAPK2 ("QGIK") and GST-RSK2 ("Q/K-G/K") were also prepared by PCR. pK3H·RSK2 was generated as described previously (10). pK3H·RSK2/MK2, encoding the HA-tagged RSK2/MK2 chimera, was generated by PCR amplification of pK3H·RSK2 from the BstBI site to 3' of the codons for amino acid 717, at which point a BamHI site was incorporated. A PCR fragment of MAPKAPK2, encoding the 35-amino acid tail flanked by BamHI and EcoRI sites, was also generated by PCR. The RSK2 BstBI/BamHI fragment and the MAPKAPK2 BamHI/EcoRI fragment were ligated at the BamHI sites and subcloned into BstBI/EcoRI-digested pK3H·RSK2 to create pK3H·RSK2/MK2. The sequences were verified by automated sequencing. Oligonucleotides used are available on request. pEFMYC·MAPKAPK2, encoding MYC-tagged WT-MAPKAPK2, was generously provided by Christopher J. Marshall (Chester Beatty Laboratories, London, United Kingdom). pcDNA3· FLAG2-2·p38 was created by subcloning the BamHI/XbaI FLAG-p38 fragment from pGST·FLAG-p38 (generously provided by Roger J. Davis, University of Massachusetts Medical School, Worcester, MA) into BamHI/XbaI-digested pcDNA3·FLAG2 (a modified pcDNA3 vector, Invitrogen (generously provided by David L. Brautigan, University of Virginia, Charlottesville, VA)). Purification of GST fusion proteins was performed according to a protocol supplied for the GST Gene Fusion System, Amersham Pharmacia Biotech.

Interaction of MAPK and GST Fusion Proteins

Glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) (25-µl bed volume) were copiously washed with phosphate-buffered saline. The washed beads were rotated with 1000 pmol (~30 µg/0.1 ml) of GST fusion protein in 25 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol for 1 h at 4 °C. The washed GST-bound beads were incubated with purified ERK2 or p38 MAPK (as indicated in figure legends) in 0.2 ml of lysis buffer A (50 mM Tris, pH 7.4, 1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM Na3VO4, 1 µM microcystin LR, 1 mM phenylmethylsulfonyl fluoride, 1 CompleteTM protease inhibitor mixture tablet/50 ml (Roche Molecular Biochemicals)) for 1.5 h at 4 °C with gentle mixing. The beads were pelleted by centrifugation. The beads were washed once with 0.2 ml of lysis buffer A and twice with 1 ml of phosphate-buffered saline. SDS sample buffer was added to the washed beads and boiled for 5 min and processed for Western analysis.

Transfection, Immunoprecipitation, and Kinase Assay

Interaction of WT-RSK2 or RSK2/MK2 Chimera with MAPK-- BHK-21 (C-13) cells were grown as described previously (10). Cells were plated at 1.8 × 106 cells/150-mm dish and were co-transfected 24 h later with a total of 26 µg of DNA/150-mm dish as described previously (5) (13 µg of pK3H·RSK2, pK3H·RSK2/MK2, or pK3H plus 13 µg of pcDNA3·FLAG2-2·p38 or pUSE·ERK (Upstate Biotechnology)). 45 h post-transfection, cells were serum-deprived in the presence of 10 µM SB 203580 for 3 h. Two 150-mm dishes per condition were scraped into 700 µl of lysis buffer B (50 mM Tris, pH 8 at 4 °C, 150 mM NaCl, 1% Nonidet P-40, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM Na3VO4, 1 µM microcystin LR). Cells were lysed by incubation on ice for 30 min. Supernatant was clarified by centrifugation at 12,000 × g for 10 min at 4 °C and precleared by incubation for 30 min with a 20-µl bed volume of Gamma Bind Plus-Sepharose beads (Amersham Pharmacia Biotech). HA-RSK2 and HA-RSK2/MK2 proteins were immunoprecipitated at 4 °C from precleared supernatant with 25 µg of 12CA5 antibody for 1 h. Gamma Bind Plus-Sepharose beads (30-µl bed volume) were incubated with the supernatant for 1 h on a Nutator rocker. The beads were pelleted by brief centrifugation, washed once with 500 µl of lysis buffer B, washed twice with 500 µl of phosphate-buffered saline, and then processed for Western analysis.

In Vivo Activation-- BHK-21 (C-13) cells were transfected with 25 µg of pK3H·RSK2, pK3H·RSK2/MK2, pK3H, or pEFMYC·MAPKAPK2 as described previously (5). 45 h post-transfection, cells were serum-deprived in the presence of 50 µM PD 98059, 10 µM SB 203580, or Me2SO (vehicle) for 3 h. The cells were treated with EGF (100 ng/ml) for 30 min and NaCl (300 mM) for 15 min (or the appropriate vehicle) and were harvested as described. HA-tagged WT-RSK2 and RSK2/MK2 chimera were immunoprecipitated with 25 µg of 12CA5, and MYC-tagged WT-MAPKAPK2 was immunoprecipitated with 25 µg of 9E10 as described. The tagged proteins were eluted from the antibody by incubation with HA peptide (686 µM) or MYC peptide (629 µM) for 16 h in an Eppendorf Thermomixer at 4 °C with shaking at 1300 rpm. Eluted WT-RSK2, RSK2/MK2 chimera, or WT-MAPKAPK2 (3 µl) was incubated in kinase mix (47 µl; 25 mM Hepes, pH 7.4, 5 mM beta -glycerophosphate, pH 7.4, 3.75 mM EGTA, 1.5 mM dithiothreitol, 30 mM MgCl2, 6 µM protein kinase A inhibitor peptide, 6 µM protein kinase C inhibitor peptide, 150 µM Na3VO4, 1 µM microcystin LR, 300 µM S6 peptide (RRRLSSLRA) for WT-RSK2 and RSK2/MK2 chimera or 75 µM glycogen synthase peptide (KKLNRTLSVA) for WT-MAPKAPK2, 150 µM ATP, and [gamma -32P]ATP (~2000 cpm/pmol)) at 30 °C for 10 min. Each assay was performed in triplicate. Phosphate incorporation into peptide substrate was determined using phosphocellulose P-81 paper as described previously (10). Duplicates of eluted proteins were processed for Western analysis with anti-HA or anti-MYC antibody. The intensity of each immunoblot band from numerous exposures of film was quantitated in the linear range of detection, and the relative amount of tagged protein was determined using NIH Image 1.61. Specific activity was normalized for the amount of immunoprecipitated protein (pmol/min/unit HA-tagged or MYC-tagged enzyme) and was plotted as percent increase in activation relative to basal kinase activity.

Phosphorylation of the Estrogen Receptor-- BHK-21 (C-13) cells were co-transfected with 13 µg of HEGO DNA (estrogen receptor) and 13 µg of either pK3H·RSK2, pK3H·RSK2/MK2, pK3H·RSK2/MK2-K100A, or pK3H DNA as described. 45 h post-transfection, all cells were serum-deprived in the presence of 50 µM PD 98059, and a subset of cells was also treated with 10 µM SB 203580 or Me2SO (vehicle) for 3 h. Cells were then treated with NaCl (300 mM) or vehicle for 15 min. Two 150-mm dishes per condition were scraped into 700 µl of lysis buffer C (50 mM Tris, pH 8 at 25 °C, 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 10 mM Na2MoO4, 2 mM Na3VO4, 10 mM MgCl2, 20 mM beta -glycerophosphate, 10% glycerol, 2 µg/ml each leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 µM microcystin LR, and 100 units/ml DNase). Cells were lysed as above. Supernatant was clarified and precleared as above. The ER was immunoprecipitated at 4 °C from precleared supernatant with anti-ER (EVG alpha 78) antibody for 1 h. Gamma Bind Plus-Sepharose beads (30-µl bed volume) were incubated with the supernatant for 1 h on a Nutator rocker. The beads were pelleted by brief centrifugation and washed three times with 1 ml of wash buffer 1 (50 mM Tris, pH 7.2 at 25 °C, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, 10 mM Na2MoO4, 1 mM Na3VO4, 20 mM beta -glycerophosphate, 10% glycerol, and 1 µM microcystin LR) and once with 1 ml of wash buffer 2 (10 mM Tris, pH 7.2 at 25 °C, 0.1% Nonidet P-40, 20 mM NaF, 1 mM Na3VO4, 10% glycerol, and 1 µM microcystin LR). The samples were electrophoresed on a 7% SDS-polyacrylamide gel, processed for Western analysis, and immunoblotted with anti-phospho-Ser-167 ER antibody.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To determine whether the putative docking sites interact with MAPK, GST fusion proteins containing the identified regions of MSK1/2, MNK1/2, and MAPKAPK2 (Fig. 2A) were incubated with kinase-defective ERK2 (K52R) or inactive p38 MAPK. The isolated carboxyl-terminal tails of the MAPKAPKs formed complexes with MAPK (Fig. 2B), whereas a GST fusion protein containing a randomized RSK1 ERK docking site, RSK1-(RANDOM), and a GST fusion protein containing the carboxyl-terminal tail of RSK1 lacking the ERK docking site, RSK1-(672-715), did not interact with either ERK or p38 MAPK. The data indicate that these regions of the MAPKAPKs are indeed interaction sites for the upstream activators.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2.   Interaction between GST fusion proteins and purified MAPK. A, the sequence of MAPKAPK tails fused to GST is shown. The ERK docking site of RSK1 is indicated by the box. The putative MAPK docking sites of the other MAPKAPKs are underlined. Also shown are the sequences of the randomized ERK docking site of RSK1 (RSK1 (RANDOM)), and the carboxyl-terminal tail of RSK1 truncated amino-terminal to the ERK docking site (RSK1 ()). B, 1000 pmol of GST fusion proteins were bound to glutathione beads. The beads were then incubated with 40 pmol of purified kinase-defective ERK2 (for anti-ERK immunoblot) or inactive p38 MAPK (for anti-p38 MAPK immunoblot). Washed beads were subjected to SDS-polyacrylamide gel electrophoresis and examined for the presence of MAPK by Western analysis. Anti-GST immunoblot demonstrates the presence of equivalent amounts of fusion protein in each reaction.

RSK1-(716-735) specifically interacts with ERK (10). Here we demonstrate that MAPKAPK2-(377-400) and MSK1-(734-750) specifically interacted with p38 MAPK, whereas MSK2-(721-737), MNK1-(385-415), and MNK2-(385-412) interacted with both ERK and p38 MAPK. Thus, the isolated MAPK docking sites demonstrate a similar specificity for ERK or p38 MAPK as reported for the full-length proteins. MSK1 is activated by both ERK and p38 MAPK (14). However, under these conditions the isolated MSK1 MAPK docking site, MSK1-(734-750), did not interact with ERK in vitro.

To examine whether the p38 MAPK docking site of MAPKAPK2 acts as a docking site in the context of a full-length protein, the carboxyl-terminal 23 amino acids of RSK2 were replaced with the carboxyl-terminal 35 amino acids of MAPKAPK2 containing the putative bipartite nuclear localization signal of MAPKAPK2 (RSK2/MK2 chimera) (Fig. 3A). HA-tagged RSK2/MK2 chimera and WT-RSK2 were immunoprecipitated from BHK cells in which either ERK or p38 MAPK was co-expressed. The RSK2/MK2 chimera co-immunoprecipitated with p38 MAPK but not with ERK, whereas WT-RSK2 co-immunoprecipitated only with ERK (Fig. 3B). One explanation for interaction of the chimera with p38 MAPK in vivo is that replacement of the RSK2 tail with that of MAPKAPK2 may result in co-localization of the chimera with p38 MAPK, whereas WT-RSK2 is co-localized with ERK. To address this question, purified His-tagged ERK and His-tagged p38 MAPK were combined and incubated with immunoprecipitated WT-RSK2 or RSK2/MK2 chimera. The chimera formed a complex only with p38 MAPK, whereas WT-RSK2 preferentially bound to ERK above levels observed with empty vector immunoprecipitates (Fig. 3C). Thus, replacing the carboxyl-terminal tail of RSK2 with that of MAPKAPK2 is sufficient to switch specificity for the upstream MAPK.


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 3.   In vivo and in vitro interaction of the RSK2/MK2 chimera with MAPK. A, schematic representation of RSK2, MAPKAPK2, and the RSK2/MK2 chimera as well as the amino acid sequence of the carboxyl-terminal tails are depicted. The regions of RSK2 and MAPKAPK2, demonstrated to be specific docking sites in vitro for ERK and p38 MAPK, respectively, are underlined. The RSK2/MK2 chimera was created by replacing the carboxyl-terminal 23 amino acids of RSK2 (718LEP ... TAL740) with the carboxyl-terminal 35 amino acids of MAPKAPK2 (366DYE ... LAH400). As indicated in Figs. 2 and 3, the GST fusion protein of the MAPKAPK2 tail contained residues 377DAS ... LAH400; however, the RSK2/MK2 chimera includes 11 additional amino acids (366DYE ... KIE376). The additional residues were included to complete the putative bipartite nuclear localization signal of MAPKAPK2 (21, 22). Creation of the chimera resulted in insertion of a Gly and Ser at the juncture. The inserted residues are in bold face in the RSK2/MK2 chimera sequence. B, wild type RSK2 and RSK2/MK2 chimera were immunoprecipitated from serum-starved and SB 203580-treated BHK cells expressing p38 MAPK (for p38 MAPK immunoblot) or ERK (for ERK immunoblot). The presence of MAPK in the immunoprecipitates was examined by Western analysis. Anti-HA immunoblot is shown to demonstrate equivalent levels of WT-RSK2 and RSK2/MK2 chimera. C, wild type RSK2 and RSK2/MK2 chimera were immunoprecipitated from serum-starved and SB 203580-treated BHK cells. The immunoprecipitates were incubated with a mixture of 60 pmol of His-ERK and 60 pmol of His-p38 MAPK. The washed beads were subjected to SDS-polyacrylamide gel electrophoresis and examined for the presence of MAPK by Western analysis. NTD, amino-terminal kinase domain; CTD, carboxyl-terminal kinase domain.

Alignment of the MAPK docking sites of RSK2 and MAPKAPK2, which bind exclusively to ERK and p38, respectively, reveals a distinction in the number of contiguous basic amino acids. The RSK2 MAPK docking site contains two adjacent basic residues, whereas that of MAPKAPK2 contains five. Specificity for the upstream activator may be dictated by the number of contiguous basic amino acids within the MAPK docking sites such that the number of basic residues is inversely related to the affinity for ERK and directly related to the affinity for p38 MAPK. To address this question, the number of basic residues in the isolated MAPK docking site of MAPKAPK2 and RSK2 was decreased and increased, respectively, to determine whether specificity for the upstream activator could be altered (Fig. 4A). Reducing the number of basic residues in the p38 MAPK docking site of MAPKAPK2 to mimic the ERK docking site of RSK2 attenuated p38 MAPK binding (Fig. 4B). These data indicate the necessity of basic residues for interaction with p38 MAPK as has been reported for interaction of RSK with ERK (10, 11). However, the mutant p38 MAPK docking site of MAPKAPK2 did not interact with ERK (not shown). Therefore, whereas the basic residues are essential for interaction with the upstream activator, additional specificity determinants must exist within the isolated MAPK docking sites. However, the number of basic residues, in combination with the additional specificity determinants, may indeed be inversely proportional to ERK affinity because increasing the number of basic residues in the ERK docking site of RSK2 decreased the affinity for ERK (Fig. 4B); as in the case of the mutant p38 MAPK docking site, mutation of the ERK docking site did not increase the affinity for p38 MAPK (not shown), supporting the role for additional determinants of specificity.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 4.   Basic amino acids are required for interaction with MAPK. A, amino acid sequences of wild type and mutant MAPKAPK2 and RSK2 tails are shown. The MAPK docking sites are indicated by boxes. Lys-385, -388, and -389 and Ala-390 of the MAPKAPK2 p38 MAPK docking site were replaced with Gln, Gly, Ile, and Lys, respectively, converting MAPKAPK2-(377-400) to MAPKAPK2-("QGIK") to mimic the ERK docking site of RSK2. Similarly, Gln-728 and Gly-731 of the RSK2 ERK docking site were replaced with Lys to mimic the p38 MAPK docking site of MAPKAPK2, converting RSK2-(721-740) to RSK2-("Q/K-G/K"). B, interaction of wild type and mutant MAPKAPK2 and RSK2 tails with MAPK is shown. Assay conditions were as described in Fig. 2B.

To examine whether interaction between the RSK2/MK2 chimera, and p38 MAPK resulted in activation of the chimera in vivo, ectopically expressed WT-RSK2, RSK2/MK2 chimera and MAPKAPK2 were immunoprecipitated from BHK cells treated with inhibitors or stimulants of the MAPK pathways. The enzymes were eluted from the immune complex, and kinase activity toward S6 or MAPKAPK2 substrate peptides was measured. NaCl stimulation of the p38 pathway resulted in an ~130% increase in RSK2/MK2 chimera activity compared with the activity observed when cells were treated with SB 203580, whereas WT-RSK2 activity was unchanged by NaCl treatment (Fig. 5A). SB 203580 is an ATP analog that specifically inhibits catalytic activity of p38 MAPK (15) and eliminates activation of components downstream of p38 MAPK. SB 203580 pretreatment abrogated NaCl-stimulated activity of the RSK2/MK2 chimera (Fig. 5A). The wild type MAPKAPK2 (WT-MAPKAPK2) control was also activated by NaCl, and this stimulation was inhibited by pretreatment with SB 203580 (not shown). Thus, a stress-activated kinase was created by replacing the carboxyl-terminal 23 amino acids of RSK2 with the carboxyl-terminal 35 amino acids of MAPKAPK2.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   In vivo activation of WT-RSK2 and RSK2/MK2 chimera. A, S6 peptide kinase activity of WT-RSK2 and RSK2/MK2 chimera immunoprecipitated from BHK-21 (C-13) cells treated with SB 203580 or NaCl was determined. Basal kinase activity of the enzymes was measured from serum-starved cells incubated for 3 h in the presence of 10 µM SB 203580 to inhibit p38 MAPK activity. Stimulated kinase activity was measured from serum-starved cells treated with NaCl (300 mM) for 15 min. The immunoprecipitated enzymes were eluted from the immune complex with HA peptide and assayed in the presence of ribosomal S6 peptide (231RRRLSSLRA239). Error bars indicate S.E. (n = 5). B, S6 peptide kinase activity of WT-RSK2 and RSK2/MK2 chimera immunoprecipitated from BHK cells treated with PD 98059 or EGF was determined. Basal kinase activity of the enzymes was measured from serum-starved cells incubated for 3 h in the presence of 50 µM PD 98059 to inhibit activation of MEK. Stimulated kinase activity was measured from serum-starved cells treated with EGF (100 ng/ml) for 30 min. The immunoprecipitated enzymes were eluted from the immune complex with HA peptide and assayed in the presence of ribosomal S6 peptide (231RRRLSSLRA239). The % difference values for SB 203580 and PD 98059-treated samples were calculated by subtracting the mean value of pmol/min/unit HA or MYC antibody from individual experimental values. The % difference values for NaCl and EGF-stimulated samples were calculated by subtracting the pmol/min/unit HA or MYC antibody values for non-stimulated samples from those of stimulated samples, dividing that value by pmol/min/unit HA or MYC antibody for non-stimulated samples, and multiplying the result by 100. Error bars indicate S.E. (n = 4). Ectopically expressed MYC-tagged WT-MAPKAPK2 was immunoprecipitated from serum-starved BHK cells treated as described above. Kinase activity of WT-MAPKAPK2 was measured as described in the presence of a peptide closely related to the amino terminus of human glycogen synthase (KKLNRTLSVA) (21). Error bars indicate S.E. (n = 4).

S6 peptide kinase activity of WT-RSK2 from cells treated with EGF, which activates the ERK pathway, was ~290% greater than that from cells treated with the MAPK-ERK kinase (MEK) inhibitor PD 98059 (Fig. 5B). PD 98059 specifically inhibits activation of MEK1/2 (16), the upstream activator of ERK, thereby reducing phosphorylation and activation of the downstream components. EGF treatment increased kinase activity of the immunoprecipitated RSK2/MK2 chimera by only 76% compared with that of RSK2/MK2 chimera immunoprecipitated from cells pretreated with PD 98059. However, this activity is likely due to cross-talk between the mitogen- and stress-activated pathways because EGF treatment also resulted in a 70% increase in WT-MAPKAPK2 activity (Fig. 5B). The RSK2/MK2 chimera and WT-MAPKAPK2 EGF-stimulated activities were inhibited by pretreatment with PD 98059 (Fig. 5B) but not by pretreatment with SB 203580 (not shown). Thus, replacing the carboxyl-terminal tail of RSK2 with that of MAPKAPK2 removes the enzyme from the ERK pathway.

To examine whether expression of the RSK2/MK2 chimera resulted in p38 MAPK-regulated phosphorylation of a physiological RSK substrate, the alpha  isoform of the human estrogen receptor (ERalpha ) was co-expressed in BHK cells with either WT-RSK2 or RSK2/MK2 chimera. RSK phosphorylates Ser-167 of the ERalpha (17). A phospho-specific Ser-167 antibody revealed that overexpression of either RSK2/MK2 chimera or WT-RSK2 resulted in phosphorylation of ERalpha (Fig. 6A). ERalpha immunoprecipitated from NaCl-treated cells co-expressing the RSK2/MK2 chimera was phosphorylated 2.8-fold more on Ser-167 than ERalpha immunoprecipitated from SB 203580-treated cells co-expressing RSK2/MK2 chimera, whereas NaCl did not increase Ser-167 phosphorylation of ERalpha in cells co-expressing WT-RSK2 (Fig. 6A). Pretreatment of cells co-expressing the RSK2/MK2 chimera and ERalpha with SB 203580 eliminated the NaCl-induced phosphorylation of Ser-167 (Fig. 6A), indicating that in vivo phosphorylation of the RSK substrate by the RSK2/MK2 chimera is a p38 MAPK-regulated event. As stated above, the amino-terminal catalytic domain of RSK phosphorylates exogenous substrates. Mutation of the critical lysine (Lys-100) in the catalytic core to an alanine eliminates kinase activity. ERalpha immunoprecipitated from cells co-expressing the kinase-defective RSK2/MK2 chimera (K100A chimera) exhibited no Ser-167 phosphorylation (Fig. 6B). Therefore, the p38-MAPK-regulated phosphorylation of ERalpha requires catalytic activity of the RSK2/MK2 chimera. These data indicate that the signaling pathway in which a MAPKAPK functions in vivo is dictated by the MAPK docking site in the carboxyl-terminal tail of the MAPKAPK.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 6.   In vivo phosphorylation of estrogen receptor by WT-RSK2 and RSK2/MK2 chimera. BHK-21 (C-13) cells co-transfected with ERalpha and WT-RSK2, RSK2/MK2 chimera, or empty vector (A) and BHK-21 (C-13) cells co-transfected with ERalpha and RSK2/MK2 chimera or K100A-RSK2/MK2 chimera (B) were serum-starved in the presence of 50 µM PD 98059 to reduce activity of endogenous RSK and either 10 µM SB 203580 or Me2SO (vehicle) as described. The cells were then treated with 300 mM NaCl or vehicle for 15 min. The estrogen receptor was immunoprecipitated with anti-ER antibody, and the phosphorylation state of Ser-167 was examined using a Ser-167 phospho-specific ER antibody. Quantitation of Ser-167 phosphorylation is presented relative to phosphorylation of Ser-167 by WT-RSK2 in the presence of PD 98059 and SB 203580.

Herein we demonstrate that specific MAPK docking sites are located in the carboxyl-terminal tails of the MAPKAPKs. Determinants for specificity of interaction with the upstream MAPK are contained within the isolated MAPK docking sites. Replacing the ERK docking site of RSK with the p38 MAPK docking site of MAPKAPK2 results in an enzyme that complexes with p38 MAPK instead of ERK in vivo, relocating the enzyme from the ERK pathway to the p38 MAPK pathway and creating a stress-activated kinase that phosphorylates a RSK substrate in vivo. Thus, complex formation of MAPKs with the MAPK docking site of the MAPKAPKs is a general feature important for specific regulation of these MAPK-targeted kinases in vivo and dictates the signal transduction pathway in which a MAPKAPK is involved.

The MAPK docking site is not limited to MAPKAPKs. The transcription factor Elk-1 has overlapping but distinct interaction sites for ERK and c-Jun NH2-terminal kinase (18), and recently the tyrosine phosphatase PTP-SL was shown to contain a MAPK docking site (19). In addition, specific docking sites are not limited to MAPKs. The retinoblastoma protein has a sequence similar to the MAPK docking sites that targets it as a substrate for the proline-directed cyclin-dependent kinase (20). Each of these interaction motifs is required for efficient phosphorylation of the substrate by the proline-directed kinases. Thus, it is clear that substrate specificity of the proline-directed kinases involves docking sites in the downstream targets that are distinct from the consensus phosphorylation sequence. In addition, docking sites may enable co-localization of a specific proline-directed kinase with a specific target in signaling complexes where they collaborate to elicit physiological responses of pathway activation. Upon the elucidation of the determinants for specificity of the docking sites, the amino acid sequence would be a useful tool for identifying novel substrates.

    ACKNOWLEDGEMENTS

We thank Dr. Ian G. Macara for critical review of the manuscript. We are indebted to Corky Harrison for excellent administrative support.

    FOOTNOTES

* This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant DK41077, and Research Project Grant 99-105-01-TBE from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

|| To whom correspondence should be addressed: Box 800577, Markey Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, VA 22908-0001. Fax: 804-924-1236; E-mail: jas8j@virginia.edu.

Published, JBC Papers in Press, August 1, 2000, DOI 10.1074/jbc.M005892200

    ABBREVIATIONS

The abbreviations used are: MAPKAPK, mitogen-activated protein kinase-activated protein kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; RSK, p90 ribosomal S6 kinase; MSK, mitogen- and stress-activated protein kinase; MNK, MAPK-interacting kinase; MEK, MAPK-ERK; RSK2/MK2, RSK2/MAPKAPK2; ER, estrogen receptor; PCR, polymerase chain reaction; WT, wild type; GST, glutathione S-transferase; BHK, baby hamster kidney; EGF, epidermal growth factor; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Bjorbæk, C., Zhao, Y., and Moller, D. E. (1995) J. Biol. Chem. 270, 18848-18852
2. Fisher, T. L., and Blenis, J. (1996) Mol. Cell. Biol. 16, 1212-1219
3. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689
4. Leighton, I. A., Dalby, K. N., Caudwell, F. B., Cohen, P. T. W., and Cohen, P. (1995) FEBS Lett. 375, 289-293
5. Poteet-Smith, C. E., Smith, J. A., Lannigan, D. A., Freed, T. A., and Sturgill, T. W. (1999) J. Biol. Chem. 274, 22135-22138
6. Waskiewicz, A. J., Flynn, A., Proud, C. G., and Cooper, J. A. (1997) EMBO J. 16, 1909-1920
7. Fukunaga, R., and Hunter, T. (1997) EMBO J. 16, 1921-1933
8. Deak, M., Clifton, A. D., Lucocq, J. M., and Alessi, D. R. (1998) EMBO J. 17, 4426-4441
9. Pierrat, B., da Silva Correia, J., Mary, J. L., Tomas-Zuber, M., and Lesslauer, W. (1998) J. Biol. Chem. 273, 29661-29671
10. Smith, J. A., Poteet-Smith, C. E., Malarkey, K., and Sturgill, T. W. (1999) J. Biol. Chem. 274, 2893-2898
11. Gavin, A. C., and Nebreda, A. R. (1999) Curr. Biol. 9, 281-284
12. Traish, A. M., al-Fadhli, S., Klinge, C., Kounine, M., and Quick, T. C. (1995) Steroids 60, 467-474
13. Traish, A. M., and Pavao, M. (1996) Steroids 61, 549-556
14. Scimeca, J. C., Nguyen, T. T., Filloux, C., and Van Obberghen, E. (1992) J. Biol. Chem. 267, 17369-17374
15. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233
16. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489-27494
17. Joel, P. B., Smith, J., Sturgill, T. W., Fisher, T. L., Blenis, J., and Lannigan, D. A. (1998) Mol. Cell. Biol. 18, 1978-1984
18. Yang, S. H., Whitmarsh, A. J., Davis, R. J., and Sharrocks, A. D. (1998) EMBO J. 17, 1740-1749
19. Zúñiga, A., Torres, J., Ubeda, J., and Pulido, R. (1999) J. Biol. Chem. 274, 21900-21907
20. Adams, P. D., Li, X., Sellers, W. R., Baker, K. B., Leng, X., Harper, J. W., Taya, Y., and Kaelin, W. G. (1999) Mol. Cell. Biol. 19, 1068-1080
21. Stokoe, D., Caudwell, B., Cohen, P. T. W., and Cohen, P. (1993) Biochem. J. 296, 843-849
22. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H. F., and Marshall, C. J. (1998) Curr. Biol. 8, 1049-1057

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. White, C. A. Pargellis, J. M. Studts, B. G. Werneburg, and B. T. Farmer II
Molecular basis of MAPK-activated protein kinase 2:p38 assembly
PNAS, April 10, 2007; 104(15): 6353 - 6358.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. L. Parra, M. Buxade, and C. G. Proud
Features of the Catalytic Domains and C Termini of the MAPK Signal-integrating Kinases Mnk1 and Mnk2 Determine Their Differing Activities and Regulatory Properties
J. Biol. Chem., November 11, 2005; 280(45): 37623 - 37633.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
P. P. Roux and J. Blenis
ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions
Microbiol. Mol. Biol. Rev., June 1, 2004; 68(2): 320 - 344.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
G. C. Scheper, J. L. Parra, M. Wilson, B. van Kollenburg, A. C. O. Vertegaal, Z.-G. Han, and C. G. Proud
The N and C Termini of the Splice Variants of the Human Mitogen-Activated Protein Kinase-Interacting Kinase Mnk2 Determine Activity and Localization
Mol. Cell. Biol., August 15, 2003; 23(16): 5692 - 5705.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. F. Waas and K. N. Dalby
Transient Protein-Protein Interactions and a Random-ordered Kinetic Mechanism for the Phosphorylation of a Transcription Factor by Extracellular-regulated Protein Kinase 2
J. Biol. Chem., April 5, 2002; 277(15): 12532 - 12540.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B.-e Xu, S. Stippec, F. L. Robinson, and M. H. Cobb
Hydrophobic as Well as Charged Residues in Both MEK1 and ERK2 Are Important for Their Proper Docking
J. Biol. Chem., July 6, 2001; 276(28): 26509 - 26515.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/41/31588    most recent
M005892200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, J. A.
Right arrow Articles by Sturgill, T. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, J. A.
Right arrow Articles by Sturgill, T. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS