©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase (*)

(Received for publication, January 6, 1996; and in revised form, February 14, 1996)

Megan M. McLaughlin(§) (1) Sanjay Kumar(§) (2) Peter C. McDonnell (2) Stephanie Van Horn (3) John C. Lee (4) George P. Livi (1) Peter R. Young (2)(¶)

From the  (1)Departments of Gene Expression Sciences, (2)Molecular Immunology, (3)Molecular Genetics, and (4)Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.


INTRODUCTION

One response to a variety of cellular stimuli involves a series of protein kinase steps known as the mitogen-activated protein (MAP) (^1)kinase cascade(1, 2, 3) . Several genetically distinct MAP kinase pathways have been defined in yeast and at least three exist in mammalian cells(1, 2) . The mammalian MAP kinases include the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the CSBP/p38/RK/Mpk2 kinases(2) . These kinases are activated by distinct upstream dual specificity kinases (MAP kinase kinases), which phosphorylate both threonine and tyrosine in a regulatory TXY (Thr-Xaa-Tyr, where X is any amino acid) loop present in all MAP kinases(4) . Once activated, these MAP kinases phosphorylate their substrates on serine and/or threonine residues with attendant effects on their activity. For example, phosphorylation of c-Jun and ATF2 by JNK (5, 6) stimulates their transcriptional activity.

CSBP (also known as p38, RK, and mpk2) (7, 8, 9) is the mammalian homologue of the yeast Hog1 protein, which is required for growth of yeast in high osmolarity media(10) , and it can partially complement a hog1 deficiency in yeast(8, 11) . CSBP is activated in mammalian cells by environmental or chemical stress such as hyperosmolarity, UV light, heat shock, arsenite, and endotoxin or cytokines such as interleukin-1 and tumor necrosis factor (TNF)(7, 8, 9, 12, 13) . In response to stress, CSBP kinase activity is activated through phosphorylation by at least two MAP kinase kinases, MKK3 and MKK4 (also known as SEK)(14, 15) . Of the in vitro substrates of CSBP, which include MAPKAP kinase-2(9, 12, 16) , myelin basic protein(7, 11) , and ATF2(13) , only MAPKAP kinase-2 is known to be an in vivo substrate, since pretreatment of cells with SB203580, a specific inhibitor of CSBP, blocks the activation of MAPKAP kinase-2. In turn, MAPKAP kinase-2 phosphorylates the small heat shock proteins HSP25/27 in vitro and in vivo(9, 12, 16) . Inhibitors of CSBP also block the production of inflammatory cytokines from lipopolysaccharide-stimulated human monocytes (7) and interleukin-1-stimulated endothelial cells(17) , and more recently CSBP has been implicated in the apoptosis of neurons upon growth factor removal(18) .

Given the many signals that activate CSBP, and its potential involvement in several cellular responses, we were interested in discovering further activators and substrates. In this paper, we used human CSBP as the bait in a yeast two-hybrid screen (19) to identify a novel serine-threonine protein kinase, MAPKAP kinase-3, which binds to and is an in vivo and in vitro substrate of CSBP.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

COS and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) in a humidified 5% CO(2) environment. Transient transfections were performed using Lipofectamine reagent according to the manufacturer's recommendations (Life Technologies, Inc.).

Plasmid Construction for Two-hybrid and Mammalian Expression

Plasmids pGBT9, pGAD424, pTD1, pVA3, and pLAM5` were purchased from Clontech Laboratories. Plasmid pTD1 encodes a GAL4-AD-SV40 large T-antigen fusion protein (in pGAD3F), and pVA3 encodes a GAL4-BD-p53 fusion protein (in pGBT9). These proteins were used as a positive control in the two-hybrid assay(20, 21) . pLAM5` encodes a GAL4-BD-lamin C fusion protein and was used to eliminate false positives. The 1.2-kb XhoI-Asp718I fragment from p137NBU-CSBP2 (11) was blunt end-cloned into the SmaI site of pGBT10 (^2)to create pGBT10-CSBP2. (pGBT10 is a modification of pGBT9 (22) where the SmaI site is in the +1 position when compared to its position in pGBT9.) The in-frame fusion of pGBT10-CSBP2 was confirmed by sequencing on an automated DNA sequencer (Applied Biosystems, Inc.). Replacement of the 904-base pair BglII fragment from pGBT10-CSBP2 with the 886-base pair BglII fragment from p138NBU-CSBP2(D168A) (11) created pGBT10-CSBP2(D168A). Mammalian vectors for expressing epitope tagged CSBP cDNAs were engineered by polymerase chain reaction amplifying the coding sequence of CSBP1, CSBP2, and CSBP2(D168A) using a 5` primer encoding the FLAG epitope (International Biotechnology, Inc.) and a 3` primer from pBS-CSBP (11) and subcloned into CDN(23) . Similarly, a mammalian expression vector encoding hemagglutinin (HA-12CA5) (24) epitope-tagged MAPKAP kinase-3 was constructed by polymerase chain reaction using a 5` primer encoding the HA epitope. The transient expression of epitope-tagged proteins in both COS and HeLa cells was confirmed by immunoblotting with anti-HA (Boehringer Mannheim) and anti-FLAG (International Biotechnologies, Inc.) or anti-CSBP antiserum(12) .

Yeast Two-hybrid Screen

A GAL4-AD/human leukocyte cDNA fusion library was purchased from Clontech. Transformations of HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3,112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(GAL4 17-mers)(3)-CYC1-lacZ) (25) were performed by a modification of the lithium acetate method as recommended by Clontech. Expression of stable fusion proteins from both CSBP constructs was detected with a rabbit polyclonal antiserum to the DNA-binding domain (BD) of yeast GAL4 (Upstate Biotechnologies, Inc.). Selection of library proteins interacting with CSBP2(D168A) was carried out on synthetic complete (SC) media lacking tryptophan, leucine, and histidine (SC-Trp-Leu-His) and containing 10 mM 3-aminotriazole (Sigma). Colony lift beta-galactosidase filter assays were performed as recommended by Clontech. Plasmid pCL1 (encoding MAPKAP kinase-3) was rescued from yeast by electroporation of DH5alpha and sequenced as above.

Expression of MAPKAP Kinase-3 as a GST Fusion Protein

The 1.39-kb EcoRI insert from pCL1 was ligated to EcoRI-digested pGEX-5X-1 (Pharmacia Biotechnology, Inc.). The resulting plasmid pGEX-5X-CL1 was introduced into Escherichia coli strain BL21 (Pharmacia) and the expression of protein was induced by the addition of 0.1 mM isopropyl-beta-D-thiogalactosidase for 2 h. The glutathione S-transferase (GST) or GST-MAPKAP kinase-3 fusion protein was purified by glutathione-Sepharose (GSH-Sepharose) affinity chromatography according to vendor's directions. The integrity and purity of the 67-kDa GST-MAPKAP kinase-3 was determined to be >95% as judged by SDS-PAGE and Coomassie staining.

Immunoprecipitation and Kinase Assay

Mammalian cells expressing epitope-tagged proteins were activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 0.4 M sorbitol or 20 ng/ml TNF treatment. In some cases cells were pretreated with 10 µM SB203580(7, 16) . The cells were washed twice in phosphate-buffered saline and solubilized on ice in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 25 mM beta-glycerophosphate, 20 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl flouride, 1 µg/ml leupeptin, 5 units/ml aprotinin) and centrifuged at 15,000 times g for 20 min at 4 °C. Endogenous or epitope-tagged (FLAG for CSBP and HA for MAPKAP kinase-3) proteins were precipitated from cell lysates using appropriate antibodies for 2 h at 4 °C. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Hepes, pH 7.4, 25 mM MgCl(2), 25 mM beta-glycerophosphate, 100 µM sodium orthovanadate, 2 mM dithiothreitol), and the immune complex kinase assays were initiated by the addition of 25 µl of kinase buffer containing 1-5 µg of substrate and 50 µM [-P]ATP (20 Ci/mmol). After 30 min at 30 °C, the reaction was stopped by the addition of SDS sample buffer and the phosphorylated products analyzed by SDS-PAGE and autoradiography. In some kinase assays, purified GST-Erk1 expressed in E. coli or p44 purified from sea star (Upstate Biotechnologies, Inc.) were used.

Binding of Endogenous CSBP or FLAG-CSBPs to GST- or HA-MAPKAP Kinase-3

GST-MAPKAP kinase-3 bound to GSH-Sepharose (5 µg of protein/20 µl of beads) was incubated at 4 °C for 2 h with gentle rotation with either sorbitol-stimulated COS cell (expressing epitope-tagged CSBP1, CSBP2 or CSBP2(D168A)) or HeLa cell lysates (100 µg) with and without sorbitol stimulation (expressing endogenous CSBP). The beads were washed six times with 1 ml of lysis buffer, and CSBP protein bound to beads was detected by immunoblotting with anti-FLAG (International Biotechnologies, Inc.) or anti-CSBP specific antiserum(7) . For co-precipitation studies in vivo, HA-tagged MAPKAP kinase-3 and FLAG-tagged CSBP2 were co-transfected into HeLa cells. Cell lysates were immunoprecipitated with anti-HA antibody (Boehringer Mannheim) and immunoblotted with anti-FLAG antibody to detect co-precipitated FLAG-CSBP.


RESULTS AND DISCUSSION

Cloning of a cDNA Encoding a Protein Interacting with CSBP

We conducted a yeast two-hybrid screen to identify proteins that interact with CSBP. When full-length wild-type CSBP2 was constructed as a GAL4-BD fusion, it activated the HIS3 reporter in HF7c in the absence of an interacting protein. Since CSBP2 expressed in yeast is active in the absence of an exogenous stimulus (11) , we suspected that this activation may have been related to the high basal kinase activity. Therefore, a catalytically inactive form of the enzyme, CSBP2(D168A)(11) , was engineered as a GAL4-BD fusion, and this did not activate the HF7c HIS3 reporter when co-transformed with the GAL4-AD plasmid. We then co-transformed this GAL4-BD-CSBP2(D168A)-encoding plasmid with a GAL4-AD-human leukocyte cDNA fusion library into HF7c and plated the transformants on SC-Trp-Leu-His media to directly select for cells containing both plasmids as well as for interacting two-hybrid proteins. A total of 87 positives were obtained from this primary screen, but only one strain tested positive for activation of both reporter genes (i.e. His and blue color for lacZ). The GAL4-AD library plasmid isolated from this strain, pCL1 (for CSBP, leukocyte 1), was retransformed into HF7c in the presence of either control or the GAL4-BD-CSBP(D168A)-encoding plasmids. Activation of the HIS3 and lacZ reporters occurred only when pCL1 was present in combination with the plasmid encoding GAL4-BD-CSBP2(D168A) but not with the control plasmids encoding GAL4-BD or GAL4-BD-human lamin C fusion protein.

Analysis of Predicted CSBP Interacting Protein Sequence

Upon sequencing, a possible initiation codon, following the predictions of Kozak(26) , was found at the 14th codon following the 5` EcoRI site, and this was followed by an complete open reading frame encoding a 382-amino acid protein with a calculated mass of 42.8 kDa. A search of GenBank indicated that the pCL1-encoded protein was novel and closely related to the serine-threonine protein kinase MAPKAP kinase-2. The predicted amino acid sequence of the 1.39-kb pCL1 cDNA insert and its alignment with the two known isoforms of human MAPKAP kinase-2 are shown in Fig. 1. Because it shares 70% amino acid identity with the two known isoforms of human MAPKAP kinase-2 and 31% amino acid identity with another MAP kinase substrate, p90 (also known as MAPKAP kinase-1), we called the new protein MAPKAP kinase-3. Interestingly, MAPKAP kinase-3 does not appear to retain the Pro-Pro-Pro-Xaa-Pro-Pro sequence found in the N-terminal domain of MAPKAP kinase-2, which has been suggested to be a binding site for SH3 domains(27) , but the putative nuclear localization signal found near the C terminus of one of the two MAPKAP kinase-2 splice variants (27) is conserved.


Figure 1: Sequence and alignment of human MAPKAP kinases-2 and -3. The predicted amino acid sequence of MAPKAP kinase-3 and alignment with MAPKAP kinase-2A (27) and MAPKAP kinase-2B (35) . The alignment was performed using MEGALIGN (DNASTAR, Inc.). Roman numerals indicate various kinase subdomains(4) . The proline-rich motif in the N terminus and the putative nuclear localization signal at the C terminus are boxed. Residues phosphorylated by CSBP/p38 in MAPKAP kinase-2, as well as the autophosphorylation sites (33) are shown by an asterisk and a dot, respectively, below the sequence.



Northern blot analysis showed a predominant mRNA of 3.5 kb and minor mRNA of 2 kb in most tissues except brain, with highest abundance in heart and skeletal muscle (data not shown).

In Vitro Association of MAPKAP Kinase-3 with CSBP

The identification of MAPKAP kinase-3 through its interaction with CSBP2 in a yeast two-hybrid screen suggested that it should bind CSBP in vitro. This was confirmed by incubating bacterially expressed and purified GST-MAPKAP kinase-3 with COS cell lysates expressing transfected CSBP1, CSBP2, CSBP2(D168A), or HeLa cell lysate expressing the endogenous CSBP (Fig. 2A), followed by precipitation and analysis by SDS-PAGE and immunoblot. GST-MAPKAP kinase-3 was able to bind and precipitate all three CSBPs from COS cells and endogenous CSBP from HeLa cells, indicating that both spliced forms of CSBP bind to MAPKAP kinase-3 independent of kinase activity. The lower amount of CSBP1 associated with GST-MAPKAP kinase-3 was due to a lower expression of CSBP1 in transfected COS cells (data not shown). After compensation for the expression level, the binding efficiencies for CSBP1, CSBP2, and CSBP2(D168A) are comparable. We were also able to show that activated epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) isolated from transfected COS cells could precipitate GAL4-AD-MAPKAP kinase-3 but not GAL4-AD alone from yeast lysates (data not shown).


Figure 2: GST-MAPKAP kinase-3 binds CSBP from COS and HeLa cells. A, GST or GST-MAPKAP kinase-3 (5 µg) loaded Sepharose beads (20 µl) were mixed with COS cell lysates (100 µg) expressing FLAG-tagged CSBP1, CSBP2, or CSBP2(D168A), respectively. After incubation for 2 h at 4 °C, beads were pelleted and washed extensively with lysis buffer and analyzed by immunoblotting with anti-FLAG antibody. Lane 7 (labeled C) represents a control COS cell lysate expressing CSBP immunoprecipitated with anti-CSBP. The same experiment was also repeated with HeLa lysate (100 µg) with or without salt activation as a source of endogenous CSBP, and blotted with anti-CSBP antibody (B) or with anti-phosphotyrosine antibody (C). The position of molecular mass markers (kDa) is indicated on the left.



The presence of small amounts of a slower migrating form of CSBP in precipitates (e.g.Fig. 2A), which corresponds to the activated, tyrosine-phosphorylated form of the enzyme, suggests that both activated and unactivated forms of CSBP can bind. This was further demonstrated by the experiments illustrated in Fig. 2(B and C), where GST-MAPKAP kinase-3 bound and precipitated both tyrosine-phosphorylated and non-tyrosine-phosphorylated CSBP from HeLa cells treated with or without sorbitol. In additional experiments, we are able to show that purified recombinant CSBP binds to purified GST-MAPKAP kinase-3, indicating that the association is direct and does not depend on any other proteins present in the yeast or mammalian cell lysates (data not shown).

MAPKAP Kinase-3 Is an in Vitro Substrate of CSBP, and HSP27 Is an in Vitro Substrate of MAPKAP Kinase-3

We next tested if MAPKAP kinase-3 could act as a substrate of CSBPs by performing immune complex kinase assays with activated CSBPs and either GST-MAPKAP kinase-3 or control GST. Both CSBP1 and CSBP2 from transfected COS cells and endogenous CSBP from HeLa cells phosphorylated GST-MAPKAP kinase-3 but not GST (Fig. 3A, lanes 1-4 and 7), indicating that MAPKAP kinase-3 is indeed a substrate of CSBP. In contrast, the catalytically inactive CSBP2(D168A) did not show any phosphorylation of GST-MAPKAP kinase-3 (Fig. 3A, lane 6). As in the co-precipitation experiments, the differences in kinase activity between COS-expressed CSBP1, CSBP2, and endogenous HeLa CSBP are most likely due to the varying level of CSBP expression. Indeed, we have yet to find any differences in substrate preference between CSBP1 or CSBP2. (^3)As for MAPKAP kinase-2(9, 27) , MAPKAP kinase-3 was also phosphorylated in vitro by activated p44 (Fig. 3B, lane 2) but not by unactivated Erk1 (Fig. 3B, lane 1).


Figure 3: MAPKAP kinase-3 is a substrate of CSBP, and HSP27 is a substrate of MAPKAP kinase-3 in vitro. A, E. coli-expressed GST or GST-MAPKAP kinase-3 (100 µg/ml) was used a substrate in an immune complex kinase assay with either CSBP1, CSBP2, or CSBP2(D168A) from transfected and activated COS cells or endogenous CSBP from HeLa cells as indicated. HSP27 (120 µg/ml) was included in lanes 8 and 9, and GST-MAPKAP kinase-3 was omitted from lane 9. The position of molecular mass markers (kDa) is indicated on the left. B, 50 ng of purified GST-Erk1 expressed in E. coli (lane 1) or 50 ng of purified p44 from sea star (lane 2) was used to phosphorylate GST-MAPKAP kinase-3.



Since Hsp27 is a known substrate of MAPKAP kinase-2 in vitro and in vivo, we wanted to determine if it was also a substrate of MAPKAP kinase-3. Inclusion of HSP27 in the CSBP/GST-MAPKAP kinase-3 immune complex kinase reaction resulted in its phosphorylation (Fig. 3, lane 8), whereas CSBP alone or unactivated MAPKAP kinase-3 did not phosphorylate Hsp27 (Fig. 3, lane 9; data not shown), indicating that Hsp27 is a substrate of MAPKAP kinase-3.

MAPKAP Kinase-3 Associates with and Is a Substrate of CSBP in Vivo

To determine if stress and cytokine activation of the CSBP pathway leads to MAPKAP kinase-3 activation, we transfected HeLa cells with vector expressing HA-tagged MAPKAP kinase-3 and treated them with sorbitol or TNF in the presence or absence of SB203580, a specific inhibitor of CSBP kinase activity(7, 16) . SB203580 has been shown to specifically inhibit the CSBP/p38 kinase activity both in vitro and in vivo and does not inhibit either ERK or JNK and various other kinases tested(7, 16) . MAPKAP kinase-3 was isolated from these cells using anti-HA antibodies, and an immune complex kinase assay was performed with HSP27 as a substrate. As shown in Fig. 4A, both sorbitol and TNF treatment stimulated MAPKAP kinase-3 activity (Fig. 4A, lanes 4 and 6), which was almost completely blocked by pretreatment with 10 µM SB203580 (Fig. 4A, lanes 5 and 7). In contrast, treatment of cells with PMA (a potent activator of the ERK pathway) did not result in MAPKAP kinase-3 activation (Fig. 4A, lanes 1-3), even though ERK isolated from these cells was active as judged by an anti-phosphotyrosine blot (data not shown). These results suggest that stress- and cytokine-activated CSBP activates MAPKAP kinase-3 in vivo.


Figure 4: Sorbitol- and TNF-mediated cell stimulation results in CSBP and MAPKAP kinase-3 activation. A, HeLa cells were transfected with HA-tagged MAPKAP kinase-3 and pretreated with 10 µM SB203580 and/or treated with 50 ng/ml PMA, 0.4 M sorbitol or 20 ng/ml TNF for 10 min as indicated. After activation, the cells were lysed and HA-MAPKAP kinase-3 was immunoprecipitated with anti-HA antibody and an immune complex kinase assay was performed with HSP27 as a substrate (indicated by an arrow). B, HeLa cells were either co-transfected with FLAG-CSBP2 and HA-MAPKAP kinase-3 (lanes 1 and 2) or with vector alone (lanes 3 and 4), activated with 0.4 M sorbitol for 10 min, and immunoprecipitated with anti-HA antibody. The immunoprecipitate was analyzed by immunoblot using anti-FLAG antibody to detect co-precipitated FLAG-CSBP (indicated by an arrow). Arrowheads indicate the heavy and light chain of anti-HA antibody used for immunoprecipitation.



To confirm the association of CSBP and MAPKAP kinase-3 in vivo, we co-expressed FLAG-tagged CSBP2 and HA-tagged MAPKAP kinase-3 in HeLa cells. Immunoprecipitation of MAPKAP kinase-3 with anti-HA antibody resulted in co-precipitation of CSBP in cells transfected with both cDNAs but not from cells transfected with vector alone, as determined by immunoblotting with anti-FLAG antibodies (Fig. 4B, lanes 1-4). Like the data in Fig. 2B, the amount of CSBP co-precipitated from sorbitol-activated cells was lower than that from unactivated cells, suggesting weaker association of the CSBP-MAPKAP kinase-3 complex. These data indicate that MAPKAP kinase-3 is functionally similar to MAPKAP kinase-2, in that they are both in vivo substrates of CSBP/p38, and can phosphorylate Hsp27 in vitro. It is not clear whether both kinases contribute to phosphorylation of hsp27 in vivo.

The finding of association between CSBP and its substrate MAPKAP kinase-3 is reminiscent of that between ERK2 and p90 (also known as MAPKAP kinase-1)(28) , in which the two kinases were found to be in a stable 110-kDa complex within unactivated Xenopus oocytes. Upon activation, ERK dissociates from the complex and is predominantly monomeric. Similarly, in the present case a stable complex between CSBP and MAPKAP kinase-3 exists in vivo, and the preliminary data in Fig. 2B and Fig. 4B suggest some dissociation of the activated enzymes, although further experiments will be needed to establish this.

There are several reports of MAP kinases binding to other proteins. ERK has been reported to interact with the transcription factor Elk1 (29, 30) and the high and low affinity nerve growth factor receptors (31) by co-immunoprecipitation from mammalian cells, and the protein kinase MEK1 in a yeast two-hybrid screen(32) . JNK binds to the transcription factors c-Jun and ATF2 in vitro(5) . Formation of these complexes may serve to restrict cross-talk between different MAP kinase pathways. For example, both ERK and CSBP can activate MAPKAP kinase-2 and -3 in vitro, but only CSBP appears to activate these kinases in vivo(9, 27, 33) . JNK, another stress- and cytokine-activated MAP kinase, did not phosphorylate MAPKAP kinase-3 in vitro (data not shown). Thus, MAPKAP kinase-3 appears to be a specific substrate of CSBP.

Recently, the sites of MAPKAP kinase-2 phosphorylation by CSBP/RK in vitro and in vivo were determined (33) and are illustrated in Fig. 1. The three key phosphorylation sites, Thr-222, Ser-272 and Thr-334, any two of which must be phosphorylated for maximal activation of MAPKAP kinase-2 activity, are conserved in MAPKAP kinase-3. An additional in vivo autophosphorylation site is also conserved (Thr-338), while a second site is changed from Ser to Thr (Ser-9), and a third is not conserved (Thr-25). Another component of MAPKAP kinase-2 activation conserved in MAPKAP kinase-3 is an autoinhibitory alpha-helix near the C terminus, which mimics the substrate and when deleted produces a constitutively active MAPKAP kinase-2(34) . It is likely that these conserved features play a role in the activation of MAPKAP kinase-3.

Given the apparent similarity in activation and substrate activity of MAPKAP kinase-2 and MAPKAP kinase-3, it will be of interest to determine if these two closely related kinases play different roles in stress and cytokine stimulated cells. The availability of purified CSBP and MAPKAP kinase-3 and their cDNAs will allow further characterization of their binding interface.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43784[GenBank].

§
These two authors contributed equally to this work and should both be considered as co-first authors.

To whom correspondence should be addressed: Dept. of Molecular Immunology/UE0548, SmithKline Beecham Pharmaceuticals, P. O. Box 1539, King of Prussia, PA 19406-0939; Tel.: 610-270-7691; Fax: 610-270-7962.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; AD, activation domain; BD, binding domain; CSBP, CSAID(TM)-binding protein; GST, glutathione S-transferase; HA, hemagglutinin; HSP, heat shock protein; JNK, c-Jun N-terminal kinase; MAPKAP kinase, mitogen-activated protein kinase-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; kb, kilobase(s).

(^2)
E. Rheaume, personal communication.

(^3)
S. Kumar, unpublished studies.


ACKNOWLEDGEMENTS

We thank our colleagues, Eric Rheaume for pGBT10, Seth Fisher for purified CSBP and Joyce Mao, Donald Bennett and Ganesh Sathe for oligonucleotide synthesis and sequencing, and Prof. P. Cohen for a preprint.


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