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Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

Background: DUSP9/MKP-4 dephosphorylates and inactivates MAP kinases. Results: The kinase interaction motif of DUSP9/MKP-4 is phosphorylated by protein kinase A, and this reduces its ability to interact with and inactivate MAP kinase substrates. Conclusion: DUSP9/MKP-4 represents a point of cross-talk between PKA and MAP kinase signaling. Significance: Cross-talk between distinct signal transduction pathways may be important in determining physiological response.


MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/ MKP-3, DUSP9/MKP-4 also inactivates the p38␣ MAP kinase both in vitro and in vivo.
Here we demonstrate that inactivation of both ERK1/2 and p38␣ by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55 RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38␣ MAP kinases. In addi-tion, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/ MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38␣ in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38␣ in vivo.
DUSP9/MKP-4 4 is one of a group of three cytoplasmic dualspecificity MAP kinase phosphatases that includes the prototypic ERK-specific phosphatase DUSP6/MKP-3 and DUSP7/ MKP-X (1-3). However, unlike DUSP6/MKP-3, which is highly specific in its ability to interact with and inactivate the classical ERK1/2 MAPKs (4, 5), DUSP9/MKP-4 can also interact with and inactivate p38␣ MAPK both in vitro and in vivo, indicating that this phosphatase regulates both mitogen and stress-activated MAPK signaling (6,7). Deletion of the murine Dusp9 gene causes embryonic lethality at mid-gestation. This is caused by placental insufficiency due to a failure in labyrinth development, and this end point correlates exactly with the spatial and temporal pattern of DUSP9/MKP-4 expression in the normal placenta (8). Despite a failure to observe gross changes in the levels of either ERK1/2 or p38␣ phosphorylation in placental tissues lacking DUSP9/MKP-4, disruption of either p38␣ or MEK1, an upstream activator of ERK1/2, gives rise to placental defects almost identical to those seen on loss of DUSP9/MKP-4 (9,10). This indicates that the abnormal regulation of either or both of these MAPK pathways likely contributes to placental failure in animals lacking DUSP9/MKP-4.
Tetraploid rescue experiments, which bypass the lethality caused by deletion of DUSP9/MKP-4, give rise to animals that develop normally with no obvious phenotype and are fertile. Furthermore, despite high levels of DUSP9/MKP-4 expression in the developing liver, adult kidney, and testis, these tissues develop normally in embryos lacking DUSP9/MKP-4, indicating that the essential function of this phosphatase is restricted to the extraembryonic tissues (8). More recently, DUSP9/ MKP-4 has been implicated in the regulation of insulin signaling in murine models of obesity and stress-induced insulin resistance (11,12). A possible link between this gene and susceptibility to type 2 diabetes in humans is also suggested by the recent identification of a type 2 diabetes risk locus near DUSP9 in a genome-wide association study, the first such locus to be identified on the X chromosome (13).
MKPs recognize and bind their cognate MAPK substrates through an arginine-rich kinase interaction motif (KIM) located in the amino-terminal non-catalytic domain of the protein. Furthermore, MAPK binding via this motif causes conformational changes at the active site of the enzyme leading to catalytic activation of MKPs in vitro (1,2). Consistent with its activity toward both ERK2 and p38␣, DUSP9/MKP-4 interacts with both of these MAPKs in yeast two-hybrid assays, and the catalytic activity of DUSP9/MKP-4 toward para-nitrophenyl phosphate (p-NPP) in vitro is increased on incubation with either recombinant ERK2 or p38␣ (6,14). Here we demonstrate that the ability of DUSP9/MKP-4 to recognize both ERK1/2 and p38␣ is mediated by a conserved KIM comprising essential arginine residues at positions 52 and 53. Furthermore, DUSP9/ MKP-4 is unique among the cytoplasmic MKPs in containing a conserved cAMP-dependent protein kinase (PKA) consensus phosphorylation site (Ser-58) immediately carboxyl-terminal to the KIM. This site can be modified by PKA in vitro, and phosphorylation abolishes the interaction of the amino-terminal domain of DUSP9/MKP-4 with both ERK2 and p38␣ in glutathione S-transferase (GST) pulldown assays. Consistent with a role for this modification in regulating the activity of DUSP9/MKP-4 toward these MAPKs in vivo, phospho-mimetic (glutamic acid) substitution of Ser-58 also reduces the ability of DUSP9/MKP-4 to inactivate both ERK1/2 and p38␣ MAPKs when expressed in mammalian cells. Finally, using a phospho-specific antibody raised against this site, we demonstrate that endogenous DUSP9/MKP-4 is modified on Ser-58 in response to the PKA agonist forskolin and also in murine placental tissue in vivo. We conclude that DUSP9/ MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and both mitogen-and stressactivated MAPK signaling.

EXPERIMENTAL PROCEDURES
Reagents-Forskolin was purchased from Sigma. Okadaic acid was obtained from Calbiochem. Monoclonal antibodies against myc and hemagglutinin (HA) were from Cancer Research UK. Antibodies against phospho-ERK, ERK, phospho-p38, p38, protein kinase A catalytic subunit (PKAc), and phospho-cyclic AMP response element-binding protein (CREB)/activating transcription factor 1 (ATF1) were purchased from Cell Signaling Technology. The anti-tubulin antibody was purchased from Santa Cruz. The sheep polyclonal antiserum (#302) raised against murine DUSP9/MKP-4 and the anti-GST rabbit polyclonal antibody have been described previously (6,15). All cell culture reagents were from Invitrogen.
Expression and Purification of Recombinant Proteins-Recombinant histidine-tagged proteins were expressed in the E. coli strain Rosetta DE3. All subsequent purification and refolding steps were performed exactly as described previously (21).
Generation of Antisera-A polyclonal antiserum (#657) was raised in sheep at the Scottish Antibody Production Unit (SAPU, Lanarkshire, UK) using recombinant human DUSP9/ MKP-4 protein as antigen. Phospho-specific antibodies recognizing either human or murine DUSP9/MKP-4 phosphorylated on Ser-58 were also raised in sheep (SAPU) using either peptide RLRRGSLSVRA or RLRRGSMSVRS corresponding to residues 53-63 of human or murine DUSP9/MKP-4, respectively. The phosphoserine residue is in bold and underlined. The antisera were affinity-purified as described previously (22).
Protein Phosphatase Assays-Phosphatase activities and catalytic activation of wild-type and mutant forms of DUSP9/ MKP-4 were assayed using p-NPP hydrolysis exactly as described previously (23).
For GST pulldown assays, E. coli were grown and induced as before. 3-ml (pGEX.PTP-SL-(147-288)) or 9 ml (pGEX.MKP-4-1-267wt and pGEX.MKP-4-1-267 S58A) culture volumes were centrifuged, and the pellets were resuspended in 600 l of PBS, 0.1% Triton X-100. Cell suspensions were then sonicated and centrifuged, and the supernatants were divided into two aliquots. 30 l of glutathione-Sepharose was then added to each aliquot, and proteins were incubated for 15 min at 4°C with shaking. Proteins were washed 4 times with PBS and once with PKA reaction buffer containing 0.2 mM ATP before incubation in 45 l of PKA reaction buffer containing 0.2 nM ATP either with or without 0.5 units/l PKAc (Promega) for 3 h at 25°C with shaking. Proteins were then washed once with PBS at 4°C. HEK293 cells were lysed in a buffer containing 100 mM Tris-HCl, pH 7.5, 300 mM NaCl, 2% octylphenoxypoly-ethoxyethanol (Igepal), 100 mM PMSF, 0.2 mg/ml aprotinin 0.5 M NaF, 200 mM Na 4 P 2 O 7 . 1.5 ml of this lysate was then added to each reaction and incubated for 2 h at 4°C with shaking. After incubation, proteins were washed 4 times with HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM Na 4 P 2 O 7 ) and once with PBS at 4°C before the addition of SDS sample buffer and analysis by SDS page and Western blotting.
Yeast Two-hybrid Assays-Assays were performed according to the manufacturer's instructions (Matchmaker 3 kit, Clontech) exactly as described previously (18).
Kinase Assays-The activity of wild-type and mutant forms of DUSP9/MKP-4 toward either ERK2 or p38␣ was determined by co-transfection of COS-1 cells with expression vectors encoding either HA-tagged ERK2 or p38␣ together with increasing amounts of an expression vector encoding either wild-type or mutant forms of DUSP9/MKP-4. After transfection, cells were exposed to an appropriate activating stimulus (15% fetal calf serum for ERK2 or 10 g/ml anisomycin for p38␣) for 30 min followed by cell lysis. Proteins were then analyzed by SDS-PAGE and Western blotting. HA-tagged MAP kinases were immunoprecipitated before assay of kinase activity toward myelin basic protein exactly as described previously (6).
Cell Culture and Treatments-Immortalized mouse embryo fibroblasts (MEFs) and COS-1 cells were cultured and transfected exactly as described previously (6,24). To stimulate PKA activity, cells were treated with 10 M forskolin for 30 min either with or without a 30-min preincubation with 1 M okadaic acid to inhibit protein phosphatase activity. Cells were then lysed, and proteins were either immunoprecipitated or analyzed directly by SDS-PAGE and Western blotting.
Immunoprecipitation and Analysis of Phospho-DUSP9/MKP-4-Freshly dissected murine placental tissue or cultured MEFs were lysed in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.2 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100). Lysates were then sonicated and cleared by centrifugation, and the protein concentration was determined using Bradford reagent (Bio-Rad). Sheep polyclonal antiserum against murine DUSP9/MKP-4 (#302, 3 g/immunoprecipitation) was incubated with protein G-Sepharose beads (Sigma, 10 l/immunoprecipitation) overnight at 4°C in an excess of phosphate-buffered saline (PBS). Beads were then washed with 10 volumes of sodium borate, pH 9.0, at room temperature, and coupling was performed by incubation with dimethyl pimelimidate (20 mM) for 30 min. Reactions were terminated by washing the beads in 0.2 M ethanola-mine for 2 h, and any loosely bound IgG was removed by washing briefly in 50 mM glycine, pH 2.5. The pH was corrected with 0.2 M Tris, pH 8.0, and the beads were washed extensively in PBS.
Coupled beads were incubated overnight with 500 g of placental or MEF lysate at 4°C with inversion. Beads were then washed four times in radioimmune precipitation assay buffer containing protease and PhosStop phosphatase inhibitors (Roche Applied Science) before the addition of LDS sample buffer (Invitrogen) supplemented with 0.1% ␤-mercaptoethanol and heating to 95°C for 15 min. Proteins were analyzed by SDS-PAGE and Western blotting. Murine DUSP9/MKP-4 protein was detected using the sheep polyclonal antiserum (#657) raised against human DUSP9/MKP-4 protein. DUSP9/MKP-4 phosphorylation was monitored using the antiserum raised against the murine DUSP9/MKP-4 Ser-58 phosphopeptide. For phosphatase treatments the final two washes of the immunoprecipitation protocol were not carried out. Instead, all samples were washed twice in 1ϫ PMP metallophosphatase buffer (New England Biolabs) supplemented with 1 mM MnCl 2 . -Protein phosphatase (2000 units) was then added. and the reactions were incubated at 30°C for 2 h with occasional agitation. After the addition of LDS sample buffer (Invitrogen) supplemented with 0.1% ␤-mercaptoethanol and heating to 95°C for 15 min, proteins were analyzed by SDS-PAGE and Western blotting as described above.

RESULTS
A Conserved KIM Mediates the Inactivation of Both ERK1/2 and p38␣ by DUSP9/MKP-4-Amino acid sequence alignment of DUSP9/MKP-4, DUSP7/MKP-X, and DUSP6/MKP-3 reveals that DUSP9/MKP-4 contains a potential KIM in which two arginine residues (Arg-52 and Arg-53 in DUSP9/MKP-4) essential for the interaction of DUSP6/MKP-3 with ERK1 and -2 are conserved (Fig. 1A). Although mutation of Arg-52, -53, and -55 to alanine does not affect the intrinsic catalytic activity of DUSP9/MKP-4 toward p-NPP in vitro, it does prevent the catalytic activation of DUSP9/MKP-4 by recombinant ERK2 and p38␣ MAPKs (Fig. 1, B and C), indicating that the KIM mediates a functional interaction with these kinases. To confirm this, we first used a yeast two-hybrid assay to examine the interaction of DUSP9/MKP-4 with ERK2, JNK, and p38␣ MAPKs. Wild-type DUSP9/MKP-4 clearly interacts with ERK2 and p38␣ and only weakly with JNK. In contrast, the R52A/ R53A/R55A mutant (DUSP9-KIM) shows no significant level of interaction with all three MAPKs (Fig. 1, D and E). These results are consistent with our previous data, which demonstrated that DUSP9/MKP-4 could dephosphorylate both ERK1/2 and p38␣ but not JNK when expressed in mammalian cells (6). Finally, we transfected increasing amounts of wild type and the KIM mutant of DUSP9/MKP-4 into COS-1 cells and assayed the phosphorylation of both ERK1/2 and p38␣ by Western blotting. Expression of wild-type DUSP9/MKP-4 significantly reduced the phosphorylation of both ERK1/2 and p38␣, whereas the KIM mutant is unable to dephosphorylate either kinase (Fig. 1, F and G). We conclude that the ability of DUSP9/MKP-4 to interact with and dephosphorylate both ERK1/2 and p38␣ MAPKs in vivo is mediated by a conserved KIM.
The KIM within DUSP9/MKP-4 Contains a PKA Phosphorylation Site-Examination of our amino acid sequence alignment reveals that the KIM within DUSP9/MKP-4 is unique among the three cytoplasmic MKPs in containing a second diarginine motif. Furthermore, this latter motif forms part of a PKA consensus sequence ( 55 RRXS 58 ) that is conserved in mammals (Fig. 1A). The proximity of this phospho-acceptor to the KIM suggests that modification of Ser-58 might modulate the ability of DUSP9/MKP-4 to interact with MAPK substrates. Interestingly, a subset of protein-tyrosine phosphatases (PTPs), which includes the striatal-enriched phosphatase (STEP/ PTPN5), STEP-related phosphatase (PTP-SL/PTPBR7/PT-PRR), and the lymphoid-specific phosphatase hematopoietic PTP (He-PTP/LC-PTP/PTPN7) also recognize ERK1/2 and p38␣ via a conserved KIM (25). Furthermore, in the case of striatal-enriched phosphatase, PTP-SL, and He-PTP, this motif also overlaps with a PKA consensus site, and phosphorylation of this site attenuates the binding of these enzymes to their MAPK substrates (26 -28). To explore the possibility that DUSP9/MKP-4 might also be regulated by PKA, we expressed the MAPK binding domain of DUSP9/MKP-4 (residues 1-267) as a GST fusion protein in which the Ser-58 phospho-acceptor was either intact or mutated to alanine. These proteins were then incubated in vitro with PKAc in the presence of [␥-32 P]ATP and analyzed by SDS-PAGE and autoradiography. Wild-type GST-DUSP9/MKP-4 1-267 clearly shows high levels of 32 P incorporation after incubation with PKAc ( Fig. 2A). In contrast, much lower levels of incorporation were seen with either GST alone or the S58A mutant of GST-DUSP9/MKP-4 1-267, demonstrating that this residue is the PKA target. To determine whether this modification might affect the ability of DUSP9/MKP-4 to bind to either ERK1/2 or p38␣, GST-DUSP9/MKP-4 fusion proteins were phosphorylated by PKAc in vitro as above in the presence of cold ATP, incubated with Rat-1 cell lysates, and precipitated with glutathione-Sepharose. As a positive control, a GST fusion of the wild-type MAPK binding domain of PTP-SL (residues 147-288) was used. Samples were analyzed by SDS-PAGE, and the presence of the MAP kinases ERK1/2 or p38␣ was detected by Western blotting. Clearly, all three GST fusion proteins can interact with both ERK and p38␣ in the absence of PKA. In contrast, incubation of these fusion proteins with PKA in the presence of ATP and Mg 2ϩ abolished the interaction of both wild-type GST-DUSP9/ MKP-4 1-267 and GST-PTP-SL-(147-288) with both MAPKs. In contrast, GST-DUSP9/MKP-4 1-267, in which the Ser-58 phospho-acceptor site was mutated to alanine, retained its ability to bind both ERK1/2 and p38␣ even in the presence of PKA (Fig. 2B). We conclude that Ser-58 can be phosphorylated by PKA in vitro and that this modification can reduce the affinity of the MAPK binding domain of DUSP9/MKP-4 for both ERK1/2 and p38␣.
Modification of Ser-58 Reduces the Affinity of DUSP9/MKP-4 for Both ERK2 and p38␣ and Leads to Impaired Inactivation of These MAPKs-To explore the effects of PKA-mediated phosphorylation of DUSP9/MKP-4 in the context of the full-length protein, we first determined the effects of mutating the Ser-58 NOVEMBER 4, 2011 • VOLUME 286 • NUMBER 44 phospho-acceptor site to either alanine or glutamic acid on the catalytic activation of DUSP9/MKP-4 by recombinant MAPKs in vitro. Although neither mutation significantly affected the intrinsic catalytic activity of DUSP9/MKP-4 toward p-NPP in vitro, the addition of recombinant MAP kinases to these reactions clearly shows that although the activity of both wild-type and the S58A mutant of DUSP9/MKP-4 is significantly increased on the addition of either ERK2 or p38␣, the protein containing the phospho-mimetic S58E mutation shows a much reduced level of catalytic activation in response to these kinases (Fig. 3, A and B). Thus, in agreement with our GST pulldown assays (Fig. 2B), this result indicates that modification of Ser-58 impairs the ability of DUSP9/MKP-4 to interact with both ERK2 and p38␣. To explore this in a more physiological context, we first made use of the yeast two-hybrid assay. This clearly shows that although both wild-type DUSP9/MKP-4 and DUSP9/MKP-4 S58A interact with ERK2, JNK and p38␣ the phospho-mimetic S58E mutant shows a greatly reduced ability to bind to these MAPKs (Fig. 3B). Finally, we compared the ability of wild-type DUSP9/MKP-4 and the Ser-58 mutants to dephosphorylate and inactivate endogenous ERK1/2 and p38␣ MAPKs when expressed in COS-1 cells. Both wild-type DUSP9/MKP-4 and the S58A mutant efficiently dephosphorylated and inactivated both MAPKs. In contrast, the S58E mutant was significantly less active toward both ERK1/2 and p38␣ (Fig. 4, A and B). We conclude that phosphorylation of Ser-58 in the context of full-length DUSP9/MKP-4 leads to a reduced binding affinity toward both ERK1/2 and p38␣ MAPKs, resulting in decreased attenuation of MAPK signaling by this phosphatase.

Phosphorylation of DUSP9/MKP-4 by PKA
Phosphorylation of DUSP9/MKP-4 Is PKA-dependent-To monitor the phosphorylation of DUSP9/MKP-4 in intact cells, we first raised antibodies against a human DUSP9/MKP-4 KIM phospho-Ser-58 peptide. After affinity purification, this antiserum was able to recognize recombinant wild-type human DUSP9/MKP-4 protein but not mutant DUSP9/MKP-4 S58A protein after incubation with PKAc and ATP in vitro (data not shown). However, this antiserum did not recognize murine DUSP9/MKP-4 under similar conditions. Examination of the amino acid sequence of the KIM peptide in murine DUSP9/ MKP-4 revealed four conservative amino acid changes including the substitution of methionine by leucine at the residue immediately carboxyl-terminal to the serine phospho-acceptor. A second antiserum raised against the murine phosphopeptide recognized recombinant murine DUSP9/MKP-4 only after incubation with PKAc and ATP in vitro (data not shown). To verify that DUSP9/MKP-4 could be phosphorylated in vivo, expression vectors encoding either myc-tagged wild type or the S58A mutant of human DUSP9/MKP-4 were transfected into COS-1 cells. After 24 h cells were either left untreated or exposed to the PKA agonist forskolin before analysis by SDS-PAGE and Western blotting. Activation of the PKA pathway clearly results in Ser-58 phosphorylation of wild type but not the S58A mutant of DUSP9/MKP-4 (Fig. 5A). Similar results were obtained on expression of either wild type or the S58A mutant of murine DUSP9/MKP-4 in COS-1 cells using the antibody raised against the murine DUSP9 phosphopeptide (Fig.  5B).
Endogenous DUSP9/MKP-4 Is Phosphorylated in Response to PKA Activation-DUSP9/MKP-4 mRNA and protein expression is tissue-specific. The major sites of expression during early development are in the liver and extraembryonic tissues. In contrast, DUSP9/MKP-4 is absent in the adult liver but is found in the testis and kidney (8). A survey of commonly used laboratory cell lines revealed that DUSP9/MKP-4 is expressed in MEFs where both mRNA and protein can be detected by RT-PCR and Western blotting, respectively (data not shown). To establish that endogenous DUSP9/MKP-4 can be phosphorylated on Ser-58, MEFs were either left untreated or exposed to forskolin both in the absence and presence of the serine/ threonine phosphatase inhibitor okadaic acid. Cells were lysed, and DUSP9/MKP-4 protein was then immunoprecipitated before analysis by SDS-PAGE and Western blotting using our anti-murine phospho-Ser-58 antiserum. We clearly detected phospho-Ser-58 DUSP9/MKP-4 in cells exposed to forskolin, and this occurred concomitantly with activation of PKA as evidenced by increased phosphorylation of the PKA targets CREB and ATF1. Furthermore, levels of Ser-58 phosphorylation are significantly increased in the presence of okadaic acid, indicat- fusion proteins were incubated either in the absence (ϪPKAc) or presence (ϩPKAc) of PKA. HEK293 cell lysates were then added and incubated for 2 h, and GST fusion proteins were then pulled down using glutathione-Sepharose before analysis by SDS-PAGE and Western blotting. MAP kinases were detected using antibodies against either ERK1/2 (B) or p38␣ (C). The presence of GST fusion proteins was verified using an antibody against GST (B and C, lower panels). In the first of each panel, total lysate (20 g) was loaded. NOVEMBER 4, 2011 • VOLUME 286 • NUMBER 44

Phosphorylation of DUSP9/MKP-4 by PKA
ing that phosphorylation of this site is subject to regulation by type 1/2 serine/threonine phosphatases (Fig. 6A). Modification of endogenous DUSP9/MKP-4 was also observed when a constitutively active form of the catalytic subunit of PKA (PKA C␣) was expressed in these cells (Fig. 6B), and the incubation of DUSP9/MKP-4 immunoprecipitated from forskolin-treated cells with -phosphatase reversed the phosphorylation of Ser-58 (Fig. 6C). Finally, we obtained placentas from mouse embryos at day 13.5 and subjected tissue lysates to immunopre-   cipitation and Western blotting. We readily detected phospho-Ser-58 DUSP9/MKP-4 in these lysates, and again this signal was lost on incubation of the immunoprecipitates with -phosphatase (Fig. 6D). We conclude that endogenous DUSP9/MKP-4 can be modified in response to activation of PKA in cultured cells. Furthermore, this modification is present at significant levels in normal murine extraembryonic tissues, where this phosphatase performs an essential function in regulating MAPK signaling during early development.

DISCUSSION
Here we demonstrate that DUSP9/MKP-4 recognizes its cognate MAPK substrates ERK1/2 and p38␣ via a conserved KIM. However, this docking sequence is unique among this family of dual-specificity MAPK phosphatases in containing a second di-arginine motif that forms part of a conserved consensus site for PKA phosphorylation. We demonstrate that the Ser-58 phospho-acceptor within this motif is efficiently phosphorylated by PKA both in vitro and in vivo and that modification of Ser-58 has the potential to attenuate the interaction of DUSP9/MKP-4 with both ERK1/2 and p38␣ MAP kinases resulting in a reduced ability of DUSP9/MKP-4 to negatively regulate these signaling pathways. These results strongly suggest that DUSP9/MKP-4 represents a point of cross-talk between the cAMP and MAPK signaling pathways at which PKA activity would positively reinforce signaling through both the classical Ras/ERK1/2 and p38 MAPKs.
The cAMP and MAPK signaling pathways have long been known to interact. Initial observations that cAMP can inhibit proliferation in a variety of cell types can be explained by the ability of PKA to inhibit C-Raf activity (29). Subsequent work has uncovered additional mechanisms of cross-talk between cAMP signaling and the Ras/MAPK pathway by which ERK1/2 activity and cell proliferation are stimulated in response to cAMP. The latter effects appear to be cell type-specific and may also involve modulation of Raf isoform activity but can also be mediated by the interaction of cAMP signaling at multiple levels in MAPK pathways (29). These include the switching of G-protein-coupled receptors from G s -mediated stimulation of adenyl cyclase to G i -coupled activation of ERK1/2 (30). Thus the ability of PKA to phosphorylate DUSP9/MKP-4 would represent an additional level of positive interaction between cAMP and MAPK signaling pathways in DUSP9/MKP-4-expressing tissues such as placenta, kidney, and testis.
Although our work identifies DUSP9/MKP-4 as the first member of the dual-specificity MKPs for which substrate binding may be regulated in response to PKA signaling, this mode of FIGURE 6. Endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in both MEFs and murine placental tissue. A, immortalized MEFs were either left untreated or exposed to forskolin (10 M) either in the absence or presence of the phosphatase inhibitor okadaic acid (1 M). Cells were then lysed, and endogenous DUSP9/MKP-4 was immunoprecipitated (IP) using a polyclonal antibody (#302) against murine DUSP9/MKP-4 followed by SDS-PAGE and Western blotting using a polyclonal antibody (#657) raised against human DUSP9/MKP-4 and an antibody raised against a murine-specific Ser-58 phosphopeptide (upper two panels). Input lysates were also analyzed by SDS-PAGE and Western blotting using antibodies against phospho-CREB/ATF1, or as a loading control, tubulin (lower two panels). B, Ser-58 phosphorylation is enhanced by expression of a constitutively active mutant of PKA. Immortalized MEFs were transfected with either empty vector (Vec) or an expression vector encoding a constitutively active PKA C␣ subunit. Cells were then lysed, and endogenous DUSP9/MKP-4 was immunoprecipitated using a polyclonal antibody (#302) against murine DUSP9/MKP-4 followed by SDS-PAGE and Western blotting using a polyclonal antibody (#657) raised against human DUSP9/MKP-4 and an antibody raised against a murine-specific Ser-58 phosphopeptide (upper two panels). Input lysates were also analyzed by SDS-PAGE and Western blotting using antibodies against phospo-CREB/ATF1, PKA C␣, or as a loading control, tubulin (lower three panels). C, immortalized MEFs were either left untreated or exposed to forskolin (10 M). Cells were then lysed, and endogenous DUSP9/MKP-4 was immunoprecipitated using a polyclonal antibody (#302) against murine DUSP9/MKP-4. Immunoprecipitates were then incubated either in the absence (Ϫ) or presence (ϩ) of -phosphatase (PPase) before analysis by SDS-PAGE and Western blotting using a polyclonal antibody (#657) raised against human DUSP9/MKP-4 and an antibody raised against the murine Ser-58 phosphopeptide (upper two panels). Input lysates were also analyzed by SDS-PAGE and Western blotting using antibodies against phospo-CREB/ATF1 and, as a loading control, tubulin (lower two panels). D, endogenous murine DUSP9/MKP-4 was immunoprecipitated from placental lysates using a polyclonal antibody (Ab, #302) against murine DUSP9/MKP-4 with appropriate controls (no antibody and antibody but no lysate), and the samples were then divided into two aliquots and incubated either in the absence (Ϫ) or presence (ϩ) of -phosphatase (PPase). Proteins were then analyzed by SDS-PAGE and Western blotting using a polyclonal antibody (#657) raised against recombinant human DUSP9/MKP-4 and an antibody raised against the murine Ser-58 phosphopeptide as indicated.
regulation has been described in a small subset of PTPs, which selectively target ERK1/2 and p38␣ in neuronal and hematopoietic tissues (26,27). To date, the physiological significance of this modification is unclear. The neurotransmitter dopamine leads to the phosphorylation of striatal-enriched phosphatase via activation of D1 receptors and PKA in brain striatal tissue slices (28), and transient phosphorylation of the KIM within He-PTP was observed after T-cell antigen receptor ligation (31). Presumably KIM phosphorylation will affect the ability of these enzymes to inhibit the ERK and p38 MAP kinases, and our results suggest that this mechanism of pathway cross-talk is a conserved, but not universal, feature of protein phosphatases, which are dedicated to the regulation of MAPK signaling pathways in mammalian cells.
Unlike many dual-specificity MKPs, which are mitogen-or stress-inducible genes, DUSP9/MKP-4 mRNA and protein are constitutively expressed in murine fibroblasts. Furthermore, we show that Ser-58 is rapidly modified in the presence of forskolin. Post-translational modification of the KIM would allow the activity of DUSP9/MKP-4 toward its MAPK substrates to be regulated very rapidly without the need for new mRNA and protein synthesis. Furthermore, our observation that PKA-inducible phosphorylation of endogenous DUSP9/MKP-4 on Ser-58 is significantly enhanced in the presence of okadaic acid suggests that this modification might be reversed by a type 2 serine/threonine phosphatase such as PP2A, thus allowing dynamic regulation of DUSP9/MKP-4 activity.
Finally, we have demonstrated that endogenous DUSP9/ MKP-4 is constitutively phosphorylated in murine placental tissue where this phosphatase plays an essential role in the regulation of MAPK signaling during early development (8). This suggests that the regulation of DUSP9/MKP-4 substrate binding by KIM phosphorylation represents a physiologically relevant mechanism of regulation. Our phospho-DUSP9/MKP-4specific antiserum should allow a more extensive analysis of the tissue and cell distribution of phospho-DUSP9/MKP-4 both during development and under physiological conditions where cAMP signaling many influence cell and tissue fate.