Cooperation of ERK and SCFSkp2 for MKP-1 Destruction Provides a Positive Feedback Regulation of Proliferating Signaling

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339–342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser296/Ser323 domain, which was not affected by substituting Ala for Ser at Ser359/Ser364. The DEF and Ser296/Ser323 sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser359/Ser364 sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser296 MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser296 MKP-1 bound to SCFSkp2 ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCFSkp2 cooperation, thereby sustaining ERK activation. The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339–342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser296/Ser323 domain, which was not affected by substituting Ala for Ser at Ser359/Ser364. The DEF and Ser296/Ser323 sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser359/Ser364 sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser296 MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser296 MKP-1 bound to SCFSkp2 ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCFSkp2 cooperation, thereby sustaining ERK activation. Members of the family of mitogen-activated protein kinases (MAPKs), 2The abbreviations used are: MAPKsmitogen-activated protein kinasesERK1/2extracellular signal-regulated kinase-1/2JNKsc-Jun N-terminal kinasesMKKsmitogen-activated protein kinase kinasesMKPs/DUSPsdual-specificity mitogen-activated protein kinase phosphatasesKIMkinase interaction motifDEFdocking site forERKFXFPIEGimmediate-early geneSCFSkp2Skp1/Cul1/F-box protein Skp2E3ubiquitin-protein isopeptide ligaseWTwild-typeGSTglutathione S-transferasesiRNAsmall interfering RNAWCEwhole cell extractALLNN-acetyl-Leu-Leu-norleucinalHAhemagglutininE1ubiquitin-activating enzyme. including extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 kinases, are important intracellular signaling molecules regulated by phosphorylation in response to a wide variety of extracellular stimuli such as growth factors and environmental stresses (1Lewis T.S. Shapiro P.S. Ahn N.G. Adv. 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Each activated MAPK specifically targets a variety of proteins such as downstream kinases and transcription factors that regulate expression of particular genes and their activators/modulators and phosphatases whose feedback turns off the signaling. The strength and duration of MAPK activation, as well as the stimulus and cell type, distinctly affect cellular outcomes such as cell cycle progression and cell proliferation, differentiation, survival, and apoptosis (7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4236) Google Scholar, 8Roovers K. Assoian R.K. BioEssays. 2000; 22: 818-826Crossref PubMed Scopus (424) Google Scholar). Activation and inactivation of MAPKs must therefore be tightly controlled with high specificity and efficiency to achieve appropriate cellular responses. mitogen-activated protein kinases extracellular signal-regulated kinase-1/2 c-Jun N-terminal kinases mitogen-activated protein kinase kinases dual-specificity mitogen-activated protein kinase phosphatases kinase interaction motif docking site for FXFP immediate-early gene Skp1/Cul1/F-box protein Skp2 ubiquitin-protein isopeptide ligase wild-type glutathione S-transferase small interfering RNA whole cell extract N-acetyl-Leu-Leu-norleucinal hemagglutinin ubiquitin-activating enzyme. The identification of specific docking domains in non-catalytic regions of MAPKs and their interacting proteins provides a principal mechanism for controlling signaling specificity and efficiency (9Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 10Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (280) Google Scholar). Most MAPK-interacting proteins have a conserved domain called the kinase interaction motif (KIM) or D/δ kinase-docking domain containing a cluster of basic residues followed by an LXL site or surrounded by hydrophobic amino acids (9Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 10Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (280) Google Scholar). The KIM domain exhibits high affinity interactions with an acid-rich region termed the common docking domain in the C termini of all three MAPKs (11Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (681) Google Scholar). Differences in the amino acid sequences of these common docking and KIM domains in the MAPKs and their substrates further fine-tune docking specificities (9Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 10Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (280) Google Scholar). A second MAPK docking site called the docking site for ERK, FXFP (DEF) motif was originally found in the LIN-1/AOP/Elk-1/SAP-1 subfamily of ETS transcription activators (12Jacobs D. Beitel G.J. Clark S.G. Horvitz H.R. Kornfeld K. Genetics. 1998; 149: 1809-1822Crossref PubMed Google Scholar, 13Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (441) Google Scholar). Upon activation, phospho-ERK2 exposes a hydrophobic pocket for DEF motif interactions (14Lee T. Hoofnagle A.N. Kabuyama Y. Stroud J. Min X. Goldsmith E.J. Chen L. Resing K.A. Ahn N.G. Mol. Cell. 2004; 14: 43-55Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). The DEF motifs and KIM domains can function independently or additively in directing distinct MAPKs to phosphorylate transcription factors at a specific Ser or Thr residue followed by Pro ((S/T)P) (15Fantz D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Galanis A. Yang S.H. Sharrocks A.D. J. Biol. Chem. 2001; 276: 965-973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (761) Google Scholar, 18Murphy L.O. MacKeigan J.P. Blenis J. Mol. Cell. Biol. 2004; 24: 144-153Crossref PubMed Scopus (269) Google Scholar, 19Vinciguerra M. Vivacqua A. Fasanella G. Gallo A. Cuozzo C. Morano A. Maggiolini M. Musti A.M. J. Biol. Chem. 2004; 279: 9634-9641Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). All mammalian MKPs identified thus far contain an N-terminal KIM domain and a phosphatase active-site HCX5R motif (5Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (718) Google Scholar, 6Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (710) Google Scholar, 10Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (280) Google Scholar). Among them, MKP-1/CL100/DUSP1 and MKP-3/Pyst1/DUSP6 are prototypes whose functional associations with MAPK signals have been well characterized. MKP-1 is a nuclear enzyme encoded by an immediate-early gene (IEG), and its transcription is rapidly induced in response to many of the stimuli that activate MAPKs (20Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (571) Google Scholar, 21Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1027) Google Scholar). By contrast, MKP-3 is localized mainly in the cytosol, and its expression is not inducible by either mitogens or cellular stresses (22Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (373) Google Scholar, 23Muda M. Boschert U. Dickinson R. Martinou J.C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), but can be stimulated by agents promoting neuronal differentiation (23Muda M. Boschert U. Dickinson R. Martinou J.C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). MKP-3 phosphatase activity is allosterically elevated upon selective binding to ERK2 in vitro (24Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (437) Google Scholar), which may explain the highly specific inactivation of ERK by MKP-3 in vivo (22Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (373) Google Scholar). Likewise, catalytic activation of MKP-1 is mediated via physical interactions with ERK2, JNK1, and p38α in vitro (25Slack D.N. Seternes O.M. Gabrielsen M. Keyse S.M. J. Biol. Chem. 2001; 276: 16491-16500Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar); this also correlates with the substrate specificity of MKP-1 in vivo (21Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1027) Google Scholar, 25Slack D.N. Seternes O.M. Gabrielsen M. Keyse S.M. J. Biol. Chem. 2001; 276: 16491-16500Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 26Hutter D. Chen P. Li J. Barnes J. Liu Y. Mol. Cell. Biochem. 2002; 233: 107-117Crossref PubMed Scopus (13) Google Scholar). Thus, distinct MAPKs can elicit a negative feedback loop to control signaling strength and duration. MKP-1 is rapidly degraded soon after its induction by mitogens in rodent fibroblasts (27Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 28Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (365) Google Scholar). Similarly, in oocytes, Xenopus CL100 (an MKP-1 homolog) is a labile protein, and sorbitol further enhances its destruction (29Sohaskey M.L. Ferrell Jr., J.E. Mol. Biol. Cell. 2002; 13: 454-468Crossref PubMed Scopus (38) Google Scholar). MKP-1 is accumulated upon proteasome inhibition in rodent fibroblasts (28Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (365) Google Scholar). Following removal of the proteasome inhibitory stress, MKP-1 is rapidly degraded, and forced expression of the ERK signal leads to MKP-1 phosphorylation and a decrease in degradation (28Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (365) Google Scholar). In sorbitol-treated oocytes, forced activation of the ERK2 signal reduces the degradation rate of exogenous Xenopus CL100 via phosphorylation (29Sohaskey M.L. Ferrell Jr., J.E. Mol. Biol. Cell. 2002; 13: 454-468Crossref PubMed Scopus (38) Google Scholar). On the other hand, upon Pb(II) exposure or overexpression of constitutively active MKK1/2 in several mammalian cell lines, ERK activation triggers MKP-1 degradation via the ubiquitin-proteasome pathway, indicating a positive feedback control (30Lin Y.-W. Chuang S.M. Yang J.-L. J. Biol. Chem. 2003; 278: 21534-21541Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The foregoing suggests that ERK may exhibit dual roles in controlling MKP-1 stability in distinct cellular environments. This has driven us to explore the molecular basis for ERK-directed MKP-1 ubiquitination and to establish whether such an event also occurs during serum stimulation of quiescent cells. Here, we show that the MKP-1 DEF motif is necessary for active ERK2 binding to initiate site-specific phosphorylation, serving as an essential recognition domain for the Skp1/Cul1/F-box protein Skp2 (SCFSkp2) ubiquitin-protein isopeptide ligase (E3), a vital E3 enzyme for S phase entry and progression (31Reed S.I. Nat. Rev. Mol. Cell. Biol. 2003; 4: 855-864Crossref PubMed Scopus (242) Google Scholar, 32Cardozo T. Pagano M. Nat. Rev. Mol. Cell. Biol. 2004; 5: 739-751Crossref PubMed Scopus (884) Google Scholar), leading to MKP-1 polyubiquitination and subsequent destruction via the 26 S proteasome. These results suggest that active ERK docking to the DEF motif and SCFSkp2 association are rate-limiting for the ubiquitin-mediated MKP-1 proteolysis that would sustain ERK signaling to facilitate G1 cells entering the cell cycle. Plasmid Construction and Mutagenesis—The plasmid pSG5-MKP-1-Myc was kindly provided by Dr. N. K. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Forward and reverse primers containing EcoRV and HindIII restriction sites were used to amplify MKP-1 cDNA from pSG5-MKP-1-Myc by PCR. This fragment was subcloned in pCMV-Tag2B (Stratagene, La Jolla, CA) to generate a FLAG-tagged wild-type (WT) MKP-1 expression vector. Similarly, the plasmids expressing glutathione S-transferase (GST)-tagged WT (GST-MKP-1) and N-terminally truncated (amino acids 1–59; GST-ΔN-MKP-1) MKP-1 cDNAs were constructed by subcloning a PCR-amplified BamHI-EcoRI fragment from pSG5-MKP-1-Myc in pGEX-4T-1 (Amersham Biosciences). Various MKP-1 and ΔN-MKP-1 substitution mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). All PCR-amplified fragments or mutagenized constructs were verified by DNA sequencing using the BigDye terminator cycle reaction kit and an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Production of Recombinant Proteins—The vectors encoding GST-tagged WT MKP-1 and mutants were expressed in Escherichia coli BL21. The GST fusion proteins were purified via binding to glutathione-Sepharose 4B (Amersham Biosciences), followed by elution in buffer containing 10 mm HEPES (pH 7.5), 15 mm glutathione, 150 mm NaCl, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. In Vitro Binding and Phosphorylation—Equal amounts (0.2 μg) of unactive and active forms of GST-ERK2 fusion proteins (Upstate Biotechnology, Inc., Lake Placid, NY) were reacted overnight with glutathione-Sepharose beads at 4 °C. Various GST-ΔN-MKP-1 fusion proteins (2 μg) were treated with thrombin (0.04 units) to release GST fragments and allowed to interact with bead-attached GST-ERK2 fusion proteins at4 °C for 3 h in binding buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride). Similarly, unactive and active forms of ERK2 were reacted with bead-attached GST-ΔN-MKP-1 mutants. Following three washes with 50 mm HEPES (pH 7.5), 150 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, and 10% glycerol, the associated proteins were released by boiling the beads for 5 min and resolved by SDS-PAGE, followed by Western blotting. Bead-attached GST-tagged active ERK2 was also reacted with various ΔN-MKP-1 mutants and 20 mm ATP in the presence or absence of 1 μCi of [γ-32P]ATP. The phosphorylation reaction was performed at 30 °C for 30 min in a total volume of 30 μl of kinase reaction buffer (20 mm HEPES (pH 7.6), 20 mm MgCl2, 2 mm dithiothreitol, 0.1 mm Na3VO4,and 1 mm NaF). Next, the reaction mixture was boiled, and the phosphorylated protein was resolved by SDS-PAGE, followed by either autoradiography or Western blotting using anti-phospho-Ser296 MKP-1 antibody. The amount of GST-tagged active ERK2 in the reaction mixture was determined by Western blotting. Cells, Vectors, Small Interfering RNA (siRNA), and Transfection—The human embryonic kidney cell line H293 was cultured in Dulbecco's minimal essential medium containing 10% fetal calf serum as described previously (30Lin Y.-W. Chuang S.M. Yang J.-L. J. Biol. Chem. 2003; 278: 21534-21541Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Vectors expressing MKK1-CA (a constitutively active form of MKK1, ΔN3/S218E/S222D) and MKK2-CA (a constitutively active form of MKK2, ΔN4/S222E/S226D) were gifts from Dr. N. G. Ahn (University of Colorado, Boulder, CO). pcDNA-FLAG-Skp2 was kindly provided by Dr. M. Pagano (New York University School of Medicine, New York, NY). The sense strand sequences of siRNA duplexes used for Skp2 and lamin (as a control) were 5′-AAUCUAAGCCUGGAAGGCCUGdTdT-3′ and 5′-CUGGACUUCCAGAAGAACAdTdT-3′ (Dharmacon, Inc., Lafayette, CO). The expression vectors were transfected into H293 cells using Lipofectamine (Invitrogen). The siRNA duplexes (200 nm) were transferred into cells immediately before and 24 h after vector transfection using Oligofectamine (Invitrogen). The transfected vectors were allowed 2 days of expression in the presence or absence of siRNAs, and the whole cell extract (WCE) was subjected to immunoprecipitation and/or Western blotting. To reveal ubiquitinated proteins, 10 μm N-acetyl-Leu-Leu-norleucinal (ALLN; Calbiochem-Novabiochem), an inhibitor of the 26 S proteasome, was supplied for the final 3 h during transfection. Serum Stimulation—Cells were cultured in medium containing 0.1% serum for 24 or 48 h and then treated with 10–20% serum for 15 min to 3 h. To prevent de novo protein synthesis, 10 μg/ml cycloheximide (Sigma) was added to cells during serum stimulation. To reveal phosphorylated and ubiquitinated MKP-1, 10 μm ALLN was supplied for the final 10 min or 1 h during serum stimulation. To determine the effect of ERK activation on MKP-1 degradation, U0126 (Calbiochem-Novabiochem), a specific MKK1/2 inhibitor, was added 1 h before serum stimulation. Protein Stability Analysis—The half-lives of endogenous and exogenous MKP-1 proteins were determined using pulse-chase experiments. Serum-starved cells were cultured in methionine-free medium for 30 min and then pulse-labeled with 5 μCi/ml [35S]methionine for 1 h in the presence or absence of U0126 (5 μm). Immediately after labeling, the cells were washed with a chasing medium containing 30 μg/ml methionine prior to stimulation with 10% serum in the chasing medium for 0–3 h. Cells were then lysed, and MKP-1 proteins in the WCE were subjected to immunoprecipitation and subsequent electrophoresis. Isotope-labeled MKP-1 was visualized by autoradiography. Western Blot Analysis—WCE collection was performed as described previously (33Lin Y.-W. Chuang S.M. Yang J.-L. Carcinogenesis. 2003; 24: 53-61Crossref PubMed Scopus (44) Google Scholar). The BCA protein assay kit (Pierce) was employed to determine protein concentrations using bovine serum albumin as a standard. Equal amounts of proteins in the WCE were fractionated by SDS-PAGE. The protein bands were then transferred electrophoretically to polyvinylidene difluoride membranes and probed with primary antibody, followed by a horseradish peroxidase-conjugated secondary antibody. The anti-phospho-Thr202/Tyr204 ERK1/2 polyclonal antibody was from Cell Signaling Technology, Inc. (catalog no. 9101; Beverly, MA). The polyclonal antibodies against ERK2 (C-14), MKP-1 (V-15), ubiquitin (P4D1), Skp2 (H-435), hemagglutinin (HA; F-7), Skp1 (H-163), Cul1 (H-213), Cks1 (FL-79), p27 (C-19), and α-tubulin (TU-02) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG monoclonal antibody M2 was from Sigma. The anti-phospho-Ser296 MKP-1 antibody was generated from rabbits injected with phosphopeptide KQRRSIIpSPNFSFMG conjugated to keyhole limpet hemocyanin. Antibodies were stripped from polyvinylidene difluoride membranes using a solution containing 2% SDS, 62.5 mm Tris-HCl (pH 6.8), and 0.7% (w/w) β-mercaptoethanol at 50 °C for 15 min before reprobing with another primary antibody. Relative protein blot intensities were determined using a computing densitometer equipped with the ImageQuant analysis program (Amersham Biosciences). Immunoprecipitation—Cells were washed twice with ice-cold phosphate-buffered saline and harvested at 4 °C in immunoprecipitation lysis buffer as described previously (30Lin Y.-W. Chuang S.M. Yang J.-L. J. Biol. Chem. 2003; 278: 21534-21541Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Equal amounts of proteins were immunoprecipitated using antibodies against ubiquitin, FLAG, Skp2, ERK2, MKP-1, and phospho-Ser296 MKP-1 and collected with protein G-Sepharose beads at 4 °C for 16 h. The immunoprecipitate was then washed three times with cold lysis buffer and subjected to Western blotting. In Vitro Ubiquitination—The SCFSkp2-Cks1 E3 complex was prepared as follows. The FLAG-Skp2 vector was transfected into H293 cells and allowed expression for 2 days, and the WCE was immunoprecipitated using anti-FLAG antibody. The FLAG-Skp2 immunocomplex was then mixed with equal amounts of the Cks1 immunocomplex prepared separately from H293 cells. Following the phosphorylation reaction, WT MKP-1 and mutants (5 μg) were subjected to in vitro ubiquitination assay using ubiquitin-activating enzyme (E1; 0.5 μg; AFFINITI Research Products Ltd., Plymouth Meeting, PA), UbcH3 conjugating enzyme (1 μg; AFFINITI), SCFSkp2-Cks1 immunocomplex, and ubiquitin (50 μg; AFFINITI) in a reaction buffer containing 40 mm Tris-HCl (pH 7.6), 5 mm MgCl2, 1 mm dithiothreitol, 10% (v/v) glycerol, 10 mm phosphocreatine, 100 μg/ml creatine kinase, 0.5 mm ATP, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 2 μg/ml pepstatin. The ubiquitination reaction mixture was incubated at 30 °C for 90 min, and the ubiquitinated proteins were subjected to SDS-PAGE and probed with anti-MKP-1 antibody. The MKP-1 DEF Motif Is Essential for Active ERK Binding and Phosphorylation, Leading to Ubiquitination and Proteolysis—ERK can induce mkp-1 gene expression at the transcriptional level (34Brondello J.M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar) and enhance phosphatase activity via docking to the N-terminal KIM domain of MKP-1 (25Slack D.N. Seternes O.M. Gabrielsen M. Keyse S.M. J. Biol. Chem. 2001; 276: 16491-16500Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), suggesting a negative feedback loop to inhibit the kinase activity. By contrast, ERK can also trigger MKP-1 proteolysis, reflecting a positive feedback loop to sustain the kinase activity (30Lin Y.-W. Chuang S.M. Yang J.-L. J. Biol. Chem. 2003; 278: 21534-21541Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Given that MKP-1 contains one putative DEF motif (FNFP, residues 339–342) in the C terminus for ERK docking (Fig. 1), we explored whether this motif is essential for ERK-triggered proteolysis. To avoid the possibility of the KIM domain competing with the DEF motif for ERK docking, we constructed the GST-tagged ΔN-MKP-1 mutant, in which the N-terminal 1–59 residues were truncated, and its derivative ΔN-MKP-1(ANAP), in which the two Phe residues in the DEF motif (FNFP) were mutated to Ala. The ΔN-MKP-1 and ΔN-MKP-1(ANAP) fusion proteins were expressed in and purified from bacteria, followed by in vitro incubation with either unactive or active ERK2. Fig. 2A shows that ΔN-MKP-1 formed a complex with active ERK2, but ΔN-MKP-1(ANAP) did not; conversely, neither ΔN-MKP-1 nor ΔN-MKP-1(ANAP) associated with unactive ERK2. Furthermore, active ERK2 could phosphorylate ΔN-MKP-1 but not ΔN-MKP-1(ANAP) (Fig. 2B). These in vitro results suggest that the MKP-1 DEF motif is essential for active ERK2 docking and phosphorylation.FIGURE 2Mutated DEF motif in MKP-1 impairs active ERK2 docking and phosphorylation in vitro. A, glutathione-Sepharose bead-attached GST-ERK2 unactive or active) was mixed with ΔN-MKP-1 (+) or ΔN-MKP-1(ANAP) (upper panels). Alternatively, bead-attached GST-ΔN-MKP-1 or GST-ΔN-MKP-1(ANAP) was mixed with unactive or active ERK2 (lower panels). The mixtures were kept at 4 °C for 3 h, and protein binding was resolved by GST pull-down and subsequent Western blotting using the indicated antibodies. Equal amounts of proteins used for the pull-down assay were also analyzed by Western blotting (indicated as loading). B, bead-attached GST-tagged active ERK2 was reacted with ΔN-MKP-1 (+) or ΔN-MKP-1(ANAP) in the presence of 20 μm ATP and 1 μCi of [γ-32P]ATP for 30 min at 30 °C. The samples were then analyzed by electrophoresis, followed by autoradiography (indicated as Kinase assay). A portion of the bead-attached GST-tagged active ERK2 was analyzed by Western blotting to determine equal input. Kinase assay results were calculated by averaging four independent experiments. The Western blots shown are representative of four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To explore whether the DEF motif plays a crucial role in MKP-1 ubiquitination stimulated by ERK in vivo, we cotransfected FLAG-MKP-1 or FLAG-ΔN-MKP-1 with HA-MKK1-CA into H293 cells, allowed expression for 2 days, and treated one set of cells with an inhibitor of the 26 S proteasome (10 μm ALLN) for the final 3 h to reveal protein ubiquitination. The second set of cells was not treated with ALLN to determine protein degradation. As with FLAG-MKP-1, FLAG-ΔN-MKP-1 was polyubiquitinated and degraded during forced expression of MKK1-CA in H293 cells (Fig. 3, A and B, lanes 7–10), suggesting that the MKP-1 KIM domain is dispensable for ERK-directed ubiquitination in vivo. We next constructed a FLAG-tagged full-length MKP-1(ANAP) mutant to evaluate the physiological role of the DEF motif in ERK activation. Forced expression of MKK1-CA in H293 cells caused polyubiquitination in FLAG-MKP-1 but not in the ANAP mutant (Fig. 3A, compare lanes 5 and 6). MKK1-ERK signaling also markedly enhanced a decrease in the FLAG-MKP-1 protein level (Fig. 3B, first row, compare lanes 3 and 5), which was notably higher than that in the ANAP mutant level (Fig. 3, B, compare lanes 4 and 6; and C, left panel). These results suggest that the DEF motif is involved in ERK-directed MKP-1 degradation in vivo via the ubiquitin-proteasome pathway. Additionally, the relative phospho-ERK levels stimulated by MKK1-CA were lower in cells coexpressing either FLAG-MKP-1 or the ANAP mutant compared with those coexpressing pcDNA3 (Fig. 3, B, second row, compare lane 2 with lanes 5 and 6; and C, right panel). Identification of ERK-directed Phosphoacceptor Sites in MKP-1 for Proteolysis via the Ubiquitin-Proteasome Pathway—Previous studies have indicated that binding of ERK to the DEF motifs of several substrates, including the Elk-1, SAP-1, and c-Fos transcription factors and the IEX-1 early response gene product, results in phosphorylation of specific (S/T) P sites positioned at DEF motif N termini and subsequent functional activation (15Fantz D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Galanis A. Yang S.H. Sharrocks A.D. J. Biol. Chem. 2001; 276: 965-973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (761) Google Scholar, 35Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (92) Google Scholar). Protein sequence analysis showed that there are four SP sites located at the N terminus (Ser296 and Ser323) and C te