A Bipartite Mechanism for ERK2 Recognition by Its Cognate Regulators and Substrates
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m303909200
ISSN1083-351X
AutoresJialin Zhang, Bo Zhou, Chao-Feng Zheng, Zhong‐Yin Zhang,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates. We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins. We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2. Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation. MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site. Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation. In addition to the shared residues, there are also residues that are unique to each target recognition. There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive. Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process. In this model, one part of the ERK2-binding proteins (e.g. the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g. the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules. Mitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates. We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins. We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2. Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation. MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site. Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation. In addition to the shared residues, there are also residues that are unique to each target recognition. There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive. Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process. In this model, one part of the ERK2-binding proteins (e.g. the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g. the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules. Mitogen-activated protein (MAP) 1The abbreviations used are: MAP kinase, mitogen-activated protein kinase; CD site, common docking site; ERK, extracellular signal-regulated protein kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal protein kinase; KIM, kinase interaction motif; MBP, myelin basic protein; MEK, MAP kinase/ERK kinase; MKP, MAP kinase phosphatase; pNPP, p-nitrophenyl phosphate; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: MAP kinase, mitogen-activated protein kinase; CD site, common docking site; ERK, extracellular signal-regulated protein kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal protein kinase; KIM, kinase interaction motif; MBP, myelin basic protein; MEK, MAP kinase/ERK kinase; MKP, MAP kinase phosphatase; pNPP, p-nitrophenyl phosphate; MOPS, 4-morpholinepropanesulfonic acid. kinase pathways are indispensable intracellular cascades that couple signals from the cell surface to the nucleus (1Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2230) Google Scholar, 2Chen Z. Gibson T.B. Robinson F. Silvestro L. Pearson G. Xu B. Wright A. Vanderbilt C. Cobb M.H. Chem. Rev. 2001; 101: 2449-2476Crossref PubMed Scopus (771) Google Scholar). The three best characterized MAP kinase cascades are the extracellular signal-regulated protein kinase (ERK) pathway that responds to stimuli that induce cell proliferation and differentiation, the c-Jun N-terminal protein kinase (JNK) pathway, and the p38 kinase pathway, both of which are activated in response to environmental stresses. The MAP kinases are the major convergence points in these signaling pathways. Each MAP kinase phosphorylates a distinct spectrum of substrates, which include key regulatory enzymes, cytoskeletal proteins, regulators of apoptosis, nuclear receptors, and many transcription factors. Such a broad array of substrates is consistent with the observation that MAP kinases control many critical cell functions. Because of the critical importance of MAP kinase in cellular signaling, the activity of the MAP kinase is tightly regulated. The activation of the MAP kinase activity requires the phosphorylation of the Thr and Tyr residues in the activation loop by the dual specificity MAP kinase/ERK kinases (MEKs) (3Ahn N.G. Seger R. Bratlien R.L. Diltz C.D. Tonks N.K. Krebs E.G. J. Biol. Chem. 1991; 266: 4220-4227Abstract Full Text PDF PubMed Google Scholar, 4Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (835) Google Scholar). MAP kinase deactivation occurs through the action of multiple protein phosphatases (5Zhou B. Wang Z.-X. Zhao Y. Brautigan D.L. Zhang Z.-Y. J. Biol. Chem. 2002; 277: 31818-31825Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Although the importance of MAP kinases in cellular signaling is well established, there is limited understanding of the molecular basis for specific MAP kinase recognition by its activators, inactivators, and substrates. Such knowledge is essential for comprehension of the ability of MAP kinases to integrate diverse biological stimuli and to transmit signals to the nucleus, in order to generate appropriate cellular responses. Recent studies (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (663) Google Scholar, 7Tanoue T. Maeda R. Adachi M. Nishida E. EMBO J. 2001; 20: 466-479Crossref PubMed Scopus (225) Google Scholar, 8Tanoue T. Yamamoto T. Nishida E. J. Biol. Chem. 2002; 277: 22942-22949Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) suggest that MAP kinases are capable of forming complexes with their cognate activating kinases, inactivating phosphatases and substrates. Our previous studies have focused on the interaction between ERK2, the founding member of the MAP kinase family, and MAP kinase phosphatase 3 (MKP3) (9Zhou B. Zhang Z.-Y. J. Biol. Chem. 1999; 274: 35526-35534Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Zhao Y. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 32382-32391Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The MKPs are dual specificity phosphatases capable of dephosphorylating both Tyr(P) and Thr(P) in the activation loop of MAP kinases (12Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (706) Google Scholar, 13Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (700) Google Scholar). MKP3 is highly specific in dephosphorylating and inactivating ERK2, with a k cat/K m for the double phosphorylated ERK2 that is 106-fold higher than those for the hydrolysis of p-nitrophenyl phosphate (pNPP) or the bisphosphorylated peptide derived from the activation loop of ERK2 (10Zhao Y. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 32382-32391Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The superiority of the double phosphorylated ERK2 to the ERK2-derived phosphopeptide most likely results from specific protein-protein interactions between ERK2 and MKP3 that are not available between the ERK2 peptide and MKP3. Interestingly, the phosphatase activity of the MKP3-catalyzed pNPP reaction can be dramatically increased in the presence of ERK2 (14Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (430) Google Scholar). Mechanistic and kinetic studies suggest that ERK2 binding to MKP3 elicit activation of MKP3 activity by facilitating the repositioning of active site residues and general acid loop closure in MKP3 (9Zhou B. Zhang Z.-Y. J. Biol. Chem. 1999; 274: 35526-35534Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Zhao Y. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 32382-32391Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Fjeld C.C. Rice A.E. Kim Y. Gee K.R. Denu J.M. J. Biol. Chem. 2000; 275: 6749-6757Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To identify structural features in MKP3 that are important for ERK2 binding and ERK2-induced activation, we carried out a systematic mutational and deletion analysis of MKP3 (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Because the activation of MKP3 by ERK2 is dose-dependent and saturable, we can determine both the dissociation constant between ERK2 and MKP3 and the extent of MKP3 activation from the concentration dependence of ERK2 on the MKP3-catalyzed pNPP reaction. Furthermore, we have developed a competitive assay to determine the binding affinity of fragments/domains of MKP3 that are important for ERK2 recognition. With these assays, we were able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP3 activation. Our results show that recognition and activation of MKP3 by ERK2 involve multiple regions of MKP3 (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In the current study, we have identified structural elements in ERK2 that are important for binding and activation of MKP3 using the activation- and competition-based assays with wild-type MKP3 and an ensemble of ERK2 mutants. In addition, we have unveiled structural features in ERK2 that mediate specific Elk1 recognition. Our results show that protein-protein interactions between ERK2 and its cognate regulators and substrates proceed with a bipartite recognition mechanism. DNA Constructs and Site-directed Mutagenesis—The coding sequence of ERK2 was subcloned into pET15b to yield the N-terminal His6-tagged ERK2. MKP3 with a C-terminal His6 tag in pET21a was described previously (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The cDNA for the constitutively active MEK1 (MEK1/G7B, i.e. MEK1/Δ44–51/S218D/M219D/N221D/S221D) in pRSETa was kindly provided by Dr. Natalie Ahn. Oligonucleotide primers CATATGACTGAGATCACCCAACC (NdeI site underlined) and GGATCCTCATGGCTTCTGGGGCCC (BamHI site underlined) were used to generate the N-terminal His6-tagged Elk1 (residues 307–428) by PCR using pGEX-2T/Elk1-(307–428) (a generous gift from Dr. Kun-Liang Guan) as a template. The PCR product was subcloned into pET14b at NdeI/BamHI. Mutant ERK2 and MKP3 were generated by PCRs according to the standard procedure of the QuickChange™ site-directed mutagenesis kit (Stratagene) using either pET15b-His6-ERK2 or pET21a-MKP3-His6 as a template. All mutants were verified by DNA sequencing. Peptide Synthesis, Protein Expression, and Purification—A synthetic peptide derived from Elk1 (residues 387–399, Ac-Arg-Arg-Pro387-Arg-Ser-Pro-Ala-Lys-Leu-Ser-Phe-Gln-Phe-Pro-Ser399-NH2), which contains an ERK2 phosphorylation site Ser-389 (16Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (578) Google Scholar) and an ERK2 docking site sequence FQFP (17Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (434) Google Scholar), was used in the competition-based assay. The Elk1 peptide was synthesized using standard protocol, purified by high pressure liquid chromatography, and characterized by matrix-assisted laser desorption ionization/time of flight mass spectrometry by Alpha Diagnostic International. The purity of the peptide was determined to be close to 100%. Wild-type and mutant N-terminal His6-tagged ERK2s, wild-type and mutant C-terminal His6-tagged MKP3s, N-terminal His6-tagged Elk1-(307–428), and N-terminal His6-tagged MEK1/G7B were expressed in Escherichia coli BL21/DE3 and purified using standard procedures of Ni2+-nitrilotriacetic acid-agarose (Qiagen) affinity purification as described previously (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Protein concentration was determined using the Bradford dye binding assay (Bio-Rad) diluted according to the manufacturer's recommendations with bovine serum albumin as standard. Determination of Dissociation Constants—The dissociation constants of ERK2 and its mutants for MKP3 were determined by the activation assay at 30 °C and pH 7.0, in 50 mm 3,3-dimethylglutarate buffer, containing 1 mm EDTA with an ionic strength of 0.15 m adjusted by addition of NaCl (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Briefly, the MKP3-catalyzed hydrolysis of the p-nitrophenyl phosphate (pNPP) reaction was initiated by the addition 0.2 μm MKP3 to a reaction mixture (200 μl) containing 40 mm pNPP (saturating concentration) and various concentrations of ERK2 or its mutants. The reaction was quenched after 20–40 min by addition 50 μl of 5 n NaOH. After quenching, 200 μl of the reaction mixture was transferred to a 96-well plate, and the amount of p-nitrophenol was determined from the absorbance at 405 nm using a Spectra MAX340 microplate spectrophotometer (Molecular Devices) with a molar extinction coefficient of 18,000 m–1 cm–1. The dissociation constant K d was calculated by fitting the absorbance at 405 nm versus ERK2 concentration data to Equation 1 using the nonlinear regression program Kaleidagraph, A=A0-(A0-A∞)(CM+CE+Kd)-(CM+CE+Kd)2-4×CM×CE2×CM(Eq. 1) where A is the absorbance at 405 nm of the sample in the presence of ERK2; A 0 is the absorbance at 405 nm in the absence of ERK2; A 0 is the absorbance at 405 nm when the concentration of ERK2 is infinite; C M is the MKP3 concentration which is fixed at 0.2 μm; C E is the ERK2 concentration during titration, and K d is the dissociation constant for ERK2 binding to MKP3. To determine the affinity of Elk1 or MEK1 for ERK2, the competitive binding assay was used (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this assay, the reaction was initiated by the addition of 0.1 μm MKP3 in a mixture (0.2 ml) containing 40 mm pNPP, 1.2 μm ERK2, and various concentrations of the Elk1 peptide, Elk1-(307–428), or MEK1 at 30 °C and pH 7.0, in 50 mm 3,3-dimethylglutarate buffer, containing 1 mm EDTA with an ionic strength of 0.15 m adjusted by addition of NaCl. The reaction was quenched by addition 50 μl of 5 n NaOH after 30 min. The data were fitted to Equation 2 by nonlinear regression analysis to obtain the dissociation constants of Elk1 and MEK1 for ERK2, AA0=A∞A0+1-A∞A0×1+KdMC0E1-2×KdM(KdL+C0L-C0E)-(KdL+C0L-C0E)2+4×C0E×KdL(Eq. 2) where Elk1 (or MEK1) and MKP3 are assumed to bind ERK2 competitively, and MKP3 concentration is much smaller than ERK2 concentration. A is the absorbance at 405 nm in the presence of Elk1 or MEK1. A 0 is the absorbance at 405 nm in the absence of Elk1 or MEK1. A 0 is the absorbance at 405 nm when the concentration of Elk1 or MEK1 is infinite. C0E is ERK2 concentration which is fixed at 1.2 μm. C0L is Elk1 or MEK1 concentration during titration. KdM is the dissociation constant for MKP3 binding to ERK2 with a value of 0.17 μm (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and KdL is the dissociation constant of Elk1 (or MEK1) for ERK2. GST Pull-down and Western Blot Analysis—The binding of MEK1 and MKP3 to ERK2 was examined by GST pull-down and Western blot analyses. GST-ERK2 (10 μg) or GST-MKP3 (10 μg) in 0.5 ml of phosphate-buffered saline (140 mm, 2.7 mm KCl, 10 mm Na2HPO4, 1.0 mm KH2PO4, 2 mm dithiothreitol, pH 7.4) was immobilized on 20 μl of glutathione-Sepharose 4B beads (Amersham Biosciences), respectively, with gentle agitation at 4 °C for 2 h. In one experiment, different amounts of MKP3 and MEK1 (0–10 μg) were mixed in 200 μl of phosphate-buffered saline containing 0.5% Triton X-100 and incubated with 20 μl of GST-ERK2 bound beads. In another experiment, different amounts of ERK2 and MEK1 (0–20 μg) were mixed in 200 μl of phosphate-buffered saline containing 0.5% Triton X-100 and incubated with 20 μl of GST-MKP3 bound beads. After incubation with gentle agitation at 4 °C for 2 h, the beads were washed with phosphate-buffered saline and then boiled in 25 μl of 2× SDS sample buffer for 5 min to release the proteins from the beads. The sample was microcentrifuged at 10,000 rpm for 2 min, and 10 μl of the supernatant was loaded on 10% SDS-polyacrylamide gel. When the electrophoresis was complete, the proteins on the gel were transferred to nitrocellulose membrane using a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-Rad) at 150 mA and at room temperature for 1 h. The membrane was blocked in 5% milk in TBS-T (20 mm Tris, 150 mm NaCl, 0.1% Tween 20, pH 7.6) for1hat room temperature and then incubated with mouse anti-His6 monoclonal antibody (sc-8036, Santa Cruz Biotechnology) overnight at 4 °C. After washing with TBS-T, the membrane was incubated with goat anti-mouse antibody conjugated with horseradish peroxidase (sc-2005, Santa Cruz Biotechnology) for 1 h at room temperature. The immunocomplexes were detected by chemiluminescence upon incubation with ECL reagents (Amersham Biosciences). The membrane was immediately expose to film for 1–5 min to visualize the His6-tagged proteins. ERK2 Kinase Assay—The kinase activity of wild-type and mutant ERK2s was monitored by a radioisotope assay in which the rate of incorporation of 32P from [γ-32P]ATP into a substrate was directly measured. Reactions were carried out in 50 μl of the kinase buffer (20 mm MOPS, 50 mm KCl, 10 mm MgCl2, 0.1 mm EDTA, and 1 mm dithiothreitol, pH 7.4), containing ERK2 at a final concentration of 0.5 μm and varied concentrations of the protein substrates, myelin basic protein (MBP, Sigma), or Elk1. Reactions were initiated by the addition 1 mm [γ-32P]ATP (PerkinElmer Life Sciences; NEG002A) (100 cpm/pmol) and allowed to proceed at 30 °C for 40 min for MBP and 25 min for Elk1. The reactions were terminated by the addition of 10 μl of 9.0% (final 1.5%) phosphoric acid. The 32P-labeled product was separated from [γ-32P]ATP using P81 phosphocellulose paper (Whatman, 2.1 cm), which binds to the protein or peptide product but not ATP and its metabolites. Detailed procedures are as follows: 30 μl of the quenched reaction mixture was spotted onto the 2.1-cm sized P81 paper strips. After washing the strips with 0.5% phosphoric acid 4 times (2 min each, 10–15 ml of 0.5% phosphoric acid per paper strip) with gentle agitation followed by 1 wash with water and 1 wash with acetone, the P81 papers were dried with a hair dryer and inserted into a 5-ml scintillation tube. Four ml of scintillation liquid was added, and the incorporation of 32P into the product was counted by liquid scintillation spectrometry. Controls were carried out in which ERK2 and the substrate were replaced by buffer. Each sample was measured in triplicate. On the basis of ERK2 concentration dependence of MKP3 activation, we developed biochemical assays that provide quantitative assessment of the importance of structural features in MKP3 for ERK2 recognition, both in terms of ERK2 binding affinity and propensity to be activated by ERK2 (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). By using these assays, we discovered that binding and activation of MKP3 by ERK2 involves multiple regions of MKP3. Obviously, structural elements in ERK2 that are important for MKP3 binding and activation can be identified by the same approach using wild-type MKP3 and various ERK2 mutants designed based on available structural and biochemical data. In the current study we provide evidence that MKP3 binding and ERK2-induced MKP3 activation require two distinct surface areas of ERK2. Our data further suggest that recognition of MKP3, MEK1, and Elk1 by ERK2 requires a conserved bipartite protein-protein recognition mechanism. Definition of ERK2 Common Docking Site for MKP3—Many ERK2-interacting proteins such as ERK2 activators (e.g. MEK1/2), inactivators (e.g. MKP3 and HePTP), and substrates (e.g. RSK1 and Elk1) contain a kinase interaction motif (KIM) characterized by a cluster of 2–3 positively charged Arg or Lys residues that are important for ERK2 binding (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (663) Google Scholar, 11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Pulido R. Zuniga A. Ullrich A. EMBO J. 1998; 17: 7337-7350Crossref PubMed Scopus (269) Google Scholar, 19Karim F.D. Rubin G.M. Mol. Cell. 1999; 3: 741-750Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 20Saxena M. Williams S. Brockdorff J. Gilman J. Mustelin T. J. Biol. Chem. 1999; 274: 11693-11700Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 21Gavin A.-C. Nebreda A.R. Curr. Biol. 1999; 9: 281-284Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 22Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 23Nichols A. Camps M. Gillieron C. Chabert C. Brunet A. Wilsbacher J. Cobb M. Pouyssegur J. Shaw J.P. Arkinstall S. J. Biol. Chem. 2000; 275: 24613-24621Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Deletion of the KIM sequence (residues 61–75) from MKP3 resulted in a 135-fold reduction in ERK2 binding affinity but did not affect the propensity of MKP3 to be activated by ERK2 (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Recent data suggest that all ERK2-interacting proteins may bind ERK2 through electrostatic interactions between the positively charged KIM motif and a common docking (CD) domain in ERK2 composed of a stretch of negatively charged amino acids (e.g. Asp-316 and Asp-319) situated opposite to the kinase catalytic cleft (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (663) Google Scholar). Residue Asp-319 in the CD domain of ERK2 is conserved in all MAP kinases from yeast to man. A dominant gain-of-function mutation of the rolled MAP kinase gene in Drosophila, termed Sevenmaker (rlsevenmaker), contains a single amino acid substitution of the analogous Asp-334 by an Asn (D334N) and activates several developmental pathways (24Brunner D. Oellers N. Szabad J. Biggs III, W.H. Zipursky S.L. Hafen E. Cell. 1994; 76: 875-888Abstract Full Text PDF PubMed Scopus (377) Google Scholar). The same mutation in mammalian ERK2 (ERK2/D319N) appears to be resistant to inactivation by MKPs in transfected cells (25Bott C.M. Thorneycroft S.G. Marshall C.J. FEBS Lett. 1994; 352: 201-205Crossref PubMed Scopus (90) Google Scholar, 26Chu Y. Solski P.A. Khosravi-Far R. Der C.J. Kelly K. J. Biol. Chem. 1996; 271: 6497-6501Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Previous studies (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (663) Google Scholar, 14Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (430) Google Scholar) indicate that ERK2/D319N displays reduced affinity for MKP3 and is unable to activate the MKP3 phosphatase activity. However, using the quantitative activation-based assay, we were able to establish the K d of ERK2/D319N for MKP3 (14.8 ± 1.2 μm), which is 87-fold larger than that of the wild-type ERK2 (11Zhou B. Wu L. Shen K. Zhang J. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; 276: 6506-6515Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). More importantly, we found that ERK2/D319N can activate MKP3 to the same level induced by the wild-type ERK2 when a saturating amount of ERK2/D319N is present in the reaction. Thus, like the KIM sequence in MKP3, Asp-319 in ERK2 plays a major role in ERK2 binding to MKP3, but it is not essential for ERK2 induced MKP3 activation. To further define the contribution of ERK2 CD domain to MKP3 binding and activation, we determined the effects of amino acid substitutions in the CD domain using the activation-based assay outlined under "Experimental Procedures." We chose amino acid residues in the vicinity of Asp-319, based on both primary and tertiary structure. All experiments were performed at pH 7.0, ionic strength of 0.15 m, and 30 °C. As summarized in Table I, the dissociation constant (K d) for ERK2 and MKP3 is 0.17 ± 0.03 μm. No significant effects were observed for ERK2 mutants Q313E, S218D, E324A, and F329L. However, when Tyr-126 and Tyr-314 were replaced by an Ala, the affinity of ERK2/Y126A and ERK2/Y314A for MKP3 was reduced by 20- and 7.8-fold, respectively. When Asp-160 and Asp-316 was replaced by an Asn, the affinity of ERK2/D160N and ERK2/D316N for MKP3 decreased 18- and 9.6-fold, respectively. Substitution of Glu-79 or Arg-133 with an Ala reduced the affinity of ERK2/E79A and ERK2/R133A for MKP3 by 5.7- and 5.1-fold, respectively. Thus, Asp-319 is the most important residue in ERK2 CD domain as large decreases (60–100-fold) in binding affinity were observed when Asp-319 was changed to Ala, Asn, Glu, or Arg. Interestingly, ERK2/E324Q exhibited a 3.7-fold higher affinity for MKP3 than that of the wild-type ERK2. Taken together, our data suggest that the CD site is likely composed of residues Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319, which form a contiguous docking surface on the back side of the ERK2 kinase active site (Fig. 1A). It is important to point out that although mutation of CD site residues decreased MKP3 binding affinity, none of them affected the ability of ERK2 to activate MKP3 (Table I).Table IContributions of residues in ERK2 CD site to MKP3 binding and activationERK2MKP3KdMKP3 activationaThis value was calculated as the ratio of maximum MKP3 activity in the presence of saturating amount of mutant versus wild-type ERK2.μ m%Wild typeWild type0.17 ± 0.04100Wild typeR64A1.24 ± 0.2594 ± 5Wild typeR64K0.18 ± 0.0591 ± 5Wild typeR65A25.8 ± 2.294 ± 5Wild typeR65K8.3 ± 1.293 ± 6Wild typeR65D7.8 ± 1.772 ± 7D319NWild type14.8 ± 1.2111 ± 8D319NR65K27.3 ± 4.6116 ± 8D319AWild type18.0 ± 3.383 ± 7D319EWild type9.5 ± 1.086 ± 5D319RWild type11.7 ± 2.772 ± 7D319RR65D15.0 ± 3.8100 ± 8Q313EWild type0.12 ± 0.0494 ± 6Y314AWild type1.33 ± 0.22111 ± 8D316NWild type1.63 ± 0.66106 ± 8S318DWild type0.26 ± 0.0383 ± 5E324AWild type0.25 ± 0.0594 ± 7E324QWild type0.046 ± 0.014104 ± 7F329LWild type0.23 ± 0.0488 ± 5E79AWild type0.97 ± 0.12116 ± 8Y126AWild type3.47 ± 0.84117 ± 8R133AWild type0.87 ± 0.1097 ± 6D160NWild type3.10 ± 0.3977 ± 5a This value was calculated as the ratio of maximum MKP3 activity in the presence of saturating amount of mutant versus wild-type ERK2. Open table in a new tab A "Hot Spot" Interaction between the KIM Sequence of MKP3 and the CD Site in ERK2—It has been suggested that the CD domain may make direct contact with the KIM sequence in ERK2-interacting molecules (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Bio
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