Differential Activation of p38 Mitogen-activated Protein Kinase Isoforms Depending on Signal Strength
2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês
10.1074/jbc.m007835200
ISSN1083-351X
AutoresGema Alonso, Concetta Ambrosino, Margaret A. Jones, Ángel R. Nebreda,
Tópico(s)Cancer Mechanisms and Therapy
ResumoWe have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38α but not p38γ. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitrousing either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38α by MKK6 correlated with more efficient binding of MKK6 to p38α than to p38γ. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38α alone (low MKK6 activity) or both p38α and p38γ (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response. We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38α but not p38γ. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitrousing either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38α by MKK6 correlated with more efficient binding of MKK6 to p38α than to p38γ. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38α alone (low MKK6 activity) or both p38α and p38γ (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response. mitogen-activated protein kinase polymerase chain reaction glutathione S-transferase polyacrylamide gel electrophoresis phosphate-buffered saline myelin basic protein Cellular responses to many external stimuli involve the activation of several types of mitogen-activated protein kinase (MAPK)1 signaling pathways. Three major subfamilies of MAPKs have been described in vertebrates. The p42/p44 ERK MAPKs are mainly activated by growth factors and other stimuli involved in cell proliferation and differentiation processes. In contrast, the SAPK/JNK and the p38 MAPKs are strongly activated in response to stress conditions and proinflammatory cytokines. Despite the diversity in function and upstream signaling events, MAPKs are always activated by a highly conserved mechanism that involves phosphorylation on both a Thr and a Tyr residue catalyzed by a MAPK kinase. The phosphorylation motif Thr-Xaa-Tyr is located in the so called activation loop or T loop whose amino acid sequence varies among different MAPK subfamilies. Accordingly, there are different activating MAPK kinases that in most cases are specific for each subgroup of MAPKs (reviewed by Refs. 1Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar, 3Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar) The p38 MAPK subfamily plays important roles in cytokine production and the stress response (reviewed by Ref. 4Ono K. Han J. Cell Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar). Recent reports have also demonstrated additional functions for p38 MAPKs, for example, in the inhibition of cell cycle progression, in developmental processes such as egg polarity and wing morphogenesis in Drosophila, and in the differentiation of several vertebrate cell types including neurons, adipocytes and myoblasts (reviewed in Ref. 5Nebreda A.R. Porras A. Trends Biochem. Sci. 2000; 25: 257-260Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Four p38 MAPKs have been cloned so far in higher eukaryotes: p38α/XMpk2/CSBP (6Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 7Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar, 8Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty d. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 9Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2413) Google Scholar), p38β/p38β2 (10Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 11Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), p38γ/SAPK3/ERK6 (12Lechner C. Zahalka M.A. Giot J.-F. Moller N.P.H. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (276) Google Scholar, 13Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (353) Google Scholar, 14Mertens S. Craxton M. Goedert M. FEBS Lett. 1996; 383: 273-276Crossref PubMed Scopus (134) Google Scholar), and p38δ/SAPK4 (15Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar, 16Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 17Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.-Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). These four proteins are 60–70% identical in their amino acid sequence and are all activated by the MAPK kinase MKK6 (15Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar, 18Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar). Another MAPK kinase, MKK3, has been shown to phosphorylate and activate p38α, p38γ, and p38δ but not p38β2 (11Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 19Enslen H. Brancho D.M. Davis R.J. EMBO J. 2000; 19: 1301-1311Crossref PubMed Scopus (187) Google Scholar). Moreover, p38α can also be activated in vitro by MKK4, one of the JNK/SAPK activators (20Doza N.Y. Cuenda A. Thomas G.M. Cohen P. Nebreda A.R. FEBS Lett. 1995; 364: 223-228Crossref PubMed Scopus (72) Google Scholar, 21Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1413) Google Scholar, 22Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (711) Google Scholar), and MKK4 −/− fibroblasts but not embryonic stem cells exhibit defects in p38 MAPK phosphorylation (23Ganiatsas S. Kwee L. Fujiwara Y. Perkins A. Ikeda T. Labow M.A. Zon L.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6881-6886Crossref PubMed Scopus (177) Google Scholar). p38 MAPK isoforms also differ in their susceptibility to inhibition by pyridinyl imidazoles that can inhibit p38α and p38β at submicromolar concentrations (24Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar) but have no inhibitory effect on p38γ and p38δ (25Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). Substrates of p38 MAPKs include the protein kinases MAPKAPK-2/3, MNK1, MSK, and PRAK and several transcription factors such as ATF-2, c/EBPs, and MEF2C (reviewed in Ref. 25Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). Some of these substrates are preferentially phosphorylated in vitro by one or more p38 MAPK isoforms, suggesting that these enzymes may have both overlapping and highly specific functions. Consistent with this possibility, some extracellular stimuli can selectively activate specific p38 MAPK isoforms (26Conrad P.W. Rust R.T. Han J. Millhorn D.E. Beitner-Johnson D. J. Biol. Chem. 1999; 274: 23570-23576Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 27Hale K.K. Trollinger D. Rihanek M. Manthey C.L. J. Immunol. 1999; 162: 4246-4252PubMed Google Scholar). Moreover, targeted gene disruption in mice has recently shown that p38α is essential only for placenta organogenesis, despite being ubiquitously expressed in the embryo (28Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). This suggests that p38α function might be redundant for other aspects of mammalian embryonic development. We are interested in the differential regulation of p38 MAPK isoforms. Here we show that p38α can be efficiently activated by low levels of MKK6 activity, whereas other p38 isoforms are only activated by high levels of MKK6 activity. The selectivity probably depends on sequences located at the carboxyl-terminal lobe of p38 MAPKs, which do not correspond to motifs previously proposed to be important for the recognition of MAPKs by MAPK kinases. Our results suggest a mechanism for the specific activation of p38 MAPKs depending on the level of MKK6 activity triggered by a given stimulus. To produce recombinant MalE-MKK6, the human MKK6/SKK3 cDNA (29Cuenda A. Alonso G. Morrice N. Jones M. Meier R. Cohen P. Nebreda A.R. EMBO J. 1996; 15: 4156-4164Crossref PubMed Scopus (115) Google Scholar) was cloned into the pMalc2 vector (New England Biolabs) as a PCR product obtained using a 5′ oligonucleotide that created an XbaI site immediately downstream of the ATG and a 3′ oligonucleotide that introduced aHindIII site downstream of the stop codon. A construct to produce recombinant His-MKK6 was generated by subcloning into pET15 (Novagen) the PCR product obtained using a 5′ oligonucleotide that created an NcoI site at the ATG and at the same time replaced amino acid number 3 of MKK6 by 5 His residues and a 3′ oligonucleotide that introduced an XhoI site downstream of the stop codon. For expression in Xenopus oocytes and mammalian cells, MKK6 was cloned into the vectors FTX5 (30Howell M. Hill C.S. EMBO J. 1997; 16: 7411-7421Crossref PubMed Scopus (57) Google Scholar) and pEFmlink (provided by Caroline Hill and Richard Treisman, Imperial Cancer Research Fund, London, UK), respectively. In both cases, MKK6 was amplified by PCR using oligonucleotides that created anNcoI site at the ATG and an XhoI downstream of the stop codon. The mutants MKK6-DD, MKK6-AA, MKK6-K/R, and MKK6-D/A (see Fig. 1) were generated by PCR using the above vectors as templates for the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing. The Xenopus p38α/XMpk2 cDNA (7Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar) was amplified by PCR using oligonucleotides that introduced two BglII sites flanking the open reading frame. The PCR product was digested withBglII and subcloned into BglII-digested pSP64T (Promega) for expression in Xenopus oocytes andBamHI-digested pSG5 (Stratagene) for mammalian cells. The construct to express MalE-XMpk2/p38α in bacteria has been previously described (20Doza N.Y. Cuenda A. Thomas G.M. Cohen P. Nebreda A.R. FEBS Lett. 1995; 364: 223-228Crossref PubMed Scopus (72) Google Scholar). The mutant p38α-K54R was generated by PCR using the QuikChange mutagenesis kit. A rat p38γ/SAPK3 cDNA cloned in the vectors pSG5 and pGEX4T-1 (18Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar) was provided by Michel Goedert (Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK) and was subcloned into FTX5 as an EcoRI fragment. The constructs to express human p38α, p38β2, and p38δ as recombinant proteins fused to GST (15Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar, 31Eyers P.A. Craxton M. Morrice N. Cohen P. Goedert M. Chem. Biol. 1998; 5: 321-328Abstract Full Text PDF PubMed Scopus (280) Google Scholar) were provided by Philip Cohen and associates (Medical Research Council, Dundee, UK). A plasmid to express GST-ATF-2 (amino acids 19–96) (32Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (474) Google Scholar) was provided by Gunvanti Patel and Nic Jones (Imperial Cancer Research Fund, London, UK). The mutants p38γ(AL-p38α), p38γ(CD-p38α), and p38γ(AL/CD-p38α) were generated by replacing amino acids 177–189 (activation loop, as indicated by "AL" above) and/or 313–321 (common docking, as indicated by "CD" above) of p38γ by the corresponding sequences of p38α (HTDDEMTGYVATR and QYHDPDDEP, respectively) using the QuikChange mutagenesis kit. The fusions between p38α and p38γ were prepared by overlapping PCR using as a linker the sequence MGADL (amino acids 109–113 of p38α). For the N-p38α/C-p38γ chimera, amino acids 1–108 of p38α were fused to the sequence MGADL followed by amino acids 117–367 of p38γ. For N-p38γ/C-p38α, amino acids 1–111 of p38γ were fused to MGADL followed by amino acids 114–360 of p38α. For protein purification, MalE or GST constructs were transformed intoEscherichia coli BL-21 (DE3), and their expression was induced with isopropyl-1-thio-β-d-galactopyranoside. Recombinant proteins were purified on amylose beads (MalE fusions) or glutathione beads (GST fusions) as described (33Palmer A. Gavin A.C. Nebreda A.R. EMBO J. 1998; 17: 5037-5047Crossref PubMed Scopus (290) Google Scholar). The anti-p38α antiserum used for immunoprecipitation was prepared against the carboxyl-terminal 14 amino acids of Xenopus p38α/XMpk2 as described previously (7Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar). For immunoblotting we used an anti-p38α antibody (C-20) purchased from Santa Cruz. The anti-SAPK3/p38γ and anti-MKK6 antisera were prepared in rabbits by immunization with the bacterially produced GST-SAPK3 and MalE-MKK6 recombinant proteins described above, respectively. The overexpressed, Myc-tagged MKK6 and p38γ proteins were immunoprecipitated using the 9E10 monoclonal antibody. Anti-phospho-p38 and anti-phospho-MKK6/MKK3 antibodies that specifically recognize p38 MAPKs phosphorylated both on the Thr and Tyr residues in the activation loop and the phosphorylated form of MKK3 and MKK6, respectively, were purchased from New England Biolabs. Anti-polyhistidine antibody was purchased from Sigma. Anti-MalE and anti-GST antibodies were provided by Julian Gannon (Imperial Cancer Research Fund, South Mimms, UK). HEK293, HeLa and NIH3T3 cells were routinely grown at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin and streptomycin, 2 mm glutamine under an atmosphere of 5% CO2. PC12 cells were grown in Dulbecco's modified Eagle's medium containing 6% fetal calf serum, 6% horse serum, penicillin, streptomycin, 2 mm glutamine, and 10 mm Hepes. Plates were about 70% confluent before stimulation. HEK293 cells were transiently transfected (calcium phosphate method) using 5–10 μg of plasmid DNA/10-cm plate of 70–80% confluent cells. The cells were then incubated for 36–48 h, stimulated with UV for 30 s (using a Stratalinker set at 80% of potency) and incubated for a further 30 min at 37 °C before lysis. Stage VI Xenopusoocytes were injected with 50 nl of capped mRNAs (usually 10 ng unless otherwise indicated) prepared from linearized pSP64T and FTX5 constructs using the MEGAscript in vitro transcription kit (Ambion). Injected oocytes were incubated for the indicated times and then frozen in dry ice. For preparation of lysates, oocytes were homogenized in 10 μl/oocyte of ice-cold H1 kinase buffer (80 mm sodium β-glycerophosphate, pH 7.5, 20 mmEGTA, 15 mm MgCl2, 1 mmdithiothreitol, 1 mm Pefabloc, and 1 μg/ml each of leupeptin and aprotinin). Lysates were centrifuged for 10 min at 10,000 × g, and 5 μl of the cleared supernatant was used for immunoblot analysis. The activity of the proteins expressed in oocytes was assayed by immunoprecipitation of 4–5 oocytes followed byin vitro kinase assay. Kinase assays were carried out in a final volume of 12 μl in buffer A (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 2 μmmicrocystin, and 10 μm ATP with 2 μCi of [γ-32P]ATP, 3000 Ci/mmol) containing 0.8–1 μg of substrate for 30 min at 30 °C. The reaction was stopped by the addition of sample loading buffer and boiling for 3 min. Proteins were resolved by SDS-PAGE and detected by autoradiography. Cells were washed with cold PBS and lysed (0.5 ml/10 cm plate) in IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 5 mm EGTA, 20 mmNaF, 0.1 mm sodium orthovanadate, 1 mmPefabloc, 2.5 mm benzamidine, 50 nm calyculin A, and 10 μg/ml of leupeptin and aprotinin). Lysates were transferred to ice-cold microcentrifuge tubes, vortexed, incubated for 10 min on ice, and centrifuged for 10 min at 10,000 × g to remove insoluble material. Lysates (200 μg) were incubated with 2–3 μl of antisera for 60 min at 4 °C, followed by incubation with 10 μl of protein A-Sepharose (Amersham Pharmacia Biotech) for a further 30 min. All incubations and centrifugations were carried out at 4 °C. Immunoprecipitates were washed three times with IP buffer and once with kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm magnesium acetate, 1 mm dithiothreitol, 0.1 mm sodium orthovanadate, and 25 nm calyculin A). Kinase assays were incubated for 20 min at 30 °C in 15 μl of kinase buffer containing 100 μm ATP (2–5 μCi of [γ-32P]ATP, 3000 Ci/mmol) and 1 μg of substrate. The reaction was stopped by adding the sample loading buffer and boiling the samples for 3 min. Proteins were resolved by electrophoresis in 17.5% PAGE (34Anderson C.W. Baum P.R. Gesteland R.F. J. Virol. 1973; 12: 241-252Crossref PubMed Google Scholar) and detected by autoradiography. For immunoprecipitation of the endogenous MKK6, 350 μg of lysates were incubated with 3 μl of anti-MKK6 antiserum for 1.5 h at 4 °C on a rotating wheel. Protein A-Sepharose (15 μl) was then added, and the samples were rocked at 4 °C for 1 h. The immunoprecipitates were collected by centrifugation (500 ×g, 1 min), washed four times in immunoprecipitation buffer, and washed once in kinase buffer. The kinase assay was performed as reported above. For immunoblotting, proteins (25–50 μg of lysate) were separated by SDS-PAGE and transferred to nitrocellulose (BA85, Schleicher & Schuell) using a semi-dry blotting apparatus. The membranes were blocked in TTBS (25 mm Tris, pH 8, 150 mm NaCl, 0.05% Tween-20) containing 4% nonfat milk for 1 h at room temperature. Primary antibodies were incubated in TTBS containing 1% milk (except for the anti-phospho-MKK6 antibody which was incubated in 3% bovine serum albumin) and binding was detected by horseradish peroxidase-coupled secondary antibodies (Dako) followed by ECL detection (Amersham Pharmacia Biotech). MalE-MKK6 recombinant proteins (2 μg) were bound to amylose beads (10 μl) for 1 h at 4 °C in PBS. The beads were washed three times in PBS and once in IP buffer and then mixed with 500 μg of HEK293 lysates overexpressing either p38α or p38γ. Binding was allowed to proceed for 2–3 h at 4 °C. After washing three times in IP buffer, the material bound to the recombinant proteins was separated by SDS-PAGE, and the presence of p38α and p38γ was detected by immunoblotting. For the in vitro binding experiments, GST- or MalE- fusion proteins (1–1.5 μg) were incubated with His-MKK6 (0.6–0.8 μg) in a final volume of 200 μl of IP buffer, at room temperature on the rotating wheel. After 30 min, 10 μl of glutathione-Sepharose (Amersham Pharmacia Biotech) or amylose beads (New England Biolabs) which have been preincubated in PBS containing bovine serum albumin (2 mg/ml) for 2 h at room temperature were added and incubated for 90 min at 4 °C on the rotating wheel. The beads were collected by centrifugation (500 × g, 1 min), washed four times in IP buffer, and analyzed by SDS-PAGE. The presence of bead-bound His-MKK6 was detected by immunoblotting using the anti-MKK6 and anti-polyHistidine antibodies for the GST and MalE pull-downs, respectively. The ability of the MAPK kinase MKK6 to activate the two p38 MAPK family members p38α and p38γ was investigated upon co-expression in Xenopus oocytes. For this purpose, oocytes were first injected with mRNAs encoding either wild type MKK6 or the mutants MKK6-K/R (Lys-82 in the ATP binding pocket mutated to Arg), MKK6-DD, or MKK6-AA (the two phosphorylation sites in the activation loop Ser-207 and Thr-211 changed to Glu or Ala, respectively) (Fig. 1). After overnight incubation to allow for expression of the MKK6 proteins, mRNAs encoding p38α or p38γ were injected, and samples were taken 4 and 10 h later. Oocytes were lysed, and the activity of p38α and p38γ was assayed by immune complex kinase assay, using MBP as an exogenous substrate. We found that p38γ activity was stimulated only by co-injection with the constitutively active MKK6-DD mutant (Fig. 2, lower panel, lanes 4 and 9). Surprisingly, p38α was activated by co-injection with all four MKK6 mutants, including MKK6-K/R, albeit MKK6-DD showed a faster kinetic of activation than the other MKK6 proteins (Fig. 2 A, upper panel, lanes 1–5). However, the extent of p38α activation by the four MKK6 proteins was very similar after co-expression for 10 h (Fig. 2,upper panel, lanes 7–10). We next used a different system to test the ability of different MKK6 mutants to phosphorylate and activate p38α. Myc-tagged MKK6 mutants were transiently transfected into HEK293 cells that were then either left unstimulated or stimulated with UV radiation, a potent activator of the p38 MAPK pathway (10Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 18Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar, 35Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1147) Google Scholar). The expressed MKK6 proteins (Fig. 3 A) were then immunoprecipitated, and their kinase activity was assayed using bacterially produced p38α or p38γ as a substrate (Fig. 3 B). p38γ was phosphorylated both by the constitutively active MKK6-DD mutant and by the wild type MKK6; the latter more strongly after UV-stimulation (Fig. 3 B, lower panel, lanes 2, 5, and 6). In contrast, p38α was phosphorylated by all MKK6 mutants, even when recovered from non-UV-stimulated cells (Fig. 3 B, upper panel, lanes 1–8), except by MKK6-D/A (Fig. 3 B, lanes 9 and 10; see below). The same results were observed when the MKK6 proteins were immunoprecipitated from mRNA-injected Xenopus oocytes, and their kinase activity was assayed on recombinant p38α and p38γ; all MKK6 mutants phosphorylated p38α, whereas p38γ was only efficiently phosphorylated by the constitutively active MKK6-DD mutant (data not shown). We also carried out this experiment using purified MKK6 mutant proteins expressed in E. coli (Fig. 4). In this case recombinant MKK6 mutants were incubated in vitro with recombinant p38α or p38γ in the presence of [γ-32P]ATP. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography. As in previous experiments p38γ was strongly phosphorylated by the MKK6-DD mutant (Fig. 4,lane 10) and more weakly by MKK6 wild type or the AA mutant (Fig. 4, lanes 8 and 11). However, p38α was phosphorylated by all the recombinant MKK6 mutant proteins (Fig. 4,lanes 2–5) except by MKK6-D/A (Fig. 4, lane 6). This confirms the results obtained with MKK6 immunoprecipitated from transfected HEK293 cells or mRNA-injected Xenopusoocytes. In all cases phosphorylation of recombinant p38α and p38γ correlated with an increase in the kinase activity of the proteins, measured by their ability to phosphorylate the substrates GST-ATF2 and MBP when these were included in the reaction mixture (see Figs. 7 A, 8, and 9).Figure 7Phosphorylation and activation of p38α and p38γ by increasing amounts of MKK6-DD. A, MalE-p38α and GST-p38γ (1 μg) were incubated alone or with increasing amounts of MalE-MKK6-DD (16, 80 and 800 ng) plus [γ-32P]ATP and in the absence (lanes 1–4 and 9–12) or in the presence of GST-ATF-2 (1 μg, lanes 5–8 and13–16). Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. B, p38α and p38γ mRNAs were co-injected into oocytes with different amounts of MKK6-DD mRNA (0.5, 2, and 10 ng). After 10 h of incubation, p38α and p38γ were immunoprecipitated from oocyte lysates and their kinase activities assayed using GST-ATF2 as a substrate (bottom panels). The expression levels in oocytes of the proteins MKK6-DD (top panels), p38α and p38γ (middle panels) were analyzed by immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Phosphorylation and activation of different p38 isoforms by increasing amounts of MKK6-DD. Purified MalE-p38α and GST fusion proteins of p38β2, p38γ, and p38δ (1 μg) were incubated alone or with increasing amounts of MalE-MKK6-DD (8, 40, and 800 ng) in the presence of [γ-32P]ATP and MBP (1 μg). Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. The lower panel shows the same gel stained with Coomassie Blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Phosphorylation and activation of p38α and p38γ mutants by increasing amounts of MKK6-DD. A and C, purified GST fusion proteins of the indicated p38α and p38γ mutants (1 μg) were incubated alone or with increasing amounts of MalE-MKK6-DD (4, 40, and 400 ng) in the presence of [γ-32P]ATP and MBP (1 μg). Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. Band D, purified GST fusion proteins of the same p38α and p38γ mutants (1 μg) were incubated with purified His-MKK6 (0.8 μg) and then recovered on GSH-Sepharose beads. The proteins bound to the beads were analyzed by immunoblotting using anti-GST and anti-MKK6 antibodies. See "Experimental Procedures" for details of the different mutants.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One possibility to explain the different susceptibility of p38α and p38γ to phosphorylation by MKK6 mutants could be that the two p38 MAPKs have different affinities for the MKK6 activator. To test this hypothesis recombinant MalE-MKK6 or MalE alone were bound to amylose beads and mixed with extracts of HEK293 cells overexpressing either p38α or p38γ. Binding of the p38 MAPKs to the beads was visualized by immunoblotting (Fig. 5 A). MalE-MKK6 was able to bind p38α from unstimulated or UV-stimulated cells much more efficiently than MalE alone (Fig. 5 A,upper panel, compare lanes 3 and 4with lanes 5 and 6), indicating that p38α specifically associates with MKK6. In contrast, p38γ was not detected in MalE-MKK6 pull-downs (Fig. 5 A, lower panel,lanes 5 and 6). When the same experiment was carried out using different MKK6 mutants, we found that p38α could associate with all mutants to a similar extent, whereas p38γ was never detected in MalE-MKK6 pull-downs (Fig. 5 B). Moreover, we could detect in vitro association between purified MKK6 and GST-p38α proteins (Fig. 5 C, lane 2) but not between MKK6 and GST-p38γ proteins (Fig. 5 C, lane 1). These results indicate that complex formation between MKK6 and p38α does not require additional proteins present in HEK293 cell extracts and that ther
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