Autophosphorylation Activity of the Arabidopsis Ethylene Receptor Multigene Family (2004)

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1Gamble R.L. Coonfield M.L. Schaller G.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7825-7829Crossref PubMed Scopus (256) Google Scholar). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2Xie C. Zhang J.S. Zhou H.L. Li J. Zhang Z.G. Wang D.W. Chen S.Y. Plant J. 2003; 33: 385-393Crossref PubMed Scopus (83) Google Scholar). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism. Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1Gamble R.L. Coonfield M.L. Schaller G.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7825-7829Crossref PubMed Scopus (256) Google Scholar). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2Xie C. Zhang J.S. Zhou H.L. Li J. Zhang Z.G. Wang D.W. Chen S.Y. Plant J. 2003; 33: 385-393Crossref PubMed Scopus (83) Google Scholar). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism. In bacteria, two-component signal transduction systems are involved in a variety of responses, including osmotic regulation, chemotaxis, and stress responses (reviewed in Ref. 3Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2398) Google Scholar). Canonical two-component signal transduction systems involve a sensor protein and a response regulator protein. In most cases the sensor consists of a variable amino-terminal domain located in the periplasm, two transmembrane regions, and a histidine kinase domain at the carboxyl terminus. The histidine kinase domain autophosphorylates in response to a given stimulus. The response regulator comprises a receiver domain and an effector domain. Following histidine autophosphorylation, the response regulator catalyzes the transfer of the phosphoryl group from the kinase domain of the sensor to an aspartate residue in its own receiver domain. The phosphorylation of the response regulator activates it, usually leading to a gain of DNA binding activity of its effector domain. Two-component proteins are also involved in more complex signaling pathways, termed phosphorelays. In these pathways the receptors are often hybrid proteins containing a receiver domain at the carboxyl terminus of their kinase domain. After autophosphorylation of the histidine residue in the kinase domain, the phosphoryl group is transferred intra-molecularly to the receiver domain. This phosphoryl group is subsequently transferred to a histidine-containing phosphotransfer protein and then to a response regulator protein, completing a phosphorelay (reviewed in Ref. 3Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2398) Google Scholar). Two-component and phosphorelay signaling systems exist in both prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, for example, there is only one histidine kinase sensor protein, the osmolarity receptor SLN1 (4Ota I.M. Varshavsky A. Science. 1993; 262: 566-569Crossref PubMed Scopus (362) Google Scholar). SLN1 is a hybrid histidine kinase that transfers its phosphoryl group to a histidine-containing phosphotransfer protein, YPD1, which then transfers the phosphoryl group to the response regulator SSK1 (5Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (932) Google Scholar, 6Maeda T. Takekawa M. Saito H. Science. 1995; 269: 554-558Crossref PubMed Scopus (553) Google Scholar). This phosphorelay signaling system has been shown to regulate the SSK2-PBS2-HOG1 mitogen-activated protein kinase cascade (7Posas F. Saito H. EMBO J. 1998; 17: 1385-1394Crossref PubMed Scopus (249) Google Scholar). In plants, there are several proteins with sequence similarity to histidine kinases, including phytochromes (8Schneider-Poetsch H.A. Braun B. Marx S. Schaumburg A. FEBS Lett. 1991; 281: 245-249Crossref PubMed Scopus (91) Google Scholar) and hormone receptors for ethylene (9Chang C. Kwok S.F. Bleecker A.B. Meyerowitz E.M. Science. 1993; 262: 539-544Crossref PubMed Scopus (1187) Google Scholar, 10Hua J. Chang C. Sun Q. Meyerowitz E.M. Science. 1995; 269: 1712-1714Crossref PubMed Scopus (469) Google Scholar, 11Hua J. Sakai H. Nourizadeh S. Chen Q.H.G. Bleecker A.B. Ecker J.R. Meyerowitz E.M. Plant Cell. 1998; 10: 1321-1332Crossref PubMed Scopus (438) Google Scholar, 12Sakai H. Hua J. Chen Q.H.G. Chang C.R. Medrano L.J. Bleecker A.B. Meyerowitz E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5812-5817Crossref PubMed Scopus (394) Google Scholar) and cytokinins (13Inoue T. Higuchi M. Hashimoto Y. Seki M. Kobayashi M. Kato T. Tabata S. Shinozaki K. Kakimoto T. Nature. 2001; 409: 1060-1063Crossref PubMed Scopus (688) Google Scholar). The cytokinin signal transduction pathway has been proposed to function through a phosphorelay mechanism (13Inoue T. Higuchi M. Hashimoto Y. Seki M. Kobayashi M. Kato T. Tabata S. Shinozaki K. Kakimoto T. Nature. 2001; 409: 1060-1063Crossref PubMed Scopus (688) Google Scholar, 14Hwang I. Sheen J. Nature. 2001; 413: 383-389Crossref PubMed Scopus (706) Google Scholar). Ethylene gas functions as a hormone in plants, controlling many aspects of development and stress responses (15Abeles F.B. Morgan P.W. Saltveit M.E. Ethylene in Plant Biology. 2nd ed. Academic Press, San Diego, CA1992Google Scholar). There are five ethylene receptors in Arabidopsis thaliana, and genetic and biochemical evidence suggests that they are all active in ethylene signal transduction (16Hall A.E. Chen Q.H.G. Findell J.L. Schaller G.E. Bleecker A.B. Plant Physiol. 1999; 121: 291-299Crossref PubMed Scopus (128) Google Scholar, 17Hua J. Meyerowitz E.M. Cell. 1998; 94: 261-271Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). This receptor family can be further divided into two classes. ETR1 and ERS1 are sub-family 1 receptors and have all the conserved motifs necessary for histidine kinase activity (18Parkinson J.S. Kofoid E.C. Annu. Rev. Genet. 1992; 26: 71-112Crossref PubMed Scopus (1237) Google Scholar). The subfamily 2 class includes ETR2, ERS2, and EIN4. These proteins lack most of the motifs characteristic of histidine kinases, and EIN4 is the only one in this group containing the conserved histidine that is phosphorylated in two-component and phosphorelay systems. ETR1 autophosphorylates in vitro on this conserved histidine (1Gamble R.L. Coonfield M.L. Schaller G.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7825-7829Crossref PubMed Scopus (256) Google Scholar). Like the yeast SLN1 signaling pathway, ethylene receptor signaling is thought to regulate the CTR1-SIMKK-SIMK/MMK3 mitogen-activated protein kinase cascade (19Ouaked F. Rozhon W. Lecourieux D. Hirt H. EMBO J. 2003; 22: 1282-1288Crossref PubMed Scopus (217) Google Scholar). However, because of the sequence divergence shown in Fig. 1A, it is doubtful that all the other family members can function as histidine kinases, even though they all function as ethylene receptors. Moreover, genetic data suggest that histidine autophosphorylation of ETR1 is not necessary for receptor function in ethylene signal transduction (20Chang C. Meyerowitz E.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4129-4133Crossref PubMed Scopus (57) Google Scholar, 21Wang W. Hall A.E. O'Malley R. Bleecker A.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 352-357Crossref PubMed Scopus (190) Google Scholar, 22Gamble R.L. Qu X. Schaller G.E. Plant Physiol. 2002; 128: 1428-1438Crossref PubMed Scopus (121) Google Scholar). There is significant evidence in the literature suggesting that histidine kinases can evolve into kinases that phosphorylate on serine residues. This phenomenon has been observed in the mitochondrial proteins branched chain α-ketoacid dehydrogenase kinase (23Popov K.M. Zhao Y. Shimomura Y. Kuntz M.J. Harris R.A. J. Biol. Chem. 1992; 267: 13127-13130Abstract Full Text PDF PubMed Google Scholar, 24Davie J.R. Wynn R.M. Meng M. Huang Y.S. Aalund G. Chuang D.T. Lau K.S. J. Biol. Chem. 1995; 270: 19861-19867Abstract Full Text PDF PubMed Scopus (50) Google Scholar) and pyruvate dehydrogenase kinase (25Popov K.M. Kedishvili N.Y. Zhao Y. Shimomura Y. Crabb D.W. Harris R.A. J. Biol. Chem. 1993; 268: 26602-26606Abstract Full Text PDF PubMed Google Scholar, 26Thelen J.J. Miernyk J.A. Randall D.D. Biochem. J. 2000; 349: 195-201Crossref PubMed Scopus (53) Google Scholar) as well as plant phytochromes (27Yeh K.C. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar, 28Lapko V.N. Jiang X.Y. Smith D.L. Song P.S. Protein Sci. 1999; 8: 1032-1044Crossref PubMed Scopus (66) Google Scholar) and a tobacco homologue of a subfamily 2 ethylene receptor (2Xie C. Zhang J.S. Zhou H.L. Li J. Zhang Z.G. Wang D.W. Chen S.Y. Plant J. 2003; 33: 385-393Crossref PubMed Scopus (83) Google Scholar). Here, we show that all five ethylene receptors autophosphorylate in vitro. However, ETR1 is the only family member that autophosphorylates exclusively on histidine residues. All other receptors show predominantly serine autophosphorylation under our assay conditions, and ERS1 autophosphorylates on both histidine and serine in the presence of Mn2+. However, histidine autophosphorylation is not observed when ERS1 is assayed in the presence of Mg2+ and Mn2+, suggesting that ERS1 might not have this activity in vivo unless a Mn2+ donor is present. Moreover, mutation studies show that the histidine residue conserved in histidine kinases is not required for the serine autophosphorylation of the ethylene receptors. Hence our results suggest that ethylene signal transduction in plants does not occur by a phosphorelay mechanism. Construction of Expression Plasmids—The soluble domains of the Arabidopsis thaliana ethylene receptors were amplified from cDNA clones with the following primers (engineered restriction sites are underlined): ETR1, 5′-AGCTCGGATCCGAAATGGGATTGATTCGAACTCA-3′ and 5′-ATCCAGGATCCTTACATGCCCTCGTACAGTACC-3′; ETR2, 5′-GAGCTTCCCGGGGAAGTTGGTTTGATTTTGATTAA-3′ and 5′-AGCCATCCCGGGTTAGAGAAGTTGGTCAGCTTGCAAC-3′; ETR-2ΔGAF, 5′-ATGGCGCCCGGGGACGCGTTGAGAGCGAGCCAAGC3′ and 5′-AGCCATCCCGGGTTAGAGAAGTTGGTCAGCTTGCAAC-3′; ERS1, 5′-AGTTAGGATCCGAAATGGGTCTTATTTTAACACA-3′ and 5′-TCCATGGATCCTCACCAGTTCCACGGTCTGGTTTGT-3′; ERS2, 5′-AGAGCTTAGATCTGAGGTTGGGATCATTATGAAGCA-3′ and 5′-CATGGATAGATCTTCAGTGGCTAGTAGACGGAGGAGTT-3′; and EIN4, 5′-AGCTTGGATCCGAGGTTGGATTGATGAAGAGGCA-3′ and 5′-AGGATGGATCCTCACTCGCTCGCGGTCTGCAAAGC-3′. ETR1, ERS1, and EIN4 PCR products were cut with BamHI and cloned into pESP-1 (Stratagene), ETR2 and ETR2-ΔGAF were cut with XmaI and cloned into pESP-1, and ERS2 was cut with BglII and cloned into the BamHI site of pESP-1. For the mutagenesis, we used the ExSite site-directed mutagenesis kit (Stratagene) on a BamHI-BamHI fragment containing receptor coding sequence cut from the previously described plasmids and cloned into pBSKS(+). The plasmids were methylated prior to mutagenesis. The primers used for the mutagenesis were as follows (nucleotide substitutions are underlined): ETR1-H, 5′-GAACACCGATGGCTGCGATTATTGCACT-3′ and 5′-GCATTTCAGCGTTCATAACCGCTAG-3′; ET-R2-H, 5′-CCTATGGCTTCGATACTCGGTCTTT-3′ and 5′-ACGCCTCATCCCTTCGCTCATCGTT-3′; ERS1-H, 5′-GGACACCGATGGCTGCCATCATCTCTCT-3′ and 5′-TCATCTCGGCGTTCATAACAGCTAG-3′; EI-N4-H, 5′-GGAGACCAATGGCCACAATTCTTGGTCT-3′ and 5′-TCATTCCAGCACTCATCACTTTCTG-3′; ETR1-G1, 5′-CAGCAGCAATAAATCCTCAAGAC-3′ and 5′-CAGAGTCTTTTACCTTCACTATA-3′; ERS1-G1, 5′-CGTGTGCAATTCACACACAAGAC-3′ and 5′-CTGTGTCCTTCACCTGCACAC-3′; ETR1-G2, 5′-ACTAGCACCGGAGCTTCTCGTCGCTAAAG-3′ and 5′-GCGCTTGCCCTCGCCATCTCCAAGA-3′; ERS1-G2, 5′-CAGCGGAATGGTTCCTCTGAGT-3′ and 5′-GAGCACTAGCGCTAGCTCTCTGTAAACGGTT-3′; ERS1-N1, 5′-TATGCTATTGTTAATCAGAAACGTCTGATGC-3′ and 5′-AGTTGGTAAGTCAGCAGACAGAATCAGATTCG-3′; ERS2-N1, 5′-AGGTCTTTCAAGCGATTTTGCACATGCTTGGGGTTC-3′ and 5′-TTCTATTATTACCTACGACGTAGTCAGGC-3′; ERS1-N2, 5′-CATCATGGCCAACGCTGTGAAGTTTACTAAGAAAGGCT-3′ and 5′-TTAAGAATTGTTTCCATCAGACGTTTCTCATCACC-3′; and ERS2-N2, 5′-TTGCACATGCTTGCGGTTCTAATGAATCGAAAGATC-3′ and 5′-AATCGCTTCAAAGACCTTCTTATCACCTACG-3′. The reverse primers were phosphorylated, and the cycle parameters for the mutagenesis followed the manufacturer's guidelines. Mutagenesis was confirmed by sequencing and the mutated BamHI-BamHI fragment was returned to the expression vector. Expression of Recombinant Proteins—The recombinant constructs were transformed into Schizosaccharomyces pombe SP-Q01 (Stratagene) according to the ESP® yeast protein expression and purification system (Stratagene) protocol. Colonies that grew on agar plates of Edinburgh minimal media supplemented with thiamine were selected for screening. Colonies were grown in Edinburgh minimal media for 8 h and lysed with the Yeast-Buster kit (Novagen), according to manufacturer's protocol. Protein blots of total lysate with a goat anti-GST antibody (Amersham Biosciences) were used to determine expression levels of positive clones. Expressing clones were grown in 50 ml yeast extract supplemented media for 18 h (3.0 ≤ A600 ≤ 4.0) and were used to inoculate 50 ml of yeast extract supplemented media to an A600 of 0.4. After a 5-h growth period (A600 ≈ 1.0) the cells were washed twice with 50 ml of sterile water and resuspended in 500 ml of Edinburgh minimal media (A600 ≈ 0.1). The culture was incubated at 30 °C for 18–22 h (1.8 ≤ A600 ≤ 2.2). Proteins were extracted by vortexing at 4 °C with 1× PBST (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, 1% (v/v) Triton® X-100) with glass beads (425–600 μm, Sigma) and protease inhibitors: 1 mm phenylmethylsulfonyl fluoride (Sigma), 1 μg/ml aprotinin (Sigma), 1 μg/ml chymostatin (Sigma), 10 μl/ml protease inhibitor mixture for fungal and yeast extracts (Sigma). Recombinant proteins were purified from clarified lysate on a glutathione-Sepharose 4B (Amersham Biosciences) column, which was washed with 1× phosphate-buffered saline (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4). GST 1The abbreviations used are: GST, glutathione S-transferase; PVDF, polyvinylidene difluoride; Hsp, heat shock protein.-tagged proteins were eluted with elution solution (10 mm reduced glutathione (Sigma), 50 mm Tris-HCl, pH 8.0, 20% (v/v) glycerol). For proteins purified without the GST tag, instead of elution solution columns were eluted with thrombin (Amersham Biosciences) in 1× phosphate-buffered saline. Eluted proteins were concentrated with Centriplus YM-50 (Amicon) and the buffer was exchanged for storage solution (50 mm Tris-HCl, pH 8.0, 25% (v/v) glycerol). Purification of recombinant protein was confirmed by protein blot, using goat anti-GST (Amersham Biosciences) or mouse anti-FLAG® (Stratagene) antibodies. Proteins were aliquoted and stored at –80 °C. Autophosphorylation Assay—50 pmol of purified recombinant protein were assayed in 50 mm Tris, pH 7.5, 10 mm MgCl2 (or MnCl2), 2 mm dithiothreitol, 10% (v/v) glycerol, 0.5 mm [γ-32P]ATP (1 Ci/mmol ≈ 1500 cpm/pmol). The reaction buffer with both Mg2+ and Mn2+ contained 0.15 mm MnCl2 and 10 mm MgCl2+. Mg2+ and Mn2+ concentrations in solution were calculated using a BASIC version of the COMICS program by Perrin and Sayce (29Perrin D.D. Sayce I.G. Talanta. 1967; 14: 833-842Crossref PubMed Scopus (577) Google Scholar) using the stability constants described in Ref. 30O'Sullivan W.J. Smithers G.W. Methods Enzymol. 1979; 63: 294-336Crossref PubMed Scopus (187) Google Scholar. The reaction buffer for the autophosphorylation of calcium-dependant protein kinase α contained 0.12 mm CaCl2 and 10 mm MgCl2. Reactions were incubated for 60 min at 25 °C and stopped by adding 5× loading dye (250 mm Tris-HCl, pH 6.8, 500 mm dithiothreitol, 10% (w/v) SDS, 0.5% (w/v) bromphenol blue, 50% (v/v) glycerol) and boiling for 3 min. Reactions were run on 8% SDS-PAGE and blotted to PVDF membrane (Hybond-P, Amersham Biosciences) using a three-solution semidry protein blotting protocol for 30 min at 16 V optimized to a lower pH to avoid loss of phosphoester linkages. Following is the blotting setup in brief, from anode to cathode: one sheet of filter paper (Whatman) wet with anode 1 solution (300 mm Tris, pH 9.5, 10% (v/v) methanol), two sheets wet in anode 2 solution (25 mm Tris, pH 9.5, 10% (v/v) methanol), PVDF membrane, gel, and three sheets wet in cathode solution (25 mm Tris, pH 8.5, 20% (v/v) methanol, 0.3% (w/v) glycine). Phosphate incorporation was visualized by autoradiography. Acid/Base Stability Assay—Autophosphorylation reactions were performed as above in triplicate for each treatment. After blotting, PVDF membranes were incubated for 16 h at room temperature in 1 m HCl, 3 m NaOH, or 100 mm Tris-HCl, pH 7.0. Protein bands were cut from the membrane and counted in scintillation fluid. The average for the counts of the acid and base treatments was normalized with respect to the counts for the control treatment (Tris-HCl). Phosphoamino Acid Analysis—Autophosphorylation reactions were performed in 50 mm Tris pH 7.5, 10 mm MgCl2 (or MnCl2), 2 mm dithiothreitol, 10% (v/v) glycerol, 0.2 μm [γ-32P]ATP (5000 Ci/mmol). After blotting, protein bands were cut from PVDF membranes and hydrolyzed in 100 μl 6 n HCl (Pierce) for 1 h at 110 °C. Membrane was removed and hydrolyzed amino acids were lyophilized and resuspended in pH 1.9 buffer (2.2% (v/v) formic acid, 7.8% (v/v) acetic acid) containing 100 μg/ml each phosphoamino acid standard (Ser-P, Thr-P, Tyr-P, Sigma). Bi-dimensional thin layer electrophoresis was performed as described in Liu et al. (31Liu G.Z. Pi L.Y. Walker J.C. Ronald P.C. Song W.Y. J. Biol. Chem. 2002; 277: 20264-20269Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and plates were visualized by autoradiography. All Five Ethylene Receptors Autophosphorylate in Vitro—The ethylene receptors show four distinct domains: a membrane spanning domain that is the ethylene binding site (32Schaller G.E. Bleecker A.B. Science. 1995; 270: 1809-1811Crossref PubMed Scopus (453) Google Scholar), a GAF domain that is a secondary and binding site for Biochem. Sci. 22: Full Text PDF PubMed Scopus Google Scholar), a kinase domain with sequence similarity to histidine kinases and a receiver domain as in response regulator proteins (reviewed in Ref. 3Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2398) Google Scholar). The receiver domain is from two of the ethylene ERS1 and ERS2 J. Chang C. Sun Q. Meyerowitz E.M. Science. 1995; 269: 1712-1714Crossref PubMed Scopus (469) Google Scholar, 11Hua J. Sakai H. Nourizadeh S. Chen Q.H.G. Bleecker A.B. Ecker J.R. Meyerowitz E.M. Plant Cell. 1998; 10: 1321-1332Crossref PubMed Scopus (438) Google Scholar). proteins for in vitro the soluble domains of the Arabidopsis ethylene receptors were cloned into as described under These constructs the GAF domain, the kinase domain, and the receiver domain, when the was in the protein. The soluble domains of all five ethylene receptors were in S. each with a GST to their amino terminus protein with most ethylene receptors and not be removed even of the ETR2 that the protein was the heat shock protein not The is usually removed by of Mg2+ and to the J. 1991; PubMed Google Scholar), which with the in vitro autophosphorylation activity of the ethylene receptors. has not been shown to have kinase it was not removed from the reaction recombinant receptors were for autophosphorylation in vitro as described under and results are shown in Fig. previously (1Gamble R.L. Coonfield M.L. Schaller G.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7825-7829Crossref PubMed Scopus (256) Google Scholar), ETR1 required Mn2+ for autophosphorylation and not function in the presence of ERS1 and ERS2 in the presence of Mg2+ or Mn2+, ETR2 and EIN4 a activity in the presence of of proteins from the beads to of the purified which be on a protein with the GST not of these products also as phosphorylated bands in the autophosphorylation because of their of autophosphorylation activity or as to the The recombinant EIN4 protein was and not be purified in EIN4 clones were with results in the autophosphorylation assay not It is to that ERS2, ETR2, and EIN4 were able to phosphorylate in the presence of Mg2+ and to a in the presence Mn2+. phosphorylation by the ethylene receptors might not be because has been shown to with receptors and kinases to stress responses in (reviewed in Ref. J. Sci. 2002; PubMed Google Scholar). shown in Fig. GST phosphorylation in that phosphorylation is on the ethylene receptors in the reaction levels of incorporation were observed for GST in the presence of Mn2+ which are by most of the receptors show levels of incorporation in the presence of we do not our results are by a kinase from yeast the GST Moreover, autophosphorylation was also observed when ERS1 was purified without the GST Fig. that the site of phosphorylation is to the the phosphorylation for the protein not It also be observed that is phosphorylated in the Hence the phosphorylation is also not by a kinase from yeast and is to subfamily 2 receptors. We also the GAF domain is required for autophosphorylation by using an ETR2 this domain Fig. shown in Fig. ETR2-ΔGAF the autophosphorylation as the ETR2 This suggests that the GAF domain is not the site of the phosphorylated residue in the kinase domain because ERS1 and ERS2 do not receiver domains J. Chang C. Sun Q. Meyerowitz E.M. Science. 1995; 269: 1712-1714Crossref PubMed Scopus (469) Google Scholar, 11Hua J. Sakai H. Nourizadeh S. Chen Q.H.G. Bleecker A.B. Ecker J.R. Meyerowitz E.M. Plant Cell. 1998; 10: 1321-1332Crossref PubMed Scopus (438) Google Scholar). of Acid in determine the nature of the phosphorylated amino acid, proteins were incubated in acid or base as described under histidine residues that are to acid and to on serine or phosphoester that are and Moreover, phosphorylation is to both acid and aspartate phosphorylation is in both acid and base B. S. Methods Enzymol. 1991; PubMed Scopus Google Scholar). has been previously (1Gamble R.L. Coonfield M.L. Schaller G.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7825-7829Crossref PubMed Scopus (256) Google Scholar), autophosphorylation of ETR1 in the presence of Mn2+ in a base phosphorylated residue under our assay with histidine autophosphorylation incorporation was from ETR1 reactions containing We used a protein kinase as a positive control for acid stability because it autophosphorylates on serines and threonines in the presence of Mg2+ M.J. PubMed Scopus Google Scholar), and results are shown in Fig. shown in Fig. phosphorylated ERS1, ETR2, EIN4, and ERS2 acid stability in the presence of Mg2+ a phosphoester In the presence of Mn2+, ERS1 to both acid and suggesting that this protein can and phosphoester in the presence of this The subfamily 2 class of ethylene receptors only phosphoester of the in the reaction mixture Our for the acid/base assay was 100 all of the were from triplicate and each recombinant protein was assayed at levels of incorporation the were from ETR2 reactions containing Mn2+, ETR2-ΔGAF and EIN4 in the buffer incorporation for Bi-dimensional thin layer electrophoresis was used to determine the phosphoester was on or shown in Fig. ERS1 only on serine residues in the presence of All receptors were in the presence of Mg2+ or Mn2+, and all ETR1 predominantly on serine residues not ETR1 not show significant phosphorylation on or in the presence of Mn2+ and phosphorylation also predominantly on serine residues not of phosphorylation were observed for ERS2 in the presence of Mn2+ the of the sites of ERS1 we for autophosphorylation in the presence of both Mg2+ and Mn2+. the of Mg2+ is to that of Mn2+ (reviewed in Ref. M.J. A. Rev. 1991; Scopus Google Scholar), the autophosphorylation reaction was performed this into the used for the autophosphorylation reaction the calculated concentrations of Mg2+ and