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• W2090972634 abstract "The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified bydifferential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade. The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified bydifferential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade. mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase c-Jun amino-terminal kinase E3 identified by differential display translocated promoter region homology to E6-AP carboxyl terminus ubiquitin-protein isopeptide ligase cyclopentyl epidermal growth factor cytomegalovirus myelin basic protein nucleoside diphosphate kinase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid glutathione S-transferase The mitogen-activated protein kinase (MAPK)1 or extracellular signal-regulated kinase (ERK) pathway is an evolutionarily conserved signaling pathway that regulates many cellular processes, including proliferation, differentiation, gene transcription, and cellular migration (1Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar). Activation of the Ras oncogene stimulates the membrane recruitment and activation of the Raf protein kinases (2Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (775) Google Scholar, 3Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 4Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (836) Google Scholar), which in turn phosphorylate and activate MAPK kinase or ERK kinases (MEK) 1 and 2. These in turn phosphorylate and activate ERK1 and ERK2 (5Her J.H. Lakhani S. Zu K. Vila J. Dent P. Sturgill T.W. Weber M.J. Biochem. J. 1993; 296: 25-31Crossref PubMed Scopus (106) Google Scholar). When in the inactive state, ERKs are anchored in the cytoplasm due to a basal association with the MEKs. Following activation, the ERKs migrate to various cellular locations, including the nucleus, microtubules, focal contacts, and others (6Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 7Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10952-10956Crossref PubMed Scopus (257) Google Scholar, 8Gonzalez F.A. Seth A. Raden D.L. Bowman D.S. Fay F.S. Davis R.J. J. Cell Biol. 1993; 122: 1089-1101Crossref PubMed Scopus (282) Google Scholar, 9Fincham V.J. James M. Frame M.C. Winder S.J. EMBO J. 2000; 19: 2911-2923Crossref PubMed Scopus (286) Google Scholar) where they phosphorylate a variety of substrates. These diverse phosphorylations allow the ERKs to orchestrate a complex but coordinated response to extracellular signals. The pattern and timing of substrate selections determines the specific biological outcome.The identification and characterization of the direct targets of a particular signaling pathway is paramount to understanding the function of that pathway within the cell. The Ras to ERK pathway is a key regulator of many cellular processes. Although there are many substrates of the ERKs that have been identified (10Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 11Lewis T.S. Hunt J.B. Aveline L.D. Jonscher K.R. Louie D.F. Yeh J.M. Nahreini T.S. Resing K.A. Ahn N.G. Mol. Cell. 2000; 6: 1343-1354Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), the diverse roles of the ERKs within the cell suggest that many unknown substrates remain to be identified. Identification of the kinase that directly phosphorylates a particular substrate can be difficult due to the overlap of consensus phosphorylation sequences, redundancy of kinases that can phosphorylate a particular site, differences between in vivo and in vitro specificity, and the diversity of kinases that can be present in a reaction, even in “pure” protein preparations. Several different approaches to determine novel MAPK substrates have been used (12Manning B.D. Cantley L.C. Science's STKE. 2002; (http://www.stke.org/cgi/content/full/OC_sigtrans;2002/162/pe49)PubMed Google Scholar). These include two-hybrid analysis (13Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (777) Google Scholar,14Maekawa M. Nishida E. Tanoue T. J. Biol. Chem. 2002; 277: 37783-37787Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), proteomic approaches under conditions of ERK activation (11Lewis T.S. Hunt J.B. Aveline L.D. Jonscher K.R. Louie D.F. Yeh J.M. Nahreini T.S. Resing K.A. Ahn N.G. Mol. Cell. 2000; 6: 1343-1354Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), phosphorylation of column fractions from cells (15Knebel A. Morrice N. Cohen P. EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (208) Google Scholar), and a solid phase phosphorylation assay (16Garcia J. Ye Y.B. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar).To identify additional novel substrates of ERK2, we employed a method developed by Shokat and coworkers (17Liu Y. Shah K. Yang F. Witucki L. Shokat K.M. Chem. Biol. 1998; 5: 91-101Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 18Shah K. Liu Y. Deirmengian C. Shokat K.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3565-3570Crossref PubMed Scopus (353) Google Scholar) to specifically label direct substrates of a particular protein kinase in a mixture of cellular proteins. Protein kinases contain similar ATP binding domains, including conserved residues that come into close contact with theN-6 position of ATP. Mutation of the ATP binding site at one or both of these conserved positions to a smaller amino acid residue can allow the mutant protein kinase, but not other cellular protein kinases, to utilize analogs of ATP that contain a bulky substituent on the N-6 position. When radiolabeled ATP analog is used in a kinase reaction with the mutant kinase and a mixture of cellular proteins, only direct substrates of the mutant kinase become radiolabeled. This method has been successfully used to identify direct substrates for the Src tyrosine kinase (19Shah K. Shokat K.M. Chem. Biol. 2002; 9: 35-47Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and for the MAPK JNK (20Habelhah H. Shah K. Huang L. Burlingame A.L. Shokat K.M. Ronai Z. J. Biol. Chem. 2001; 276: 18090-18095Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).We used an engineered mutant of ERK2 to search for new ERK substrates that physically associate with the MAPK. As validation of our method, our assay resulted in the labeling and identification of Rsk1, a known ERK2 substrate (7Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10952-10956Crossref PubMed Scopus (257) Google Scholar). We also identified two novel ERK2 substrates, the ubiquitin ligase EDD (E3 identified bydifferential display) and the nucleoporin Tpr (translocated promoter region). EDD is the human homolog of the Drosophila genehyperplastic discs and was originally identified as a gene up-regulated in response to progestin (21Callaghan M.J. Russell A.J. Woollatt E. Sutherland G.R. Sutherland R.L. Watts C.K.W. Oncogene. 1998; 17: 3479-3491Crossref PubMed Scopus (93) Google Scholar). EDD is believed to serve as an E3 ubiquitin ligase due to the presence of a HECT domain (homology to E6-AP carboxylterminus) in its carboxyl terminus that binds ubiquitin through a single cysteine residue (21Callaghan M.J. Russell A.J. Woollatt E. Sutherland G.R. Sutherland R.L. Watts C.K.W. Oncogene. 1998; 17: 3479-3491Crossref PubMed Scopus (93) Google Scholar). Honda et al. (22Honda Y. Tojo M. Matsuzaki K. Anan T. Matsumoto M. Ando M. Saya H. Nakao M. J. Biol. Chem. 2002; 277: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) have identified the topoisomerase II beta binding protein TopBP1 as a ubiquitination target of EDD. Tpr is a nucleoporin that is a component of the nuclear basket of the nuclear pore complex (23Byrd D.A. Sweet D.J. Pante N. Konstantinov K.N. Guan T.L. Saphire A.C.S. Mitchell P.J. Cooper C.S. Aebi U. Gerace L. J. Cell Biol. 1994; 127: 1515-1526Crossref PubMed Scopus (99) Google Scholar, 24Cordes V.C. Reidenbach S. Rackwitz H.R. Franke W.W. J. Cell Biol. 1997; 136: 515-529Crossref PubMed Scopus (190) Google Scholar). Functional studies suggest a role for Tpr in the nuclear export of proteins with a nuclear export signal (25Frosst P. Guan T. Subauste C. Hahn K. Gerace L. J. Cell Biol. 2002; 156: 617-630Crossref PubMed Scopus (130) Google Scholar).In this report we describe the engineering of ERK2 to allow it to utilize an analog of ATP. Based on sequence alignment of ERK2 with other protein kinases and the crystal structure of ERK2, we generated alanine and glycine mutations of ERK2 at isoleucine 82 and glutamine 103, singly or in combination, to generate an ERK2 with a larger ATP binding pocket. Mutation of these residues did not significantly inhibit ERK2 kinase activity in vitro or signaling in vivo. ERK2 mutants with a glutamine to glycine mutation at position 103 were able to efficiently utilize several ATP analogs to phosphorylate the ERK substrate Elk1 (26Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1104) Google Scholar) in vitro. Using ERK2 Q103G and [γ-32P]cyclopentyl-ATP, we were able to radiolabel EDD, Tpr, and Rsk1 as ERK2-associated substrates in COS-1 cells. We further validated these results by demonstrating that EDD was phosphorylated by ERK2 in vitro and in an MEK-dependent manner in cells in response to EGF. This work describes the generation of a molecular tool for the identification of many more targets of this important signaling cascade.RESULTSBased on the crystal structure of ERK2 bound to ATP (31Zhang J.D. Zhang F.M. Ebert D. Cobb M.H. Goldsmith E.J. Structure. 1995; 3: 299-307Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and sequence alignment of the ATP binding site of several protein kinases (18Shah K. Liu Y. Deirmengian C. Shokat K.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3565-3570Crossref PubMed Scopus (353) Google Scholar), we observed that both isoleucine 82 and glutamine 103 are at conserved positions that come into close contact with theN-6 position of adenine in ATP. We hypothesized that mutation of one or both of these residues to either alanine or glycine would generate a larger ATP binding pocket enabling the mutant kinase to accept and utilize ATP analogs with bulky side groups at theN-6 position. We therefore generated five ERK2 mutants that contained either single or double mutations of these residues to either alanine or glycine. The five mutants generated were: ERK2 I82A; ERK2 Q103A; ERK2 Q103G; ERK2 I82A/Q103A; and ERK2 I82A/Q103G (Fig.1A). All ERK2 constructs encoded an amino-terminal FLAG tag.We first examined whether mutation of these residues affected the ability of ERK2 to phosphorylate its substrates in vitro andin vivo. ERK2 or an ERK2 mutant was transfected into COS-1 cells, and the cells were stimulated with EGF. In vitrokinase assays were performed with immunoprecipitated ERK2 and the ERK2 mutants using myelin basic protein (MBP) and [γ-32P]ATP as substrates. ERK2 and the mutants had roughly equivalent levels of kinase activity (Fig. 1B), although alanine mutations at isoleucine 82 somewhat inhibited ERK2 kinase activity toward MBP. Similar experiments using serum stimulation or co-transfected mutationally activated MEK1 (MEK1 S218D/S222D), demonstrated that these mutants could be phosphorylated and activated by activated MEK1 (data not shown).To determine if the ERK2 mutants could signal to a physiological ERK2 substrate in vivo, GAL4-Elk1 luciferase assays were performed. This system utilizes a plasmid containing a luciferase gene with five GAL4 binding sites in the promoter and a plasmid encoding a fusion of the DNA-binding domain of GAL4 and the ERK-responsive transactivation domain of Elk1 (28Roberson M.S. Misrapress A. Laurance M.E. Stork P.J.S. Maurer R.A. Mol. Cell. Biol. 1995; 15: 3531-3539Crossref PubMed Google Scholar). MEK1 S218D/S222D was co-transfected to activate both endogenous and transfected ERK. Co-transfection of ERK2 or an ERK2 mutant stimulated luciferase expression in a dose-dependent manner (Fig. 1C). These data demonstrate that mutation of one or both residues in the ATP binding site of ERK2 did not substantially affect the localization, substrate recognition, or kinase activity of ERK2.We next needed to identify the optimal pairing between an ATP binding site mutant and an ATP analog. To do this, we screened each of our ERK mutants with a panel of seven ATP analogs. We first determined the ability of each analog to inhibit the capacity of ERK2 or an ERK2 mutant to phosphorylate MBP with [γ-32P]ATP. If an ATP analog was able to compete with [γ-32P]ATP for an ERK2 mutant, incorporation of radiolabeled phosphate into MBP would be inhibited. ERK2 or an ERK2 mutant was immunoprecipitated from transfected COS-1 cells stimulated for 10 min with EGF. The FLAG immunoprecipitates were aliquoted equally and used in a kinase reaction containing MBP, 10 μm ATP, and 10 μCi of [γ-32P]ATP, with or without 100 μm ATP analog. The reactions were resolved on a gel and transferred, and incorporation of 32P into MBP was quantified by Cerenkov counting. Comparison of reactions containing an ATP analog (Fig.2, lanes 2–8 in each panel) to control reactions without analog (Fig. 2, lane 1 in each panel) demonstrated that none of the analogs inhibited the ability of wild-type ERK2 to utilize ATP. By contrast, many of the ATP analogs had varied abilities to compete with [γ-32P]ATP for phosphate incorporation into MBP, suggesting that some of the ERK2 mutants were able to utilize or at least bind to the ATP analogs. Mutation of isoleucine 82 alone had little effect on the susceptibility of ERK2 to inhibition by an analog. Mutation of glutamine 103 to alanine increased the susceptibility of ERK2 to inhibition by an analog, whereas mutation of this residue to glycine had the greatest effect on the ability of an analog to inhibit 32P incorporation into MBP. The Q103G mutation (either by itself or in conjunction with the I82A mutation) demonstrated a significant reduction of 32P incorporation into MBP when kinase reactions were performed in the presence of analog. These results suggested that residue 103 of ERK2 was the critical residue in controlling the capacity of ERK2 to accept an ATP analog with a bulkyN-6 substituent. In particular, a substantial inhibition of32P incorporation into MBP by ERK2 Q103G was observed with addition of either N-6-benzyl-ATP (98.8% inhibition,lane 2, middle panel, right side),N-6-cyclopentyl-ATP (95.2% inhibition, lane 3,middle panel, right side),N-6-(3,3-dimethyl)butyl-ATP (98.4% inhibition, lane 6, middle panel, right side),N-6-(2-phenethyl)-ATP (97.8% inhibition, lane 7,middle panel, right side), andN-6-(1-methyl)butyl-ATP (94.1% inhibition, lane 8, middle panel, right side).Figure 2Analog inhibition assay. FLAG-ERK2 plasmids were transfected into COS-1 cells. The cells were serum-starved and stimulated with 10 ng/ml EGF for 10 min. FLAG-ERKs were immunoprecipitated, and each was aliquoted into eight sets of duplicate kinase reactions. Kinase reactions were performed with MBP, 10 μm ATP, 10 μCi/reaction [γ-32P]ATP, without (lane 1) or with (lanes 2–8) 100 μm ATP analog. ATP analogs were: N-6-benzyl ATP (lane 2), N-6-cyclopentyl-ATP (lane 3), N-6-(p-methyl)benzyl-ATP (lane 4), N-6-(1,3-dimethyl)butyl-ATP (lane 5),N-6-(3,3-dimethyl)butyl-ATP (lane 6),N-6-(2-phenethyl)-ATP (lane 7), andN-6-(1-methyl)butyl-ATP (lane 8). The reactions were performed at 30 °C for 10 min, run on a gel, and transferred to nitrocellulose. MBP bands were excised and quantitated by Cerenkov counting. The values were normalized to a reaction without ATP analog (lane 1), which was set at 100%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)There are two likely explanations for the inhibition of 32P incorporation into MBP by the addition of excess unlabeled ATP analog. First, the ATP analog could be utilized as an ATP source by the mutant ERK2 kinase to phosphorylate MBP, thus effectively competing with the [γ-32P]ATP for usage by the mutant ERK2. Alternatively, the kinase could bind the analog but not be able to utilize it, thus effectively blocking [γ-32P]ATP from the ATP binding site of the kinase. To differentiate between these two possibilities, we screened ERK2, ERK2-Q103G, and ERK2-I82A/Q103G for their ability to phosphorylate GST-Elk1 with either ATP or an ATP analog. The reactions were resolved on a gel and immunoblotted with an antibody that specifically recognizes phospho-S383 Elk1, the major site of phosphorylation by ERK2. Incubation of GST-Elk1 in the absence of ERK2 did not result in S383 phosphorylation (lane 1). All three ERK2 proteins were able to phosphorylate GST-Elk1 with normal ATP (Fig.3, lane 2 in all p-Elk1 panels). However, wild-type ERK2 could not use any of the ATP analogs to phosphorylate GST-Elk1 (Fig. 3, top panel,lanes 3–9). These data support the previous result that none of the analogs could inhibit wild-type ERK2 from using [γ-32P]ATP to phosphorylate MBP.Figure 3ERK2 phosphorylation of Elk1 with ATP analogs. FLAG-ERK2 plasmids were transfected into COS-1 cells, and the cells were serum-starved before stimulation with 10 ng/ml EGF for 10 min. FLAG-ERKs were immunoprecipitated, aliquoted, and used in kinase reactions with GST-Elk1 and either 100 mm unlabeled ATP (lane 2) or 100 mm unlabeled ATP analog (lanes 3–9). The reaction in lane 1 contained GST-Elk1 and ATP but no ERK. ATP analogs were:N-6-benzyl-ATP (lane 3),N-6-cyclopentyl-ATP (lane 4),N-6-(p-methyl)benzyl-ATP (lane 5),N-6-(1,3-dimethyl)butyl-ATP (lane 6),N-6-(3,3-dimethyl)butyl-ATP (lane 7),N-6-(2-phenethyl)-ATP (lane 8), andN-6-(1-methyl)butyl-ATP (lane 9). The reactions were incubated at 30 °C for 10 min, resolved on a gel, and transferred to nitrocellulose. The membranes were then immunoblotted with antibodies to phospho-S383 Elk1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Conversely, both ERK2-Q103G and ERK2-I82A/Q103G were able to effectively use several ATP analogs to phosphorylate GST-Elk1. Overall, ERK2-Q103G appeared to use the analogs more effectively than ERK2-I82A/Q103G, correlating with the inhibition assay in Fig. 2. ERK2-Q103G effectively used all of the ATP analogs tested with the exception of N-6-(p-methyl)benzyl-ATP. Although this analog appeared somewhat effective in the inhibition assay, it was the least efficient of the seven and was most likely an example of an analog that simply blocks the ERK2 ATP binding site. Based on the inhibition assay and the Elk1 phosphorylation assay, we decided to use ERK2-Q103G (henceforth designated ERK2-QG) along with cyclopentyl ATP (henceforth designated cpATP) (Fig. 3, middle panel, lane 4) in our attempts to identify novel ERK2 substrates.Detection of Direct Substrates of ERK2 with [γ-32P]cpATPTo identify relevant substrates of ERK2, one would like to introduce labeled ATP analog into the live cells. However, cells are impermeable to ATP, and addition of labeled ATP analog to digitonin-permeabilized cells results in hydrolysis of the analog ATP within 1 min (32Chaudhary A. Brugge J.S. Cooper J.A. Biochem. Biophys. Res. Commun. 2002; 294: 293-300Crossref PubMed Scopus (20) Google Scholar). Furthermore, the intracellular concentration of ATP is 3 mm (33Weber M.J. Edlin G. J. Biol. Chem. 1971; 246: 1828-1833Abstract Full Text PDF PubMed Google Scholar), making it difficult to achieve analog concentrations sufficient to compete with ATP for kinases. Therefore, it was necessary to apply this technique to cell lysates or fractions. We chose to express the ERK2 and ERK2-QG (mutant ERK2) in COS-1 cells and then immunoprecipitate ERKs under non-stringent conditions where we would expect many ERK-binding proteins to remain associated. The immunoprecipitates were then used in a kinase reaction with either [γ-32P]ATP or [γ-32P]cpATP. In addition to ERK2 and ERK2-QG, other co-immunoprecipitated kinases would be capable of utilizing [γ-32P]ATP to phosphorylate co-immunoprecipitated substrates. However, reactions with ERK2-QG and [γ-32P]cpATP should result in the phosphorylation of only those proteins that are direct ERK substrates. Therefore, we should detect fewer radiolabeled substrates in the reactions with [γ-32P]cpATP as compared with reactions with [γ-32P]ATP.ERK2 or ERK2-QG from unstimulated or EGF-stimulated cells were immunoprecipitated, and associated proteins were labeled in an in vitro kinase reaction using either [γ-32P]ATP or [γ-32P]cpATP. The reactions were resolved on SDS-PAGE, silver-stained, and subjected to autoradiography (Fig.4A). ERK2 and ERK2-QG proteins were expressed at similar levels and were activated efficiently after EGF stimulation (Fig. 4A, silver stain). Both ERK2 and ERK2-QG used [γ-32P]ATP very efficiently, generating similar patterns of phosphorylated substrates on SDS-PAGE (Fig.4A, autoradiogram). Phosphorylation of substrates in the absence of EGF stimulation was most likely due to the basal activity of ERK2. There were noticeable differences in the substrates recognized by ERK2 prior to and after EGF stimulation (Fig. 4A, autoradiogram; compare lane 1 with 2, lane 3 with 4, and lane 7 with 8). These differences are probably due to (i) differences in the association of substrates with ERK2 depending on the phosphorylation status of ERK2; (ii) dissociation of phosphorylated substrates from ERK2 after EGF stimulation in vivo; or (iii) prior phosphorylation of the substrate in vivo during the 10-min EGF stimulation.Figure 4Detection of ERK2 substrates. FLAG-ERK2 or FLAG-ERK2-QG plasmids were transfected into COS-1 cells. Cells were serum-starved for 4–5 h followed by stimulation with EGF (10 ng/ml) for 10 min. ERK2 and ERK2-QG were immunoprecipitated, and kinase reactions were performed with [γ-32P]ATP or [γ-32P]cpATP. A, kinase reactions were resolved on 10% SDS-PAGE followed by silver staining and autoradiography. ATP or cpATP labels on the top indicate kinase reactions carried out with [γ-32P]ATP or [γ-32P]cpATP, respectively. ERK2 and dually phosphorylated ERK2 (pERK2) are marked by arrows. B, ERK2 in vitro kinase reactions with [γ-32P]ATP or [γ-32P]cpATP analyzed on pH 4–7 linear (L) two-dimensional gels. C, ERK2-QG kinase reactions with [γ-32P]ATP or [γ-32P]cpATP analyzed on pH 4–7 L two-dimensional gels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When the kinase reactions were performed with [γ-32P]cpATP, there was a marked difference in the pattern of labeled substrates between reactions with ERK2 and ERK2-QG. Neither wild-type ERK2 nor its associated proteins could efficiently utilize [γ-32P]cpATP. The background phosphorylation with wild-type ERK2 that was detected with [γ-32P]cpATP was most likely due to trace contamination with [γ-32P]ATP (∼0.1%, data not shown) that occurred when generating [γ-32P]cpATP. At this level of electrophoretic resolution, few differences were detected in the substrates recognized by ERK2-QG in reactions with [γ-32P]ATP versus[γ-32P]cpATP (Fig. 4A, autoradiogram; compare lanes 3 and 4 with lanes 7 and8). However, when the resolution of the gel systems was increased by using two-dimensional gels (Fig. 4, B andC), the advantages of the analog approach became evident.ERK2 in vitro kinase reactions with [γ-32P]ATP or [γ-32P]cpATP resolved by two-dimensional gel electrophoresis are shown in Fig. 4B. These results further demonstrated that ERK2 and its associated proteins could not utilize [γ-32P]cpATP. In vitro kinase reactions using ERK2-QG were also analyzed on two-dimensional gels (Fig. 4C). Although the phosphorylation patterns between the [γ-32P]ATP and the [γ-32P]cpATP reactions were somewhat similar, we detected fewer substrates with [γ-32P]cpATP. The use of [γ-32P]cpATP decreased the overall level of phospho-proteins while enhancing ERK2-QG-specific phosphorylations. These results clearly demonstrate the advantage of using [γ-32P]cpATP for specific detection of the direct substrates of ERK2.Identification of Novel ERK2 SubstratesAfter optimizing the conditions for the specific detection of direct substrates of ERK2-QG with [γ-32P]cpATP, we scaled up the procedure to detect co-immunoprecipitating substrates by mass spectrometry. Proteins co-immunoprecipitating with ERK2-QG were eluted along with ERK2 from M2 beads with FLAG peptide. Kinase reactions were then performed with [γ-32P]cpATP, and the reactions were resolved on an 8% SDS-PAGE (Fig. 5A). Two radiolabeled silver-stained bands, one at ∼80 kDa and the other at ∼250 kDa, were excised from the lane with a non-EGF-stimulated (serum-free) sample (Fig. 5A, lane 2) and were sequenced by mass spectrometry. Eleven peptides (16.3% coverage) corresponding to the ERK2 substrate Rsk1 (34Moller D.E. Xia C.H. Tang W. Zhu A.X. Jakubowski M. Am. J. Physiol. 1994; 266: C351-C359Crossref PubMed Google Scholar) were detected in the 80-kDa band. The isolation of a known ERK2 substrate validated our methodology for detecting ERK2 substrates. Mass spectrometry analysis of the 250-kDa band revealed 14 peptides (7% total coverage) corresponding to the E3 ubiquitin ligase EDD (E3 identified by differential display). EDD is the human homolog of the Drosophila gene hyperplastic discsand was originally identified as a gene up-regulated in response to progestin (21Callaghan M.J. Russell A.J. Woollatt E. Sutherland G.R. Sutherland R.L. Watts C.K.W. Oncogene. 1998; 17: 3479-3491Crossref PubMed Scopus (93) Google Scholar). EDD is a 300-kDa protein and contains a region that has extensive homology to HECT domain ubiquitin ligases (21Callaghan M.J. Russell A.J. Woollatt E. Sutherland G.R. Sutherland R.L. Watts C.K.W. Oncogene. 1998; 17: 3479-3491Crossref PubMed Scopus (93) Google Scholar).Figure 5Identification of Rsk1, EDD, and Tpr. pCDNA3.1 or ERK2-QG plasmids were transfected into COS-1 cells (four 150-mm dishes/lane, 5–6 × 106 cells/dish at the time of transfection). Cells were serum-starved, stimulated with EGF, and lysed with hypotonic lysis buffer followed by immunoprecipitation with anti-FLAG-M2-agarose beads. Proteins were eluted with FLAG peptide and kinase reactions carried out with [γ-32P]cpATP. A, proteins were resolved on a 8% SDS-PAGE visualized by silver staining and subjected to autoradiography. Bands corresponding to 80-kDa Rsk1 and 250-kDa EDD are indicated by arrows. B, proteins were resolved on 12% SDS-PAGE, silver-stained, an" @default.
• W2090972634 created "2016-06-24" @default.
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• W2090972634 creator A5015873058 @default.
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• W2090972634 date "2003-04-01" @default.
• W2090972634 modified "2023-10-14" @default.
• W2090972634 title "Identification of Novel ERK2 Substrates through Use of an Engineered Kinase and ATP Analogs" @default.
• W2090972634 cites W1528493806 @default.
• W2090972634 cites W1566737177 @default.
• W2090972634 cites W1693778124 @default.
• W2090972634 cites W1927958267 @default.
• W2090972634 cites W1963805331 @default.
• W2090972634 cites W1970320023 @default.
• W2090972634 cites W1970564598 @default.
• W2090972634 cites W1976662277 @default.
• W2090972634 cites W1991760443 @default.
• W2090972634 cites W1993444261 @default.
• W2090972634 cites W2000082154 @default.
• W2090972634 cites W2000131007 @default.
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• W2090972634 cites W2021021216 @default.
• W2090972634 cites W2022138447 @default.
• W2090972634 cites W2031108838 @default.
• W2090972634 cites W2032225851 @default.
• W2090972634 cites W2032666422 @default.
• W2090972634 cites W2038447145 @default.
• W2090972634 cites W2046813978 @default.
• W2090972634 cites W2059624730 @default.
• W2090972634 cites W2065549444 @default.
• W2090972634 cites W2070023258 @default.
• W2090972634 cites W2091265469 @default.
• W2090972634 cites W2097569409 @default.
• W2090972634 cites W2104237107 @default.
• W2090972634 cites W2107180907 @default.
• W2090972634 cites W2109458412 @default.
• W2090972634 cites W2111563081 @default.
• W2090972634 cites W2112184006 @default.
• W2090972634 cites W2117669008 @default.
• W2090972634 cites W2136133297 @default.
• W2090972634 cites W2139043430 @default.
• W2090972634 cites W2154766134 @default.
• W2090972634 cites W2162322742 @default.
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