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• W2079156404 abstract "We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (withno lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of ∼230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases. We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (withno lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of ∼230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases. cAMP-dependent kinase kinase suppressor of Ras polymerase chain reaction mitogen-activated protein extracellular signal-regulated protein kinase ERK kinase kilobase(s) myelin basic protein glutathioneS-transferase hemagglutinin The protein kinase superfamily contains over a thousand members that share a catalytic core of approximately 300 residues organized in two domains (1.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3812) Google Scholar, 2.Plowman G.D. Sudarsanam S. Bingham J. Whyte D. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13603-13610Crossref PubMed Scopus (232) Google Scholar, 3.Taylor S.S. Radzio-Andzelm E. Structure. 1994; 2: 345-355Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Conserved structural motifs within the core sequence maintain the basic fold of the catalytic domain, and fewer than 10 highly conserved residues create the functional elements of the active site (4.Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1459) Google Scholar, 5.Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-429Crossref PubMed Scopus (812) Google Scholar). Prior to the solution of the three-dimensional structure of cAMP-dependent protein kinase (PKA)1 by Knighton et al. (4.Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1459) Google Scholar), several of the residues essential for the integrity of the structure and the active site were identified primarily by a combination of multiple sequence alignment (1.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3812) Google Scholar, 6.Barker W.C. Dayhoff M.O. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2836-2839Crossref PubMed Scopus (126) Google Scholar), chemical modifications (7.Taylor S.S. Kerlavage A.R. Zoller M.J. Methods Enzymol. 1983; 99: 140-153Crossref PubMed Scopus (5) Google Scholar), and alanine scanning mutagenesis (8.Gibbs C.S. Zoller M.J. J. Biol. Chem. 1991; 266: 8923-8931Abstract Full Text PDF PubMed Google Scholar). Among these a lysine residue near the N terminus of the kinase in protein kinase subdomain II (Lys72 in PKA); this residue has frequently been mutated to eliminate the catalytic activity of protein kinases (9.Zoller M.J. Nelson N.C. Taylor S.S. J. Biol. Chem. 1981; 256: 10837-10842Abstract Full Text PDF PubMed Google Scholar). This lysine functions to anchor and orient ATP through interactions with the α and β phosphoryl groups (4.Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1459) Google Scholar, 5.Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-429Crossref PubMed Scopus (812) Google Scholar, 10.Robinson M.J. Harkins P.C. Zhang J. Baer R. Haycock J.W. Cobb M.H. Goldsmith E.J. Biochemistry. 1995; 35: 5641-5646Crossref Scopus (129) Google Scholar). Until recently all members of the protein kinase family were found to contain a lysine following a short string of hydrophobic residues in this conserved position. Kinase suppressor of Ras (KSR) contains arginine in place of this lysine but has not yet been shown to catalyze phosphorylation of protein substrates (11.Therrien M. Michaud N.R. Rubin G.M. Morrison D.K. Genes Dev. 1996; 10: 2684-2695Crossref PubMed Scopus (210) Google Scholar, 12.Therrien M. Chang H.C. Solomon N.M. Karim F.D. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 879-888Abstract Full Text PDF PubMed Scopus (340) Google Scholar). Mutation of this arginine in KSR does impair its function in reconstitution assays, suggesting that it plays a significant role in KSR function (11.Therrien M. Michaud N.R. Rubin G.M. Morrison D.K. Genes Dev. 1996; 10: 2684-2695Crossref PubMed Scopus (210) Google Scholar). Structural analysis of the mitogen-activated protein (MAP) kinase ERK2 shows that substitution of the conserved lysine with arginine causes the phosphoryl groups of ATP to be rotated away from the position necessary for phosphoryl transfer (10.Robinson M.J. Harkins P.C. Zhang J. Baer R. Haycock J.W. Cobb M.H. Goldsmith E.J. Biochemistry. 1995; 35: 5641-5646Crossref Scopus (129) Google Scholar). Thus, the function of arginine in this conserved position of KSR is uncertain. We have identified a novel protein kinase WNK1 (withno lysine (K)), which contains cysteine in place of lysine at the usual conserved location but has kinase activity as deduced from its ability to autophosphorylate and to phosphorylate an exogenous substrate in vitro. We have investigated the basis for its catalytic activity using a structural model, mutagenesis, and protein expression. WNK1 was isolated in a nested PCR cloning strategy aimed to identify novel members of the MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK) family. MAP kinases are a family of protein kinases that have been utilized to varying degrees to regulate or modulate almost all signal transduction pathways in cells (13.Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 14.English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). These enzymes themselves are regulated by cascades of at least two upstream protein kinases, a MEK and a MEK kinase. Members of the MEK (or MKK) family display considerable selectivity for their particular MAP kinase targets, thereby contributing to signaling specificity (13.Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). They activate MAP kinases by dual phosphorylation on a tyrosine and a threonine residue, and each MEK recognizes only a small subset of possible MAP kinase substrates. After the purification and cloning of the first MEK family member, MEK1 (15.Seger R. Seger D. Lozeman F.J. Ahn N.G. Graves L.M. Campbell J.S. Ericsson L. Harrylock M. Jensen A.M. Krebs E.G. J. Biol. Chem. 1992; 267: 25628-25631Abstract Full Text PDF PubMed Google Scholar, 16.Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8205-8209Crossref PubMed Scopus (152) Google Scholar), others (MKK2, MKK3, MKK4, MEK5, MKK6, and MKK7) were discovered through low stringency or PCR screens rather than by purification (17.Wu J. Harrison J.K. Dent P. Lynch K.R. Weber M.J. Sturgill T.W. Mol. Cell. Biol. 1993; 13: 4539-4548Crossref PubMed Scopus (124) Google Scholar, 18.Zheng C.-F. Guan K. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar, 19.Dérijard 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, 20.Lin A. Minden A. Martinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (711) Google Scholar, 21.Sánchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar, 22.English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 23.Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 24.Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 25.Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Not only did this approach streamline the definition of the components of known MAP kinase cascades, but it also uncovered new pathways. We continued to examine clones derived from the screen that led us to isolate cDNAs encoding MEK5 (22.English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). As described in this report, one of these clones encoded the unusual protein kinase WNK1. First strand cDNA isolated from nerve growth factor stimulated PC12 cells was used as the template in PCR reactions utilizing nested degenerate primers derived from MEK sequences (22.English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). One PCR product (product 15) was used to screen a rat forebrain cDNA library (kindly provided by Jim Boulter) at low stringency, and a weakly hybridizing clone of approximately 900 base pairs (PC12 clone 2-3) was isolated that had a short region of identity to the PCR product. This PC12 clone 2-3 was then used to rescreen the rat forebrain cDNA library at high stringency, and a group of strongly hybridizing clones were isolated. Partial sequences that encoded the N terminus and the kinase domain of WNK1 were assembled from two of these clones. A 0.5-kb WNK1 3′ probe was labeled with [α-32P]dCTP by random-priming (Amersham Pharmacia Biotech) and used to screen another rat brain cDNA library that contains longer inserts (also kindly provided by Jim Boulter). One of the clones isolated contained the complete 3′ WNK1 sequence. The full-length WNK1 cDNA was assembled from these clones. A rat adult multi-tissue Northern blot (CLONTECH) was hybridized with a random-primed (Amersham Pharmacia Biotech) 0.5-kb WNK1 5′ probe according to the manufacturer's suggestions. The same blot was stripped and reprobed with a 2.8-kb WNK1 3′ probe. This Northern blot was stripped again and hybridized with a β-actin probe to confirm the presence of mRNA in each lane. A pSK-WNK1 full-length construct was created from three overlapping cDNA clones and was used as the template for the subsequent subcloning. A 1.6-kb WNK1 fragment encoding residues 1–555 was amplified by PCR with anEcoRI site incorporated at the 5′ end and aHindIII site incorporated at the 3′ end, and this fragment was digested with EcoRI-HindIII and ligated into pGEX-KG or pCMV5-Myc vectors that had been digested withEcoRI and HindIII to create pGEX-KG-WNK1 (1–555) and pCMV5-Myc-WNK1 (1–555). To make a pGEX-KG-WNK1 full-length construct, pGEX-KG-WNK1 (1–555) was digested with HindIII, filled-in with Klenow, and then digested with SacII; pSK-WNK1 was digested with SpeI, filled-in with Klenow, and then digested with SacII. The 6.5-kb insert fragment from pSK-WNK1 was gel purified and ligated into the 5.1-kb vector fragment from pGEX-KG-WNK1 (1–555). To generate a pCMV5-Myc-WNK1 full-length construct, pSK-WNK1 was digested with SacII-SpeI and the 6.5-kb insert fragment was gel purified and ligated into the 4.8-kb SacII-XbaI digested pCMV5-Myc-WNK1 (1–555) vector backbone. All constructs and mutants were sequenced to confirm that the sequences were correct. pCEP4HA-ERK2, pSRα-HA-JNK1, pCEP4HA-p38α, and pCEP4HA-ERK5 were as described (26.Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar, 27.English J.M. Pearson G. Hockenberry T. Shivakumar L. White M.A. Cobb M.H. J. Biol. Chem. 1999; 274: 31588-31592Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Site-directed mutagenesis was carried out using the Quikchange kit (Stratagene) according to the manufacturer's recommendation. The WNK1 mutants used in this study include K233M, K256M, K259M, C250A, C250K, D368A, and the double mutant S378D/S382D. GST-WNK1 (full-length and 1–555) proteins were expressed in and purified from Escherichia coli strain BL21DE3 using the standard protocol (28.Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1640) Google Scholar). The induction conditions were: 40 μm (for 1–555) or 400 μm (for full-length) isopropyl-β-d-thiogalactopyranoside at 30 °C for 5 h. The protein concentration was estimated by comparing to serial dilutions of bovine serum albumin on the same gel stained with Coomassie Blue. Myelin basic protein (MBP) was purchased from Calbiochem. GST-c-Jun and GST-ATF2Δ were as described (29.English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The rabbit polyclonal anti-WNK1 antisera Q255 and Q256 were raised using standard methods against a WNK1 N-terminal peptide: TSKDRPVSQPSLVGSKE (30.Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (282) Google Scholar). Anti-ERK1 Y691 was as described (30.Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (282) Google Scholar). Monoclonal anti-HA antibody (12CA5) was obtained from Berkeley Antibody Company. Monoclonal anti-Myc antibody (9E10) was obtained from the Cell Culture Center. For immunoblotting, cell or tissue lysates were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose paper. Blots were then developed using enhanced chemiluminescence. Proteins were immunoprecipitated from 0.2 ml of cell lysates with 2 μl of antibody and 40 μl of protein A-Sepharose beads. Precipitates were washed three times with 20 mm Tris-HCl (pH 7.4), 1m NaCl and once with 10 mm Hepes (pH 8.0) and 10 mm MgCl2. HEK 293 cells were maintained, transfected, and harvested as described (31.Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). For cell fractionation, cells from each 60-mm dish were washed once with 1× phosphate-buffered saline, scraped, and resuspended in 200 μl of buffer A (10 mmHepes, pH 7.6, 1.5 mm MgCl2, 10 mmNaCl, 1 mm EDTA, 1 mm EGTA, and protease inhibitors 1 mm dithiothreitol, 1 μg/ml leupeptin, 10 mm benzamidine, and 1 mm phenylmethylsulfonyl fluoride). Cells were lysed with a Dounce apparatus, and the nuclei were collected by sedimentation at 4,000 rpm for 5 min in a microcentrifuge at 4 °C. A particulate fraction was collected by sedimenting the supernatant at 55,000 rpm for 30 min in a TL-100 ultracentrifuge at 4 °C. The nuclear pellet was washed with 200 μl of buffer C (20 mm Hepes, pH 7.6, 2.5% glycerol, 0.42m NaCl, 1.5 mm MgCl2, 1 mm EDTA, and 1 mm EGTA) at 4 °C for 30 min. Both the washed nuclear pellet and the particulate fraction were resuspended in 200 μl of lysis buffer (10 mm Tris-HCl, pH 7.6, 100 mm NaCl, 1% SDS, 1 mm EDTA, 1 mm EGTA, and protease inhibitors 1 mmdithiothreitol, 1 μg/ml leupeptin, 10 mm benzamidine, and 1 mm phenylmethylsulfonyl fluoride). Immunofluorescence was essentially as described (32.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskelet. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar). HEK 293 cells were grown on coverslips coated with collagen, fixed with formaldehyde, and treated with anti-Myc antibody. Cells were washed and treated with goat anti-mouse fluorescein secondary antibody followed by diamidinophenylindole staining. Coverslips were mounted onto slides, and cells were examined under a fluorescence microscope. In vitro kinase assays and phosphoamino acid analysis were performed as described (33.Robbins D.J. Cobb M.H. Mol. Biol. Cell. 1992; 3: 299-308Crossref PubMed Scopus (55) Google Scholar). A WNK1 model was made using PKA as a template. An alignment of the kinase domains of WNK1 and PKA was generated. InsightII (Molecular Biosystems) was used to change residues 50–100 of the structure of PKA to the corresponding residues found in WNK1 and create a ribbon diagram. In an attempt to isolate novel mammalian MEKs, nested degenerate PCR primers designed based on sequences conserved among MEK family members were used to amplify products from first strand cDNA isolated from PC12 cells (22.English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). One product of 150 base pairs was used to probe a rat forebrain cDNA library. A clone isolated from a low stringency screen was used to screen two rat brain cDNA libraries and several positive clones encoding a novel protein kinase named WNK1 were isolated. The full-length WNK1 cDNA containing 7.2 kb was assembled from three overlapping clones. The sequence surrounding the ATG start codon matched the Kozak consensus sequence for translation initiation, and stop codons were present upstream in all three reading frames. Although there was no poly(A) track found downstream of the stop codon in the available WNK1 sequence, there is a polyadenylation signal sequence (AATAAA) at the end of the cDNA clone. The open reading frame encoded by the WNK1 cDNA contains 2126 amino acids with a serine/threonine protein kinase domain in the N-terminal 490 residues (Fig. 1, A and B). A partial clone encoding a closely related kinase (WNK2) was also isolated. Homologs of WNK1 exist in Caenorhabditis elegans,Phycomyces, Arabidopsis, and Oryza as well as other mammals (Fig. 1 C). A human expressed sequence tag containing an open reading frame lacking the kinase domain is almost identical to the WNK1 C-terminal sequence, and two human open reading frames containing partial kinase domains show strong similarity to the WNK1 catalytic domain, indicating that they may encode parts of human WNK family members. WNK1 and its homologs are characterized by the absence of the catalytic lysine found in subdomain II of almost all known protein kinases (1.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3812) Google Scholar). In WNK1, a cysteine lies in the position of the usual lysine. WNK1 shares several sequence features with the MEK family, including the length of the activation loop (between subdomain VII and VIII), the position of potential activating phosphorylation sites (SFAKS; these sites are also conserved in the IκB kinases IKK1 and 2 (34.Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 35.DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar)), conservation in the region of protein substrate binding, and a pattern of conserved hydrophobic residues near the C terminus. However, the kinase domain of WNK1 does not clearly fall into any kinase subgroup but shows the greatest similarity to MEKK-like kinases (∼30% identity) and, to a lesser extent, to Ste20p-like kinases. The N-terminal and C-terminal noncatalytic domains of WNK1 are not similar to any other proteins in the data base. To examine the expression of WNK1 in various rat tissues, a rat multi-tissue Northern blot was hybridized with a 0.5-kb WNK1 5′ probe. Two transcripts of 11 and 9.5 kb were detected in several tissues including lung, liver, and spleen (Fig.2). The existence of two different species of mRNA suggests that an alternative splicing event might be involved in WNK1 mRNA processing. Similar results were obtained using a 3′ 2.8-kb probe (data not shown). A polyclonal antibody raised against a WNK1 N-terminal peptide recognized a protein of ∼230 kDa corresponding to the predicted size of WNK1 in rat brain and several mammalian cell lines including 293, COS-1, and INS-1 cells (Fig.3), suggesting that it is widely expressed.Figure 3Detection of endogenous WNK1 protein in cell lysates. Lysates of rat brain and several cultured cell lines were separated on a 7.5% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and blotted with anti-WNK1 antibody Q256 as described under “Materials and Methods.” GST-WNK1 (1–555) was loaded as the positive control. The arrowindicates the position of the endogenous protein on the blot. Molecular weight markers are shown. − Peptide, the antibody was not exposed to the antigenic peptide; + Peptide, the antibody was preincubated with 50 μg/ml of antigenic peptide overnight to demonstrate antibody specificity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether WNK1 is soluble or associated with nuclear or particulate fractions, HEK293 cells were fractionated and proteins in each fraction were separated by SDS-polyacrylamide gel electrophoresis and blotted with an anti-WNK1 antibody. The majority of endogenous WNK1 protein was found in the particulate fraction, suggesting that WNK1 is associated with either membranes or the cytoskeleton (Fig.4 A). To examine its cellular localization of further, a Myc-tagged WNK1 construct was transfected into 293 cells, and the expressed protein was visualized by immunofluorescence with an anti-Myc antibody. Most staining was observed outside of the nucleus, suggesting that WNK1 is cytoplasmic protein not restricted to the plasma membrane (Fig.4 B). To examine the kinase activity of WNK1, Myc-tagged WNK1 was transfected in 293 cells and then immunoprecipitated from lysed cells with an anti-Myc antibody. The immunoprecipitate showed kinase activity toward MBP as well as itself (Fig. 5 A). Although activity toward MBP was detected, autophosphorylation was more consistent than MBP phosphorylation in the immune complex kinase assay because of the high background contributed by other protein kinases. Wild type GST-WNK1 expressed in E. coli phosphorylated both itself and MBP. Mutation of the putative Mg2+ binding residue aspartate 368 to alanine generated a WNK1 protein with no detectable kinase activity. Phosphoamino acid analysis revealed that phosphorylation occurs mainly on serine residues, indicating that WNK1 is a serine/threonine kinase (Fig. 5 B). To test the endogenous activity of WNK1, the endogenous protein was immunoprecipitated with anti-WNK1 antibodies and assayed by its ability to autophosphorylate. Immunoblotting confirmed that WNK1 was immunoprecipitated (not shown). The ability of WNK1 to autophosphorylate was apparent from the incorporation of labeled phosphate into a band of approximately 230 kDa. In contrast, neither preimmune serum nor the unrelated anti-ERK1 antibody immunoprecipitated an autophosphorylating band of this size (Fig. 5 C). To identify regulators of WNK1, we tested a number of agents and stimuli to determine whether they could increase WNK1 activity in 293 cells. Endogenous WNK1 was immunoprecipitated following cell treatment, and autophosphorylation was assayed. Among the stimuli tested, 0.5m NaCl (Fig. 5 D) and less so 0.5 msorbitol (not shown) caused a reproducible increase in WNK1 autophosphorylation, suggesting that WNK1 may be involved in osmosensing pathways. No effects were detected with epidermal growth factor, the microtubule disrupting agent nocodazole, anisomycin, lysophosphatidic acid (Fig. 5 D), serum, heat shock, phorbol ester, H2O2, or okadaic acid (not shown). Because WNK1 was isolated as a possible MEK homolog and has modest similarity to the MEKK-like and Ste20p-like kinases within its catalytic domain, we examined the potential regulation of MAP kinase pathways by WNK1. The WNK1 kinase domain (residues 1–555) was expressed in mammalian cells and the activities of the known MAP kinases, HA-tagged forms of ERK2, ERK5, JNK1, or p38, were measured. Myc-tagged WNK1 (1–555) constructs, either wild type or kinase dead (D368A), were used in the majority of experiments because expression was to a much greater extent than for full-length WNK1. HA-tagged proteins were then immunoprecipitated with an anti-HA antibody and assayed using MBP, c-Jun, or ATF-2 as substrates. No obvious changes in activity of any of these kinases were observed with overexpression of either wild type or kinase-dead WNK1 (1–555) (Fig.6) or with full-length WNK1 (not shown).In vitro, GST-WNK1 displayed neither MEK nor MEKK activity. It failed to phosphorylate recombinant ERK1, ERK2, ERK5, JNK1, or p38. WNK1 also did not phosphorylate MEK1, MKK2, MKK3, MKK4, MEK5, MKK6, or MKK7; nor did it phosphorylate IκB, ribosomal protein S6, or fragments of MEKK1 (not shown). In addition, the two possible phosphorylation sites of WNK1 (SFAKS) that lie in the same relative positions as the activating sites of phosphorylation in the MEK family were mutated to aspartic acid (S378D/S382D). Although comparable mutations increase the activity of some MEK family members (e.g. MEK1), there was no detectable effect of these mutations on WNK1 activity (not shown). In summary, these results suggest that WNK1 does not directly regulate the MAP kinase pathways tested. Several residues involved in catalysis of phosphoryl transfer are highly conserved in all protein kinases, notably the catalytic lysine residue present in kinase subdomain II, which binds to ATP. Surprisingly, this apparently invariant lysine residue is replaced by a cysteine (Cys250) in WNK1. The lack of lysine at this position was confirmed in multiple independent clones of rat WNK1. More striking, this unusual difference is conserved across diverse species, suggesting functional relevance. Two possible explanations for this deviation in WNK1 were either that the catalytic lysine was located at a different position in the structure or that the WNK1 catalytic mechanism was distinct from other protein kinases. To distinguish between these possibilities, we first created a structural model of WNK1 based on the coordinates of PKA (Fig.7). Based on this model, several candidate lysine residues that might potentially function in ATP binding, Lys233, Lys256, and Lys259, were mutated to methionine. In addition, Cys250 was mutated to either alanine or lysine to determine whether it was required for catalysis. These mutants were expressed in bacteria as GST fusion proteins and assayed in vitro using MBP as substrate (Fig. 8). WNK1 C250K had greatly reduced kinase activity, suggesting that Cys250 may play some catalytic role. However, WNK1 C250A had the same activity as wild type protein. This result demonstrated that Cys250 is not required for WNK1 activity, but a lysine that has a larger and positively charged side chain may interfere with the folding or activity of the catalytic site. WNK1 K256M and K259M exhibited kinase activity similar to the wild type protein, indicating that Lys256 and Lys259 are not required for catalytic activity. In contrast, WNK1 K233M had no detectable kinase activity, indicating that Lys233, like Asp368, plays a critical role in WNK1 activity. This lysine residue, Lys233, is conserved in position in all the WNK homologs, consistent with its importance for catalytic activity. Interestingly, Lys233 replaces a glycine residue in the glycine string of subdomain I that comprises the phosphate anchor ribbon as shown in Fig.7. Mutation of this residue in PKA has relatively little impact on its kinetic properties (36.Hemmer W. McGlone M. Tsigelny I. Taylor S.S. J. Biol. Chem. 1997; 272: 16946-16954Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), consistent with the idea that glycine, although it is usually present, is not required at this position for kinase activity. From this location in the primary sequence, the side chain of Lys233 can apparently fill the position normally occupied by the lysine residue present in other protein kinases that corresponds to Cys250 in WNK1 (Fig. 7).Figure 8Analysis of WNK1 mutants. A,top panel, various GST-WNK1 mutant proteins were used in kinase assays with MBP as the substrate and MBP phosphorylation is shown. Bottom panel, data from the top panel are plotted as the percentage of maximal activity with wild type protein activity represented as 100%. WT, wild type. One of four experiments is shown. B, partial sequence alignment between PKA and WNK1. WNK1 residues that were mutated areunderlined. Identical residues between the two enzymes are shown in between the two sequences. Roman numerals indicate the kinase subdomains.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In summary, we have isolated and characterized a protein kinase represented in diverse organisms with a previously unknown placement of a key catalytic residue. Mutagenesis studies indicates that a lysine, Lys233, in the glycine ribbon is required for activity of WNK1. All other residues conserved among the protein kinases are in the expected locations in WNK1. The impaired activity of a mutant in which the putative magnesium binding residue, aspartate 368, was replaced with alanine supports the conclusion that the active site is otherwise typical of protein kinases. Modeling experiments suggest that the active site structure can be preserved despite this significant sequence deviation. Interestingly, a key lysine present in GTPases and other nucleotidases often lies quite near the glycine string (37.Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Perhaps the altered organization of the catalytic residues in WNK1-like kinases reflects a yet-to-be-discovered regulatory or functional adaptation better served by this particular sequence arrangement. We thank Jim Boulter (UCLA) for cDNA libraries, Tandi Collisson and Colleen Vanderbilt for technical assistance in the early stages of this work, Shuichan Xu (Salk Institute) for rat tissue lysates, Tara Beers-Gibson for comments about the manuscript, and Lavette James for administrative assistance." @default.
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• W2079156404 title "WNK1, a Novel Mammalian Serine/Threonine Protein Kinase Lacking the Catalytic Lysine in Subdomain II" @default.
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• W2079156404 cites W1556401308 @default.
• W2079156404 cites W1566737177 @default.
• W2079156404 cites W1572416479 @default.
• W2079156404 cites W1656231611 @default.
• W2079156404 cites W1964100540 @default.
• W2079156404 cites W1968590447 @default.
• W2079156404 cites W1969759973 @default.
• W2079156404 cites W1979520552 @default.
• W2079156404 cites W1996223704 @default.
• W2079156404 cites W1997966434 @default.
• W2079156404 cites W1998487776 @default.
• W2079156404 cites W2008857234 @default.
• W2079156404 cites W2011184275 @default.
• W2079156404 cites W2012599652 @default.
• W2079156404 cites W2020257412 @default.
• W2079156404 cites W2024940343 @default.
• W2079156404 cites W2027624022 @default.
• W2079156404 cites W2030284600 @default.
• W2079156404 cites W2035980889 @default.
• W2079156404 cites W2036985660 @default.
• W2079156404 cites W2067405360 @default.
• W2079156404 cites W2071242185 @default.
• W2079156404 cites W2072061824 @default.
• W2079156404 cites W2076059393 @default.
• W2079156404 cites W2077339073 @default.
• W2079156404 cites W2079557731 @default.
• W2079156404 cites W2088894802 @default.
• W2079156404 cites W2091455173 @default.
• W2079156404 cites W2092961902 @default.
• W2079156404 cites W2134468110 @default.
• W2079156404 cites W2157531111 @default.
• W2079156404 cites W2159979956 @default.
• W2079156404 cites W2312445582 @default.
• W2079156404 cites W4230279981 @default.
• W2079156404 cites W953142693 @default.
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