18 results on '"Steve Stippec"'
Search Results
2. Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding
- Author
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Svetlana Earnest, Aroon S. Karra, Elhadji M. Dioum, Elma Zaganjor, Kristina Lorenz, Yan Li, Adrian Turjanski, Elias Daniel Lopez, Steve Stippec, Andrea C. McReynolds, Kathleen McGlynn, and Melanie H. Cobb
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0301 basic medicine ,endocrine system diseases ,Otras Ciencias Biológicas ,Oligonucleotides ,Biology ,environment and public health ,Biochemistry ,Protein Structure, Secondary ,Article ,ERK2 ,MAP2K7 ,Ciencias Biológicas ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Protein phosphorylation ,phosphrylation ,Phosphorylation ,Threonine ,Kinase activity ,Protein kinase A ,Mitogen-Activated Protein Kinase 1 ,Regulation of gene expression ,Kinase ,DNA ,MAPK ,Rats ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Mutation ,biological phenomena, cell phenomena, and immunity ,CIENCIAS NATURALES Y EXACTAS ,hormones, hormone substitutes, and hormone antagonists ,030217 neurology & neurosurgery ,Protein Binding - Abstract
The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site. Fil: McReynolds, Andrea C.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Karra, Aroon S.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Li, Yan. The University of Texas Southwestern Medical Center; Estados Unidos. National Institute of Neurological Disorders and Stroke; Estados Unidos Fil: Lopez, Elias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Dioum, Elhadji. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Lorenz, Kristina. Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V; Alemania Fil: Zaganjor, Elma. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Stippec, Steve. The University of Texas Southwestern Medical Center; Estados Unidos Fil: McGlynn, Kathleen. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Earnest, Svetlana. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Cobb, Melanie H.. The University of Texas Southwestern Medical Center; Estados Unidos
- Published
- 2016
3. Role of WNK1/OSR1 in maintaining endothelial tight junction integrity
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Ankita Bachhawat Jaykumar, Svetlana Earnest, Melanie H. Cobb, Sakina Plumber, and Steve Stippec
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Materials science ,Tight junction ,Genetics ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2020
4. The function of WNK1/OSR1 in cell migration and angiogenesis
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Kathy McGlynn, Ankita Bachhawat Jaykumar, Svetlana Earnest, Gray W. Pearson, Steve Stippec, Melanie H. Cobb, and Sachith Gallolu Kankanamalage
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Chemistry ,Angiogenesis ,Genetics ,WNK1 ,Molecular Biology ,Biochemistry ,Function (biology) ,Biotechnology ,Cell biology - Published
- 2018
5. Assaying Protein Kinase Activity with Radiolabeled ATP
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Steve Stippec, Melanie H. Cobb, and Aroon S. Karra
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0301 basic medicine ,Cell signaling ,Immunoprecipitation ,General Chemical Engineering ,Allosteric regulation ,Biology ,medicine.disease_cause ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Adenosine Triphosphate ,medicine ,Humans ,Phosphorylation ,Protein kinase A ,Mutation ,General Immunology and Microbiology ,Kinase ,General Neuroscience ,Assay ,Cell biology ,030104 developmental biology ,Radiopharmaceuticals ,Signal transduction ,Phosphorus Radioisotopes ,Protein Kinases ,Signal Transduction - Abstract
Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.
- Published
- 2017
6. Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals
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Andrés Lorente-Rodríguez, Samarpita Sengupta, Hamid Mirzaei, Melanie H. Cobb, Xiaofeng Guo, Svetlana Earnest, Steve Stippec, and David C. Trudgian
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Sodium-Potassium-Chloride Symporters ,Immunoblotting ,Oligonucleotides ,Mechanistic Target of Rapamycin Complex 2 ,Protein Serine-Threonine Kinases ,Biology ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Osmotic Pressure ,Humans ,Immunoprecipitation ,Sorbitol ,ASK1 ,Phosphorylation ,RNA, Small Interfering ,Protein kinase A ,Protein kinase B ,Adaptor Proteins, Signal Transducing ,Phosphoinositide-3 Kinase Inhibitors ,Analysis of Variance ,Multidisciplinary ,urogenital system ,Kinase ,TOR Serine-Threonine Kinases ,fungi ,Intracellular Signaling Peptides and Proteins ,Biological Sciences ,WNK1 ,Cell biology ,Ion homeostasis ,Biochemistry ,Multiprotein Complexes ,Cotransporter ,HeLa Cells ,Signal Transduction - Abstract
The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.
- Published
- 2013
7. Interactions with WNK (With No Lysine) Family Members Regulate Oxidative Stress Response 1 and Ion Co-transporter Activity
- Author
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Katherine Luby-Phelps, Svetlana Earnest, Szu Wei Tu, Samarpita Sengupta, Melanie H. Cobb, Kyle Wedin, and Steve Stippec
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Cytoplasm ,Sodium-Potassium-Chloride Symporters ,Immunoprecipitation ,Amino Acid Motifs ,Green Fluorescent Proteins ,Hypertonic Solutions ,Immunoblotting ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Animals ,Humans ,Solute Carrier Family 12, Member 2 ,Amino Acid Sequence ,Molecular Biology ,Ion transporter ,Solute Carrier Family 12, Member 1 ,Protein-Serine-Threonine Kinases ,Kinase ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,WNK1 ,Rats ,Transport protein ,Cell biology ,Isoenzymes ,Protein Transport ,nervous system ,Hypotonic Solutions ,Microscopy, Fluorescence ,Phosphorylation ,RNA Interference ,Signal Transduction ,Fluorescence Recovery After Photobleaching ,HeLa Cells ,Protein Binding - Abstract
Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.
- Published
- 2012
8. Biological Cross-talk between WNK1 and the Transforming Growth Factor β-Smad Signaling Pathway
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Byung-Hoon Lee, Wei Chen, Steve Stippec, and Melanie H. Cobb
- Subjects
Small interfering RNA ,Smad Proteins ,SMAD ,Protein Serine-Threonine Kinases ,Biology ,Models, Biological ,Biochemistry ,Minor Histocompatibility Antigens ,Mice ,WNK Lysine-Deficient Protein Kinase 1 ,Transforming Growth Factor beta ,Transcription (biology) ,Animals ,Humans ,Phosphorylation ,RNA, Small Interfering ,Molecular Biology ,Cell Nucleus ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Fibroblasts ,Cell biology ,Ion homeostasis ,Gene Expression Regulation ,Smad2 Protein ,Signal transduction ,HeLa Cells ,Signal Transduction ,Transforming growth factor - Abstract
WNKs (with no lysine (K)), unique serine/threonine protein kinases, have been best studied in the context of cell volume regulation and ion homeostasis. Here we describe a biological link between WNKs and transforming growth factor (TGF) beta-Smad signaling. Both WNK1 and WNK4 directly bind to and phosphorylate Smad2. Knockdown of WNK1 in HeLa cells using small interfering RNA reduces Smad2 protein expression; this decrease is at least partially due to down-regulation of Smad2 transcription. In contrast, phosphorylated Smad2 significantly accumulated in the nucleus as a consequence of depletion of WNK1, resulting in Smad-mediated transcriptional responses. In addition, TGFbeta-induced target gene transcripts were increased in WNK1 small interfering RNA cells. These findings suggest WNK1 as a dual modulator of TGFbeta-Smad signaling pathways.
- Published
- 2007
9. WNK1 Activates SGK1 by a Phosphatidylinositol 3-Kinase-dependent and Non-catalytic Mechanism
- Author
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Ahmed Lazrak, Bing E. Xu, Steve Stippec, Melanie H. Cobb, and Chou Long Huang
- Subjects
DNA Mutational Analysis ,Protein Serine-Threonine Kinases ,Biology ,Mitogen-activated protein kinase kinase ,Kidney ,Biochemistry ,Sodium Channels ,Cell Line ,Immediate-Early Proteins ,MAP2K7 ,Minor Histocompatibility Antigens ,Mice ,Phosphatidylinositol 3-Kinases ,Xenopus laevis ,WNK Lysine-Deficient Protein Kinase 1 ,Catalytic Domain ,Animals ,ASK1 ,Enzyme Inhibitors ,Insulin-Like Growth Factor I ,Phosphorylation ,Epithelial Sodium Channels ,Protein kinase A ,Molecular Biology ,Phosphoinositide-3 Kinase Inhibitors ,MAP kinase kinase kinase ,urogenital system ,Akt/PKB signaling pathway ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,WNK1 ,Introns ,Electrophysiology ,Hypertension ,Mutagenesis, Site-Directed ,Oocytes ,RNA Interference ,Cyclin-dependent kinase 9 - Abstract
WNK1 (with no lysine (K) 1) is a protein-serine/threonine kinase with a unique catalytic site organization. Deletions in the first intron of the WNK1 gene were found in a group of hypertensive patients with pseudohypoaldosteronism type II. No changes in coding sequence of WNK1 were found, but its expression was increased severalfold. We have been investigating actions of WNK1 and have found that WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1, which impacts membrane expression of the epithelial sodium channel. Here we explore the role of WNK1 in SGK1 regulation. Activation of SGK1 by WNK1 is blocked by phosphatidylinositol 3-kinase inhibitors. Neither the catalytic activity nor the kinase domain of WNK1 is required; rather the N-terminal 220 residues of WNK1 are necessary and sufficient to activate SGK1. Phosphorylation of WNK1 on Thr-58 contributes to SGK1 activation. Finally, we show that WNK1 is required for the activation of SGK1 by insulin-like growth factor 1.
- Published
- 2005
10. WNK1 Activates ERK5 by an MEKK2/3-dependent Mechanism
- Author
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Youn Kyoung Lee, Bing E. Xu, Wei Zhang, Steve Stippec, Lisa Lenertz, Melanie H. Cobb, and Byung-Hoon Lee
- Subjects
Small interfering RNA ,Gene Expression ,MAP Kinase Kinase Kinase 3 ,MAP Kinase Kinase 5 ,MAP Kinase Kinase Kinase 2 ,Protein Serine-Threonine Kinases ,Mitogen-activated protein kinase kinase ,Biology ,Transfection ,Biochemistry ,Cell Line ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Epidermal growth factor ,Nitriles ,Gene expression ,Butadienes ,Humans ,Enzyme Inhibitors ,RNA, Small Interfering ,Protein kinase A ,Molecular Biology ,Immunosorbent Techniques ,Mitogen-Activated Protein Kinase 7 ,Mitogen-Activated Protein Kinase Kinases ,Epidermal Growth Factor ,MAP kinase kinase kinase ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,MAP Kinase Kinase Kinases ,WNK1 ,Molecular biology ,Recombinant Proteins ,WNK4 ,Enzyme Activation ,Mutagenesis, Site-Directed ,Mitogen-Activated Protein Kinases ,HeLa Cells - Abstract
WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.
- Published
- 2004
11. Hydrophobic as Well as Charged Residues in Both MEK1 and ERK2 Are Important for Their Proper Docking
- Author
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Bing E. Xu, Steve Stippec, Melanie H. Cobb, and Fred L. Robinson
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endocrine system diseases ,Molecular Sequence Data ,Mutant ,MAP Kinase Kinase 1 ,Protein Serine-Threonine Kinases ,Biology ,environment and public health ,Biochemistry ,Cell Line ,Structure-Activity Relationship ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,Mitogen-Activated Protein Kinase 1 ,Mitogen-Activated Protein Kinase Kinases ,Alanine ,Cell Biology ,Fusion protein ,N-terminus ,enzymes and coenzymes (carbohydrates) ,medicine.anatomical_structure ,Docking (molecular) ,Cytoplasm ,Mutation ,Phosphorylation ,biological phenomena, cell phenomena, and immunity ,Nucleus ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.
- Published
- 2001
12. Differential Effects of PAK1-activating Mutations Reveal Activity-dependent and -independent Effects on Cytoskeletal Regulation
- Author
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Andrei Khokhlatchev, Michael A. White, Steve Stippec, Jeffrey A. Frost, and Melanie H. Cobb
- Subjects
Stress fiber ,Membrane ruffling ,Focal adhesion assembly ,Cell Cycle Proteins ,Small G Protein ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Focal adhesion ,GTP-Binding Proteins ,Cell Adhesion ,Cloning, Molecular ,Cytoskeleton ,Molecular Biology ,Cell Membrane ,Cell Biology ,Peptide Fragments ,Cell biology ,Enzyme Activation ,Intercellular Junctions ,p21-Activated Kinases ,Mutagenesis ,Lamellipodium ,Protein Binding ,Binding domain - Abstract
PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.
- Published
- 1998
13. Protein kinase WNK3 regulates the neuronal splicing factor Fox-1
- Author
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Yu Chi Juang, Jon Self, Fan Yang, Xiaojun Ding, A-Young Lee, Melanie H. Cobb, Wei Chen, She Chen, and Steve Stippec
- Subjects
RNA Splicing Factors ,Immunoprecipitation ,animal diseases ,RNA-binding protein ,Electrophoretic Mobility Shift Assay ,Biology ,Protein Serine-Threonine Kinases ,Splicing factor ,Mice ,Two-Hybrid System Techniques ,parasitic diseases ,Animals ,Humans ,Protein kinase A ,DNA Primers ,Gene Library ,Multidisciplinary ,Reverse Transcriptase Polymerase Chain Reaction ,RNA ,virus diseases ,food and beverages ,Brain ,RNA-Binding Proteins ,Biological Sciences ,Deuterium ,Cell biology ,HEK293 Cells ,Biochemistry ,RNA splicing ,Phosphorylation ,population characteristics ,Phosphorus Radioisotopes - Abstract
We report an action of the protein kinase WNK3 on the neuronal mRNA splicing factor Fox-1. Fox-1 splices mRNAs encoding proteins important in synaptic transmission and membrane excitation. WNK3, implicated in the control of neuronal excitability through actions on ion transport, binds Fox-1 and inhibits its splicing activity in a kinase activity-dependent manner. Phosphorylation of Fox-1 by WNK3 does not change its RNA binding capacity; instead, WNK3 increases the cytoplasmic localization of Fox-1, thereby suppressing Fox-1–dependent splicing. These findings demonstrate a role of WNK3 in RNA processing. Considering the implication of WNK3 and Fox-1 in disorders of neuronal development such as autism, WNK3 may offer a target for treatment of Fox-1–induced disease.
- Published
- 2012
14. Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members*
- Author
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Svetlana Earnest, Staci L. Deaton, Seung Kuy Cha, Yingda Xu, Melanie H. Cobb, Samarpita Sengupta, Chih-Jen Cheng, Charles J. Heise, Yingming Zhao, Yu Chi Juang, Steve Stippec, Bing E. Xu, and Chou Long Huang
- Subjects
Epithelial sodium channel ,Nedd4 Ubiquitin Protein Ligases ,Ubiquitin-Protein Ligases ,Immunoblotting ,CHO Cells ,Protein Serine-Threonine Kinases ,Biochemistry ,Cell Line ,Immediate-Early Proteins ,Minor Histocompatibility Antigens ,Mice ,Cricetulus ,WNK Lysine-Deficient Protein Kinase 1 ,Cell Line, Tumor ,Cricetinae ,medicine ,Animals ,Humans ,Immunoprecipitation ,Phosphorylation ,Protein kinase A ,Epithelial Sodium Channels ,Molecular Biology ,biology ,Endosomal Sorting Complexes Required for Transport ,urogenital system ,Cell Biology ,WNK1 ,Amiloride ,WNK4 ,Ubiquitin ligase ,Rats ,SGK1 ,biology.protein ,medicine.drug ,Signal Transduction ,HeLa Cells - Abstract
The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.
- Published
- 2010
15. Regulation of a Third Conserved Phosphorylation Site in SGK1*
- Author
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Melanie H. Cobb, Yu Chi Juang, Wei Chen, Yue Chen, Steve Stippec, Yingming Zhao, and Bing E. Xu
- Subjects
Nedd4 Ubiquitin Protein Ligases ,Ubiquitin-Protein Ligases ,Amino Acid Motifs ,Biology ,Mitogen-activated protein kinase kinase ,Protein Serine-Threonine Kinases ,Biochemistry ,MAP2K7 ,Immediate-Early Proteins ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Humans ,ASK1 ,Protein phosphorylation ,Kinase activity ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Endosomal Sorting Complexes Required for Transport ,urogenital system ,Cyclin-dependent kinase 5 ,Cyclin-dependent kinase 2 ,Mechanisms of Signal Transduction ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Cell biology ,Enzyme Activation ,Protein Transport ,Mutation ,biology.protein ,Proto-Oncogene Proteins c-akt ,HeLa Cells - Abstract
SGK1 (serum- and glucocorticoid-induced kinase 1) is a member of the AGC branch of the protein kinase family. Among well described functions of SGK1 is the regulation of epithelial transport through phosphorylation of the ubiquitin protein ligase Nedd4-2 (neuronal precursor cell expressed developmentally down-regulated 4-2). The activation of SGK1 has been widely accepted to be dependent on the phosphorylation of Thr256 in the activation loop and Ser422 in the hydrophobic motif near the C terminus. Here, we report the identification of two additional phosphorylation sites, Ser397 and Ser401. Both are required for maximum SGK1 activity induced by extracellular agents or by coexpression with other protein kinases, with the largest loss of activity from mutation of Ser397. Coexpression with active Akt1 increased the phosphorylation of Ser397 and thereby SGK1 kinase activity. SGK1 activation was further augmented by coexpression with the protein kinase WNK1 (with no lysine kinase 1). These findings reveal further complexity underlying the regulation of SGK1 activity.
- Published
- 2009
16. WNK1: analysis of protein kinase structure, downstream targets, and potential roles in hypertension
- Author
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Charles J. Heise, Lisa Lenertz, Byung-Hoon Lee, Xiaoshan Min, Melanie H. Cobb, Bing E. Xu, Elizabeth J. Goldsmith, and Steve Stippec
- Subjects
DNA, Complementary ,MAP Kinase Signaling System ,Protein Conformation ,Pseudohypoaldosteronism ,Nerve Tissue Proteins ,Biology ,Protein Serine-Threonine Kinases ,Crystallography, X-Ray ,Ligands ,Models, Biological ,Minor Histocompatibility Antigens ,Electrolytes ,Synaptotagmins ,WNK Lysine-Deficient Protein Kinase 1 ,Calcium-binding protein ,Cell Line, Tumor ,Two-Hybrid System Techniques ,Animals ,Humans ,Protein kinase A ,Molecular Biology ,Mitogen-Activated Protein Kinase 7 ,Gene Library ,Protein-Serine-Threonine Kinases ,Membrane Glycoproteins ,MAP kinase kinase kinase ,Kinase ,Calcium-Binding Proteins ,Cell Membrane ,Intracellular Signaling Peptides and Proteins ,Brain ,Cell Biology ,WNK1 ,Protein Structure, Tertiary ,Enzyme Activation ,Ion homeostasis ,Protein kinase domain ,Biochemistry ,COS Cells ,RNA Interference ,Gene Deletion - Abstract
The WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis. Intronic deletions in the WNK1 gene result in its overexpression and lead to pseudohypoaldosteronism type II, a disease with salt-sensitive hypertension and hyperkalemia. This review focuses on the recent evidence elucidating the structure of the kinase domain of WNK1 and functions of these kinases in normal and disease physiology. Their functions have implications for understanding the biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. The WNK kinases may be able to influence ion homeostasis through its effects on synaptotagmin function.
- Published
- 2005
17. Regulation of WNK1 by an autoinhibitory domain and autophosphorylation
- Author
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Melanie H. Cobb, Byung-Hoon Lee, Xiaoshan Min, Elizabeth J. Goldsmith, Bing E. Xu, and Steve Stippec
- Subjects
Recombinant Fusion Proteins ,Molecular Sequence Data ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,Cell Line ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Catalytic Domain ,Humans ,Amino Acid Sequence ,Kinase activity ,Phosphorylation ,Molecular Biology ,Sequence Homology, Amino Acid ,Kinase ,Lysine ,Autophosphorylation ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,WNK1 ,WNK4 ,Protein kinase domain ,Mutagenesis, Site-Directed ,Protein Kinases - Abstract
WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.
- Published
- 2002
18. WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II
- Author
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Jessie M. English, Julie L. Wilsbacher, Melanie H. Cobb, Bing E. Xu, Elizabeth J. Goldsmith, and Steve Stippec
- Subjects
Models, Molecular ,DNA, Complementary ,Molecular Sequence Data ,Serine threonine protein kinase ,Mitogen-activated protein kinase kinase ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,MAP2K7 ,Minor Histocompatibility Antigens ,WNK Lysine-Deficient Protein Kinase 1 ,Catalytic Domain ,Animals ,c-Raf ,Amino Acid Sequence ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Serine/threonine-specific protein kinase ,MAP kinase kinase kinase ,Base Sequence ,Sequence Homology, Amino Acid ,Lysine ,Cell Biology ,Rats ,Mutagenesis, Site-Directed ,Casein kinase 2 - Abstract
We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 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.
- Published
- 2000
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