Radha Akella

Chloride Sensing by WNK1 Involves Inhibition of Autophosphorylation

Structural analysis reveals how chloride inhibits the activation of a kinase implicated in hypertension. Inhibited by Chloride in the Active Site Chloride ions are essential for regulation of blood pressure and electrolyte homeostasis. Kinases of the WNK family are involved in the phosphorylation-mediated regulation of chloride cotransporters and ion channels involved in salt regulation; mutations in WNK1 or WNK4 produce hypertension, hyperkalemia, and hyperchloremia. Piala et al. found that chloride ions stabilized the WNK1 kinase domain and inhibited its autophosphorylation activity and kinase activity toward substrates. Analysis of crystal structures of the kinase domain with either bromide or chloride revealed that the ion bound in the catalytic site, and mutation of the residues involved in binding the ion rendered the kinase less sensitive to inhibition of autophosphorylation activity by chloride salts. Thus, these results provide a molecular explanation for how these kinases function as sensors of intracellular chloride. WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade, leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. We found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride-binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

Determinants That Control the Specific Interactions between TAB1 and p38α

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

The third conformation of p38α MAP kinase observed in phosphorylated p38α and in solution.

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.

Precisely Ordered Phosphorylation Reactions in the p38 Mitogen-activated Protein (MAP) Kinase Cascade

Background: MAPK cascades are signaling modules that function as switch generators. Results: In vitro phosphorylation in the p38 MAPK cascade tracked by LC-MS/MS revealed specific phosphorylation intermediates at each level. Conclusion: The p38 MAPK cascade reactions occur through intermediates MEK6/ST* and p38α/TY*. Significance: The precise order of reactions may contribute to the diverse kinetic outputs of the cascades, including those with large Hill coefficients. The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

Natural Language Query in the Biochemistry and Molecular Biology Domains Based on Cognition Search

MOTIVATION With the increasing volume of scientific papers and heterogeneous nomenclature in the biomedical literature, it is apparent that an improvement over standard pattern matching available in existing search engines is required. Cognition Search Information Retrieval (CSIR) is a natural language processing (NLP) technology that possesses a large dictionary (lexicon) and large semantic databases, such that search can be based on meaning. Encoded synonymy, ontological relationships, phrases, and seeds for word sense disambiguation offer significant improvement over pattern matching. Thus, the CSIR has the right architecture to form the basis for a scientific search engine. RESULT Here we have augmented CSIR to improve access to the MEDLINE database of scientific abstracts. New biochemical, molecular biological and medical language and acronyms were introduced from curated web-based sources. The resulting system was used to interpret MEDLINE abstracts. Meaning-based search of MEDLINE abstracts yields high precision (estimated at >90%), and high recall (estimated at >90%), where synonym, ontology, phrases and sense seeds have been encoded. The present implementation can be found at http://MEDLINE.cognition.com. CONTACT Elizabeth.goldsmith@UTsouthwestern.edu Kathleen.dahlgren@cognition.com.

Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

Anatomy of a pressure sensing protein kinase

Abstract Cells respond to hydrostatic pressure to maintain cellular, organ and organism level functions, to set and respond to blood pressure, tissue perfusion, filtration rates and other processes. Pressure sensing is thought to occur at membranes where proteins can respond to mechanical cues 1,2,3. Thus, proteins implicated as direct pressure sensors have been mainly ion channels 2,4, and more recently G-protein coupled receptors5,6. Here we show, contrary to expectations, that hydrostatic pressure directly induces autophosphorylation and activation of an intracellular protein kinase, With No Lysine kinase-3 (WNK3), and to a lesser extent, WNK17. The pressure sensitivity is a property of the kinase domains alone of WNK1 and WNK3. The crystal structure of the unphosphorylated inactive WNK1 kinase domain (iWNK1) suggests that a dimer to monomer equilibrium and changes in hydration are central to pressure sensing. Aspects of this mechanism are supported by mutagenic analysis. We further show that hydrostatic pressure activates full-length WNK3 in Drosophila tubules.

Structural basis for activation of WNK kinases by hydrostatic pressure

WNK1 is a protein kinase on pathway for the regulation of cationchoride cotransporters (CCCs), important mediators of transepithelial transport and cell volume control. CCCs are regulated by chloride and cell-external osmolarity in a phosphorylation dependent manner. We demonstrated recently that the ability of WNK1 to autophosphorylate is inhibited by chloride, and that the kinase domain of WNK1 binds directly binds chloride ion. The phosphorylated form of the kinase domain of WNK1 (210-483)/S* adopts a different configuration in the activation loop, closer to a fully active kinase, revealing the conformational equilibrium nature of the chloride inhibition.