Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Intracellular Ion Control of WNK Signaling.

The with no lysine (K) (WNK) kinases are an evolutionarily ancient group of kinases with atypical placement of the catalytic lysine and diverse physiological roles. Recent studies have shown that WNKs are directly regulated by chloride, potassium, and osmotic pressure. Here, we review the discovery of WNKs as chloride-sensitive kinases and discuss physiological contexts in which chloride regulation of WNKs has been demonstrated. These include the kidney, pancreatic duct, neurons, and inflammatory cells. We discuss the interdependent relationship of osmotic pressure and intracellular chloride in cell volume regulation. We review the recent demonstration of potassium regulation of WNKs and speculate on possible physiological roles. Finally, structural and mechanistic aspects of intracellular ion and osmotic pressure regulation of WNKs are discussed.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

Nuclear speckle integrity and function require TAO2 kinase.

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

Functional divergence caused by mutations in an energetic hotspot in ERK2.

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

A Phosphorylated Intermediate in the Activation of WNK Kinases.

WNK kinases autoactivate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphorylated on the primary activating site (pWNK1) in the presence of AMP-PNP reveals a well-ordered but inactive configuration. This new pWNK1 structure features specific and unique interactions of the phosphoserine, less hydration, and smaller cavities compared with those of unphosphorylated WNK1 (uWNK1). Because WNKs are activated by osmotic stress in cells, we addressed whether the structure was influenced directly by osmotic pressure. pWNK1 crystals formed in PEG3350 were soaked in the osmolyte sucrose. Suc-WNK1 crystals maintained X-ray diffraction, but the lattice constants and pWNK1 structure changed. Differences were found in the activation loop and helix C, common switch loci in kinase activation. On the basis of these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. Our data suggest multistage activation of WNKs.

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.

How activating mutations affect MEK1 regulation and function.

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

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.

Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK.

The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was determined in the unphosphorylated inactive form and, like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK and OSR1, we have determined the crystal structures of SPAK 63-403 at 3.1 Å and SPAK 63-390 T243D at 2.5 Å resolution. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without and the shorter with an activating T243D point mutation. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1 but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1 but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of partially active SPAK T243D is consistent with a multistage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.

MAP kinase modules: the excursion model and the steps that count.

MAP kinase modules propagate diverse extracellular signals to downstream effectors. The two dual phosphorylation reactions catalyzed by the modules are thought to control the switch behavior of the pathway. Here we review recent approaches to understand these pathways through signal-to-response studies in cells and in vitro. These data are reconciled with physical models as well as predictions made on mathematical and theoretical grounds. Biochemical analysis has shown recently that the dual phosphorylation reactions catalyzed by MAP kinase modules are sequential at both levels of the cascade. The observed order of phosphorylation events suggests an excursion from the Ser/Thr kinase activity of the MAP3K into Tyr kinase activity of the central dual specificity MAP2K. How the order of events might be encoded in the structures and interactions is discussed. The ordered mechanism confirms predictions that reactions should be sequential to generate the steep signal-to-response curves and delayed responses observed in cells.

Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade.

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.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Previous observations showed that chloride and osmotic stress regulate the autophosphorylation and activity of the kinase domains of WNK1 and WNK3. Further, prior crystallography on the asymmetric dimeric of the unphosphorylated WNK1 kinase domain (WNK1/S382A, WNK1/SA) revealed conserved waters in the active site. Here we show by crystallography that PEG400 applied to crystals of dimeric WNK1/SA grown in space group P1 induces de-dimerization with a change in space group to P2 1 . Both the conserved waters, referred to here as conserved water network 1 (CWN1) and the chloride binding site are disrupted by PEG400. CWN1 is surrounded and stabilized by a pan-WNK-conserved cluster of charged residues. Here we mutagenized these charges in WNK3 to probe the importance of the CWN1 to WNK regulation. Two mutations at E314 in the Activation Loop (WNK3/E314Q and WNK3/E314A) enhanced activity, consistent with the idea that the CWN1 is inhibitory. Mutations of other residues in the cluster had similar or less activity than wild-type. PEG400 activation of WNK3 was not significantly reduced in the point mutants tested. The crystallographic and assay data support a role for CWN1 and the charged cluster in stabilizing an inactive configuration of WNKs and suggest that water functions as an allosteric inhibitor of WNKs.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Synthesis and Structural Characterization of Novel Trihalo-sulfone Inhibitors of WNK1.

With No lysine (K) [WNK] kinases are structurally unique serine/threonine protein kinases that have therapeutic potential for blood pressure regulation and cancer. A novel class of trihalo-sulfone compounds was identified by high-throughput screening. Trihalo-sulfone 1 emerged as an effective inhibitor of WNK1 with an IC50 value of 1.6 µM. Herein, we define chemical features necessary for inhibition of WNK1 using chemical synthesis and X-ray crystallography. Analogues that probed the role of specific functional groups to the inhibitory activity were synthesized. X-ray structures of trihalo-sulfone 1 and a second trihalo-sulfone 23 bound to WNK1 revealed active site binding to two of the three previously defined canonical inhibitor binding pockets as well as a novel binding site for the trihalo-sulfone moiety. The elucidation of these novel interaction sites may allow for the strategic design of even more selective and potent WNK inhibitors.

Evolution of substrate specificity within a diverse family of beta/alpha-barrel-fold basic amino acid decarboxylases: X-ray structure determination of enzymes with specificity for L-arginine and carboxynorspermidine.

Pyridoxal 5'-phosphate (PLP)-dependent basic amino acid decarboxylases from the beta/alpha-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-A resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the beta/alpha-barrel domain of one subunit and the beta-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the beta-barrel domain we refer to as the "specificity helix." Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu(314), which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5'-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.