Identification, Expression and Functional Analysis of a Receptor-like Cytoplasmic Kinase, OsRLCK1, in Rice

Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCK1, which belongs to the receptor-like cytoplasmic kinase Ⅷ subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCK1 is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCK1 recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCK1 is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.     

Function and regulation of Aurora／Ipllp kinase family in cell division

During mitosis,the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation,a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell.Defects in these processes can lead to aneuploidy or polyploidy.Aurora/Ipllp family, a class of conserved serine/threonine kinases,plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipllp family in mitosis and provides potential links between aberrant regulation of Aurora/Ipllp kinases and pathogenesis of human cancer.     

Alteration of β-secretase traffic by the receptor tyrosine kinase signaling pathway- a new mechanism for regulating Alzheimer's β-amyloid production

The receptor tyrosine kinases (RTKs) are a family of cellsurface proteins with diverse functions in proliferation, dif-ferentiation or cell-cell communication. When a specific li-gand binds to its cognate receptor, a conformational changeof this receptor due to the ligand-receptor interaction willlead to activation of the intrinsic tyrosine kinase residing inthe intracellular domain of the receptor. The activation ofthis tyrosine kinase is essential for transducing the signals toa cascade of its downstream molecules that eventually causerelated physiological responses [1]. For example, binding ofnerve growth factor (NGF) to its receptor TrkA is essentialfor the proper development, patterning, and maintenanceof the mammalian nervous system. This ligand and recep-tor interaction will lead to the formation of a crab-shapedhomodimeric TrkA structure [2], and the subsequent activa-tion of its intrinsic RTK will cause auto-phosphorylationof its own intracellular tyrosine residues. PhosphorylatedTrkA receptors recruit and increase the phosphorylationof PLC-γ and Shc, which leads to activation of either thePI3K/Akt pathway or Ras/raf/ERK pathway. In the brainOf Alzheimer's disease (AD) patients, alterations of nervegrowth factor (NGF) and its receptor TrkA have beenreported to associate with AD pathogenesis [3]. However,the underlying mechanisms remain elusive.     

Delayed information induces oscillations in a dynamical model for infectious disease

During disease outbreak,it has been observed that information about the disease prevalence induces the individuafs behavioral changes.This information is usually assumed to be generated by the density of infective individuals and active mass media.The delay in reporting of these infective individuals may have its impact on generated information.Hence,to study the impact of delay on information generation,and therefore on the disease dynamics,a delay differential equation model is proposed and analyzed.The dynamics of information with delay effect is also modeled by a separate rate equation.Model analysis is performed and a unique infected equilibrium is obtained when the basic reproduction number(R0)is greater than one,whereas the disease free equilibrium always exists.When R0<1,the disease free equilibrium is found to be locally stable independent of delay effect.The unique infected equilibrium is found to be locally stable till delay reaches a threshold value.The global stability of the unique infected equilibrium is also established under some parametric conditions by constructing a suitable Lyapunov function.The occurrence of Hopf bifurcation is observed when the delay in information crosses the threshold value.Analytically,the direction and stability of bifurcating periodic solutions is established.Further,we observed the occurrence of Hopf-Hopf bifurcation at two different delays.At first delay threshold,the endemic equilibrium loses its st ability and produces periodic oscillations via Hopf bifurcation.It further regains its stability at second delay threshold via another Hopf bifurcation.Hence,the delay effect on information shows possibility of stability switches.Numerical experiments are carried out to support the obtained analytical results.Our study infers that the disease will show persistent oscillations if there is a significant time lag in reporting of infective after the disease outbreak.Thus,the delay in dissemination of information shows rich and complex dynamics in the model and provides important insights.We also observe numerically that the saturation in information plays a significant role on stability of infected equilibrium in presence of delay.     

Phylogenetic and sequence analyses of insect transferrins suggest that only transferrin 1 has a role in iron homeostasis

Iron is essential to life,but surprisingly little is known about how iron is managed in nonvertebrate animals.In mammals,the well-characterized transferrins bind iron and are involved in iron transport or immunity,whereas other members of the transferrin family do not have a role in iron homeostasis.In insects,the functions of transferrins are still poorly understood.The goals of this project were to identify the transferrin genes in a diverse set of insect species,resolve the evolutionary relationships among these genes,and predict which of the transferrins are likely to have a role in iron homeostasis.Our phylogenetic analysis of transferrins from 16 orders of insects and two orders of noninsect hexapods demonstrated that there are four orthologous groups of insect transferrins.Our analysis suggests that transferrin 2 arose prior to the origin of insects,and transferrins/,i,and 4 arose early in insect evolution.Primary sequence analysis of each of the insect transferrins was used to predict signal peptides,carboxyl-terminal transmembrane regions,GPI-anchors,and iron binding.Based on this analysis,we suggest that transferrins 2,and 4 are unlikely to play a major role in iron homeostasis.In contrast,the transferrin 1 orthologs are predicted to be secreted,soluble,iron-binding proteins.We conclude that transferrin 1 orthologs are the most likely to play an important role in iron homeostasis.Interestingly,it appears that the louse,aphid,and thrips lineages have lost the transferrin 1 gene and,thus,have evolved to manage iron without transferrins.     

TRPC1 protects dopaminergic SH-SY5Y cells from MPP＋, salsolinol,and N-methyl-（R）-salsolinol-induced cytotoxicity

Neurotoxins and alterations in Ca2＋ homeostasis have been associated with Parkinson＇s disease （PD）, but the role of store-operated Ca2＋ entry channels is not well understood. Previous studies have shown the neurotoxicity of salsolinol and 1-methyl-4-phenylpyridinium ion on SH-SY5Y cells and cytoprotection induced by transient receptor potential protein 1 （TRPC1）. In the present study, N-methyl-（R）-salsolinol was tested for its cellular toxicity and effects on TRPC1 expression. MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-dipbenyl- tetrazolium bromide） assays, DAPI （4＇,6-diamidino-2-pheny- lindole）, fluorescein isothiocyanate-Annexin-V/propidium iodide, western blot analysis, and JC-1 labeling revealed that the three indicated drugs could induce caspase-dependent, mitochondrial-mediated apoptosis. Exposure of SH-SY5Y cells to the indicated drugs resulted in a significant decrease in thapsigargin-mediated Ca2＋ influx and TRPC1 expression. Immnnocytochemistry experiments revealed that neurotoxins treatment induced TRPC1 translocation to the cytoplasm. Taken together, our results indicate that treatment with neurotoxins may alter Ca2＋ homeostasis and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD.     

Alteration of β-secretase traffic by the receptor tyrosine kinase signaling pathway - a new mechanism for regulating Alzheimer＇s β-amyloid production

The receptor tyrosine kinases （RTKs） are a family of cell surface proteins with diverse functions in proliferation, differentiation or cell-cell communication. When a specific ligand binds to its cognate receptor, a conformational change of this receptor due to the ligand-receptor interaction will lead to activation of the intrinsic tyrosine kinase residing in the intracellular domain of the receptor. The activation of this tyrosine kinase is essential for transducing the signals to a cascade of its downstream molecules that eventually cause related physiological responses .     

Die in pieces:How Drosophila sheds light on neurite degeneration and clearance

Dendrites and axons are delicate neuronal membrane extensions that undergo degeneration after physical injuries. In neurodegenerative diseases, they often degenerate prior to neuronal death. Understanding the mechanisms of neurite degeneration has been an intense focus of neurobiology research in the last two decades. As a result, many discoveries have been made in the molecular pathways that lead to neurite degeneration and the cell-cell interactions responsible for the subsequent clearance of neuronal debris. Drosophila melanogaster has served as a prime in vivo model system for identifying and characterizing the key molecular players in neurite degeneration, thanks to its genetic tractability and easy access to its nervous system. The knowledge learned in the fly provided targets and fuel for studies in other model systems that have further enhanced our understanding of neurodegeneration. In this review, we will introduce the experimental systems developed in Drosophila to investigate injuryinduced neurite degeneration, and then discuss the biological pathways that drive degeneration. We will also cover what is known about the mechanisms of how phagocytes recognize and clear degenerating neurites, and how recent findings in this area enhance our understanding of neurodegenerative disease pathology.     

High-density lipoproteins： an emerging target in the prevention of cardiovascular disease

High-density lipoproteins （HDLs） have been well established to protect against the development of atherosclerotic cardiovascular disease. It has become apparent that in addition to the promotion of reverse cholesterol transport, HDLs possess a number of additional functional properties that may contribute to their beneficial influence on the arterial wall. A number of exciting therapeutic strategies have been developed that target HDL and its ability to protect against the development of atherosclerotic plaque. This paper will review how the promotion of the functional properties of HDL inhibits the formation of atherosclerotic plaque and stabilises lesions in patients with established disease.     

Properties of WNK1 and implications for other family members

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

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.     

Investigating specificity of the anti-hypertensive inhibitor WNK463 against With-No-Lysine kinase family isoforms via multiscale simulations

Abstract

The With-No-Lysine (WNK) kinase family plays a significant role in regulating cation-chloride cotransporters, blood pressure and body fluid homeostasis. Mutations in the gene of WNK family, especially in WNK1 and WNK4 are responsible for pseudohypoaldosteronism type II (PHAII), characterized by hypertension. The selective inhibition of WNK1 over other isoforms has created an immense challenge in the design of an ATP competitive inhibitor due to their high conservatism. In this work, we have compared the selectivity of the inhibitor WNK463, which was designed for WNK1 with other WNK family isoforms by comprehensive molecular modeling, docking and molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area method. Our calculations show that the affinity of the inhibitor decreases in the order WNK2?>?WNK1?>?WNK3?>?WNK4, in agreement with the experiment. Our study reveals that the inhibitor is most selective to WNK2 due to decreased polar solvation and configurational entropy compared to other isoforms. Furthermore, our analyses indicated that the nonpolar contribution from the hydrophobic residues and hydrogen bonds in the hinge region gatekeeper residue Met³⁰⁴ of WNK1 and its equivalent residue from other kinases played a critical role in stabilizing the inhibitor against WNK kinases. Residues Lys²³³, Met³⁰⁴, Phe³⁵⁶ and Leu³⁶⁹ of WNK1 were the essential residue differences compared to other isoforms that led to specific interactions thereby forming the basis of molecular binding pattern of binding interactions. Overall, we have identified conserved WNK-inhibitor interactions and elucidated isoform-specific interactions that could be exploited in the design of more potent and selective WNK inhibitors.

Communicated by Ramaswamy H. Sarma     

Evolutionarily conserved WNK and Ste20 kinases are essential for acute volume recovery and survival after hypertonic shrinkage in Caenorhabditis elegans

Members of the germinal center kinase (GCK)-VI subfamily of Ste20 kinases regulate a Caenorhabditis elegans ClC anion channel and vertebrate SLC12 cation-Cl^– cotransporters. With no lysine (K) (WNK) protein kinases interact with and activate the mammalian GCK-VI kinases proline-alanine-rich Ste20-related kinase (PASK) and oxidative stress-responsive 1 (OSR1). We demonstrate here for the first time that GCK-VI kinases play an essential role in whole animal osmoregulation. RNA interference (RNAi) knockdown of the single C. elegans GCK-VI kinase, GCK-3, dramatically inhibits systemic volume recovery and survival after hypertonic shrinkage. Tissue-specific RNAi suggests that GCK-3 functions primarily in the hypodermis and intestine to mediate volume recovery. The single C. elegans WNK kinase, WNK-1, binds to GCK-3, and wnk-1 knockdown gives rise to a phenotype qualitatively similar to that of gck-3(RNAi) worms. Knockdown of the two kinases together has no additive effect, suggesting that WNK-1 and GCK-3 function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that postulated for its mammalian homologs. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan and therefore likely coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis. cell volume regulation; osmotic stress; osmoregulation     

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation

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.     

WNK1 and WNK4, new players in salt and water homeostasis

Arterial hypertension is a complex trait influenced by a variety of environmental and genetic factors. Several approaches can be used to identify its susceptibility genes : one is to study rare monogenic forms of hypertension, like familial hyperkalemic hypertension (FHH). Also known as pseudohypoaldosteronism type 2 or Gordon syndrome, FHH is characterized by hypertension, hyperkalemia despite normal renal glomerular filtration rate, abnormalities which are particularly sensitive to thiazide diuretics. Mild hyperchloremia, metabolic acidosis, and suppressed plasma renin activity are associated findings. Despite its phenotypic and genetic heterogeneity, mutations in two related genes, WNK1 and WNK4, were recently identified. These genes belong to a newly identified family of serine-threonine (with no lysine [K]) kinases. Both are highly expressed in the kidney and in a variety of epithelia involved in chloride transport. It has thus been postulated that these two kinases could be implicated in a new pathway of ionic transport regulation. Several studies have very recently confirmed this hypothesis in vitro, in Xenopus oocytes or kidney cell lines. They have shown that, in the renal distal tubule, WNK4 inhibits sodium reabsorption and potassium secretion, via inhibition of NCC (thiazide-sensitive Na+-Cl- cotransporter) and K+ channel ROMK activity, respectively. Interestingly, FHH mutations have opposite effects : while they lead to loss of NCC inhibition, they increase ROMK inhibition. Moreover, they also increase paracellular permeability to chloride of MDCK cells. WNK4 also inhibits apical and basal chloride transporters present in extra-renal epithelia, such as CFEX and Na+-K+-2 Cl-, respectively. It is also interesting to note that the WNK4-mediated negative regulation of NCC activity is in turn inhibited by WNK1. By its role on several transporters, WNK4 appears as a putative key regulator of ionic transport and blood pressure.     

WNK kinases and the kidney

In the kidney, the renal tubule plays a major role in maintaining fluid and electrolyte balance. This balance is achieved by an interplay between various hormones and nerves that signal changes throughout the body and transfer these signals to transport proteins. Increased or reduced activity of these transporters helps to restore homeostasis, but can also contribute to disease (e.g. sodium retention in hypertension). In recent years, it has become clear that the signal transfer to transporters is largely mediated by kinases. Among these, WNK kinases (With No lysine=K) stand out, because they regulate the major sodium and potassium transporters in the distal nephron. Moreover, mutations in genes encoding WNK kinases result in an inherited form of salt-sensitive hypertension with hyperkalemia, illustrating their important role in sodium, potassium, and blood pressure regulation. More recently, WNK kinases were found to play a role in acquired forms of hypertension as well. Together, the evolving insight in the kinase regulation of ion transport is providing new insights in the longstanding question how salt and blood pressure are related. Here, we review the current models of how WNK kinases regulate the various transport proteins and which roles they play in health and disease.     

WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.     

WNK1 phosphorylates synaptotagmin 2 and modulates its membrane binding

WNK (with no lysine [K]) protein kinases were named for their unique active site organization. Mutations in WNK1 and WNK4 cause a familial form of hypertension by undefined mechanisms. Here, we report that WNK1 selectively binds to and phosphorylates synaptotagmin 2 (Syt2) within its calcium binding C2 domains. Endogenous WNK1 and Syt2 coimmunoprecipitate and colocalize on a subset of secretory granules in INS-1 cells. Phosphorylation by WNK1 increases the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of threonine 202, a WNK1 phosphorylation site, partially prevents this change. These findings suggest that phosphorylation of Syts by WNK1 can regulate Ca2+ sensing and the subsequent Ca2+-dependent interactions mediated by Syt C2 domains. These findings provide a biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. Interruption of this regulatory pathway may disturb membrane events that regulate ion balance.