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 glucocorticoidinduced 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. WNKs5 (with no lysine (K)) are large protein-serine/threonine kinases found in all multicellular and a few unicellular eukaryotes (1). WNK1, the first member of the family identified in mammals, was found in searches for novel components of protein kinase cascades (2). WNK1 is expressed ubiquitously, consistent with effects on many cell types (2-4). Numerous splice forms, containing from just under 2000 to almost 2400 residues, are known, including one lacking most of the kinase domain (KS-WNK1), which is enriched in kidney (5, 6).The four WNK family members are distinct from all other protein kinases in that their catalytic lysine is shifted from its usual position buried in the N-terminal part of the kinase core to a more exposed position in the glycine-rich loop (2, 7). The strict conservation of the unique catalytic core structure of the WNK family in organisms such as Chlamydomonas, Phycomyces, Arabidopsis, and mammals suggests conserved properties for these kinases.Our initial characterization of WNK1 revealed that the kinase activity is sensitive to hypertonic stress (2). The subsequent discovery that WNK1 and WNK4 are genetically linked to a rare type of hypertension, pseudohypoaldosteronism type 2 (PHA2) (4), demonstrated the importance of WNK function in man and an implicit significance of the sensitivity of WNK1 kinase activity to osmotic stress. Further characterization showed that activity is increased in response to increased and decreased ionic strength (8). The consequences of WNK mutations in PHA2 are hyperkalemia, renal tubular acidosis, and eventually hypertension (1, 9 -11). WNK1 knock-out mice do not survive, but heterozygotes have low blood pressure (12), ...
Sleep-wake disturbances are common non-motor manifestations in Parkinson Disease (PD). Complex pathophysiological changes secondary to neurodegeneration in combination with motor symptoms and dopaminergic medications contribute to development of sleep-wake disturbances. The management of sleep complaints in PD is important as this symptom can affect daily activities and impair quality of life. Deep brain stimulation (DBS) is an effective adjunctive therapy for management of motor symptoms in PD. However, its effect on non-motor symptoms including sleep-wake disturbances is not widely understood. In this article, we reviewed studies assessing the effect of DBS at various therapeutic targets on sleep-wake disturbances. Of the studies examining the role of DBS in sleep-wake disturbances, the effect of subthalamic nucleus stimulation is most widely studied and has shown improvement in sleep quality, sleep efficiency, and sleep duration. Although, studies investigating changes in sleep with stimulation of thalamus, globus pallidus interna, and pedunculopontine nucleus are limited, they support the potential for modulation of sleep-wake centers with DBS at these sites. The mechanism by which DBS at different anatomical targets affects sleep-wake disturbances in PD is unclear and may involves multiple factors, including improved motor symptoms, medication adjustment, and direct modulation of sleep-wake centers.
In the antiphospholipid syndrome (APS), patients produce antiphospholipid antibodies (aPL) that promote thrombosis and adverse pregnancy outcomes. Current therapy with anticoagulation is only partially effective and associated with multiple complications. We previously discovered that aPL recognition of cell surface β2-glycoprotein I (β2-GPI) initiates apolipoprotein E receptor 2 (apoER2)-dependent signaling in endothelial cells and in placental trophoblasts that ultimately promotes thrombosis and fetal loss, respectively. Here we sought to identify a monoclonal antibody (mAb) to β2-GPI that negates aPL-induced processes in cell culture and APS disease endpoints in mice. In a screen measuring endothelial NO synthase (eNOS) activity in cultured endothelial cells, we found that whereas aPL inhibit eNOS, the mAb 1N11 does not, and instead 1N11 prevents aPL action. Coimmunoprecipitation studies revealed that 1N11 decreases pathogenic antibody binding to β2-GPI, and it blocks aPL-induced complex formation between β2-GPI and apoER2. 1N11 also prevents aPL antagonism of endothelial cell migration, and in mice it reverses the impairment in reendothelialization caused by aPL, which underlies the non-thrombotic vascular occlusion provoked by disease-causing antibodies. In addition, aPL inhibition of trophoblast proliferation and migration is negated by 1N11, and the more than 6-fold increase in fetal resorption caused by aPL in pregnant mice is prevented by 1N11. Furthermore, the promotion of thrombosis by aPL is negated by 1N11. Thus, 1N11 has been identified as an mAb that attenuates APS-related pregnancy complications and thrombosis in mice. 1N11 may provide an efficacious, mechanism-based therapy to combat the often devastating conditions suffered by APS patients.
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.T he protein kinases oxidative stress-responsive 1 (OSR1) and its homolog the STE20/SPS1-related proline-, alanine-rich kinase (SPAK or PASK) are the mammalian members of the germ-cell kinase VI subgroup of the large Ste20 branch of the mammalian kinome. OSR1 and SPAK directly regulate the solute carrier 12 family of cation-chloride cotransporters which modulate ion homeostasis throughout the body (1, 2). OSR1/ SPAK kinase domains lie close to their N-termini and they contain two additional conserved regions named "PF1" and "PF2" [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal extension to the kinase domain and is required for enzyme activity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regulatory site as well as a second site in the PF1 region with an undefined function (6-9).The four WNK protein kinases are large enzymes notable for the alternative placement of the essential ATP-binding lysine residue in their catalytic domains, distinguishing them from other members of the protein kinase superfamily (10, 11). Initial attention was focused on these enzymes because certain mutations in two family members cause pseudohypoaldosteronism type II, a heritable form of hypertension (12). WNKs are activated by changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell volume regulation and maintenance of blood pressure (13-19). Control of cation-chloride cotransporters through OSR1 and SPAK is among the best-documented actions of WNKs in diverse tissues (5,(20)(21)(22).WNKs also regulate serum-and glucocorticoid-inducible protein k...
Background: WNK1 binds OSR1 via RFXV motifs and is on intracellular puncta. Results: C-terminal WNK1 segments bind OSR1 and localize to puncta like endogenous WNK1; changes in tonicity decrease WNK1 mobility. Conclusion: Complex interplay of protein-protein interactions, changes in tonicity, and localization control the WNK-OSR1 cascade. Significance: WNK function varies because of family member and splice form expression, protein interactions, and localization.
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 solved 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/OSR1, we have determined the crystal structures of SPAK 63–403 at 3.1 Å and SPAK 63–390 T243D at 2.5 Å resolutions. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without, and the shorter with an activating point mutation T243D. 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 the partially active SPAK T243D is consistent with a multi-stage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.
Intracranial pressure (ICP) is often obtained via external ventricular drain (EVD) placement and is discussed as a key vital sign in neuroscience. Nurses are most often delegated the task of observing, adjudicating, and documenting ICP. Cerebrospinal fluid drainage requires that the transducer connected to the EVD is open to drain, prohibiting ICP monitoring. There are no recent data to support an evidence-based standard for the period an ICP waveform should be observed, after the EVD is clamped, to be able to adjudicate a value that represents the patient's status. Therefore, the purpose of this study is to determine the optimal period for which an EVD should be closed to obtain an accurate ICP value. In a sample of 30 subjects who received continuous ICP monitoring for 15 minutes, there was no universal pattern to ICP after clamping an EVD. The conditional probability of observing a patient's highest ICP, if ICP is observed for 5 minutes, is 0.0181. The conditional probability increased to 0.0402 if ICP is observed for 10 minutes. There were no instances of ICP elevation requiring intervention. The results suggest that at least 5 minutes of ICP monitoring is safe and is required to provide an ICP value that reflects true ICP.
The WNK (With No K-Lysine) family of proteins is widely expressed and has been shown to promote blood pressure homeostasis through a variety of mechanisms. Members of this family have been reported to affect sodium/chloride cotransporters, sodium/potassium/chloride cotransporters, potassium/chloride cotransporters, the renal outer medullary potassium channel, and the epithelial sodium channel, directly and indirectly. Mutations in WNK1 and WNK4 were shown to cause pseudohypoaldosteronism type II, a Mendelian disorder characterized by hypertension, hyperkalemia, and acidosis. Because of the complexity of the renal system, it has been difficult to completely define the role of these kinases in kidney function. This article reviews current knowledge of the role of these proteins in ion homeostasis and volume control.
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