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), ...
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.
The full molecular consequences of oncogene activation during tumorigenesis are not well understood, but several studies have recently linked oncogene activation to epigenetic silencing of specific genes.1,2 Transcriptional repressor Id1 is overexpressed in many malignancies including melanoma, and Id1 targets include tumor suppressor genes TSP1, CDKN2A (p16) and CDKN1A (p21), which are frequently epigenetically silenced in cancer. We confirmed that both TSP1 and CDKN2A have abnormal promoter region DNa methylation in primary melanoma, but the mechanism by which this silencing occurs remains unknown. Here we explore the effects of stable lentiviral Id1 overexpression on the expression of these Id1 target genes in human melanoma cell lines. Overexpressed Id1 was functional and bound transcriptional activator E2A, but did not sequester E2A from gene promoters and repress gene expression. Therefore, these Id1 target genes were resistant to Id1-mediated gene silencing. Our results suggest that Id1 activation may need to occur at discrete stages in cooperation with additional gene dysregulation to repress and induce epigenetic silencing of tumor suppressor genes during melanoma progression.
The with no lysine (K) 1 (WNK1) protein kinase maintains cellular ion homeostasis in many tissues through actions on ion cotransporters and channels. Increased accumulation of WNK1 protein leads to pseudohypoaldosteronism type II (PHAII), a form of familial hypertension. WNK1 can be degraded via its adaptor-dependent recruitment to the Cullin3-RBX1 E3 ligase complex by the ubiquitin-proteasome system. Disruption of this process also leads to disease. To determine if this is the primary mechanism of WNK1 turnover, we examined WNK1 protein stability and degradation by measuring its rate of decay after blockade of translation. Here, we show that WNK1 protein degradation exhibits atypical kinetics in Hela cells. Consistent with this apparent complexity, we found that multiple degradative pathways can modulate cellular WNK1 protein amount. WNK1 protein is degraded not only by the proteasome, but also by the lysosome. Non-lysosomal cysteine proteases calpain and caspases also influence WNK1 degradation, as inhibitors of these proteases modestly increased WNK1 protein expression. Importantly, we discovered that the E3 ubiquitin ligase UBR5 interacts with WNK1 and its deficiency results in increased WNK1 protein. Our results further demonstrate that increased WNK1 in UBR5-depleted cells is attributable to reduced lysosomal degradation of WNK1 protein. Taken together, our findings provide insights into the multiplicity of degradative pathways involved in WNK1 turnover and uncover UBR5 as a previously unknown regulator of WNK1 protein stability that leads to lysosomal degradation of WNK1 protein.
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