A high dietary potassium (K + ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K + ]ex ) modulate NCC phosphorylation via a Cl − -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT Key points summary:• High dietary potassium (K + ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT).• Using several ex-vivo models, we show that physiological changes in extracellular K + , like those occurring after a K + rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells.• Whilst the increase of NCC phosphorylation upon decreased extracellular K + appears to depend on cellular Cl -fluxes, the rapid NCC dephosphorylation in response to increased extracellular K + is not Cl -dependent.• The Cl -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl -independent pathway may include additional signaling cascades.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Cordycepin (39 deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 39 hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 39 end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 39 end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

Background: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. Methods: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Results: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Conclusions: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKAdependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity. I1 mediates cAMP stimulation of NCC Significance statement:The thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT)is critical for the renal control of ion homeostasis and blood pressure. NCC phosphorylation, and hence activity, is increased by cAMP-elevating stimuli, including β-adrenergic agonists and PTH. We tested the hypothesis that the inhibitor 1 (I1) of protein phosphatase 1 (PP1) mediates the effects of cAMP-elevating hormones on NCC. Using several in vitro and ex vivo approaches, we propose a novel signaling pathway in which a PKA-dependent phosphorylation of I1 inhibits the PP1-dependent dephosphorylation of NCC. This novel pathway may contribute to the physiological regulation of NCC and the development of arterial hypertension in the context of abnormal hormonal stimulation. I1 mediates cAMP stimulation of NCC Abstract Background: The NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is critical for renal Na + reabsorption and blood pressure control. A number of cAMPelevating hormones stimulate NCC phosphorylation, and hence activity. Methods: Using a variety of experiments on mouse ex vivo kidney preparations, we t...

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4 mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.