The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.
The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na
+
and K
+
balance, blood volume, and arterial blood pressure, by acting in the aldosterone‐sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto‐oncogene PIM3 (
P
roviral
I
ntegration Site of
M
oloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin‐regulated group of kinases, is stimulated by aldosterone in vitro (mCCD
cl1
cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline
Pim3
−
/
−
mouse model, we found that these mice have an upregulated Renin‐Angiotensin‐Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na
+
‐deficient diet. Surprisingly, we did not observe any obvious salt‐losing phenotype in
Pim3
KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na
+
transport proteins. These observations suggest that the potential effects of the loss of the
Pim3
gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of
Pim3
−
/
−
mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone‐induced protein, but its precise role in aldosterone‐dependent renal homeostasis remains to be determined.
Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.
Background:MEZI, a novel oral cereblon E3 ligase modulator (CELMoD ™ ) with enhanced tumoricidal and immune-stimulatory effects compared with IMiD ® agents, induces maximal Ikaros/Aiolos degradation, leading to increased apoptosis in MM cells. Preclinically, MEZI demonstrated potent synergy with DEX, proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies (mAbs). In phase 1 of CC-92480-MM-001 (NCT03374085), the recommended phase 2 dose (RP2D) of MEZI + DEX (Mezi-d) was selected at 1 mg once daily for 21/28 days.
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