The Distal Convoluted Tubule Defining Transcription Factor, Spalt Like 3 (Sall3), is Regulated by WNK Signaling

The DCT-specific transcription factor (TF) Sall3 is essential for DCT differentiation and maintenance of fate. RNAseq and molecular studies revealed that Sal3 gene expression is induced by WNK-SPAK-dependent signaling, but it is not clear if post-transcriptional processes also regulate it. Here, we test the role of WNK signaling and the Cullin 3 (CUL3)-KLHL3 ubiquitin ligase complex in regulating Sall3 protein expression. Sall3 protein levels in the DCT rapidly increased following the activation of WNK-SPAK signaling in physiologic and genetic mouse models. To investigate the role of WNK kinases regulating Sall3 protein abundance, we performed molecular reconstitution studies in WNK knockout (KO) Human Embryonic Kidney (HEK) cells compared to Wild-type HEK cells. In contrast to robust expression of Sall3 following Sall3 transfection in WT HEK cells, transfection of Sall3 in WNK KO HEK cells resulted in minimal Sall3 protein expression. MG132, a known proteosome inhibitor, rescued Sall3 expression in the WNK KO cells but had no effect in WT cells. This finding suggests that Sall3 stability is determined by a WNK dependent proteasomal degradation pathway. Based on this observation, we hypothesized that the CUL3-KLHL3 E3 ubiquitin ligase complex, known to be involved in the WNK signaling pathway, might be responsible for this regulation. To test whether Sall3 interacts with KLHL3, we transfected WT HEK cells with Sall3 and KLHL3 with a C-terminal HA tag. An immunoprecipitation assay was performed, and western blot analysis revealed an interaction between Sall3 and KLHL3. In summary, this study examines the regulation of Sall3, a transcription factor critical for DCT differentiation, by the WNK signaling pathway. Our results reveal that WNK signaling regulates Sall3 protein expression through a proteasomal degradation pathway that may be facilitated by KLHL3 interaction, and CUL3-KLHL3 dependent ubiquitination. NIDDK This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.