OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant

Abstract

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains

“…Also, the K D for the 18-mer was 1.16 and 2.5 µM for SPAK and OSR1 respectively with traditional multicycle kinetics ( Figure 4B and 4D ). These K D values are comparable to those reported previously (Supporting Table S2) [17, 19, 20, 23] using SPR and fluorescence polarisation although most of these studies employed GST-tagged SPAK and OSR1 CCT domains, which will be dimeric. This may account for some of the differences observed in values in the literature.…”

STK39 is a novel kinase contributing to the progression of hepatocellular carcinoma by the PLK1/ERK signaling pathway

“…As shown in Figure 6 F, overexpression of STK39 in 293T cells obviously promotes the phosphorylation of NKCC1 (the phosphorylation of NKCC1 as a positive control), PLK1, CRAF and ERK1/2 43 , 44 . However, catalytically inactive STK39 (D210A) and WNK1 insensitive STK39 (T231A) could not facilitate the phosphorylation of PLK1 and ERK1/2 45 , 46 . Moreover, knockdown of STK39 in HuH7 cells significantly decreased PLK1, CRAF, MEK1/2 and ERK1/2 phosphorylation, while overexpression of STK39 in HCCLM3 cells enhanced these processes (Figure 6 G-H).…”

Osmosensing by WNK Kinases

“…The phosphatase inhibitor orthovanadate prepared as described ( Zhou and Zhang, 1999 ) was added to the protein solutions to 2 mM. The GST-OSR1(314-344) (gOSR) encompassing the phosphorylation site S325 was a gift of Clinton Taylor and Melanie Cobb ( Taylor et al. , 2018 ).…”