Mouse anti-Human OSR1 monoclonal antibody, clone 2E6

The oxidative-stress-responsive kinase 1 ( OSR1) and the STE20/SPS1-related proline/alanine-rich kinase ( SPAK) are key enzymes in a signalling cascade regulating the activity of Na+/ K+/2Cl(-) co-transporters ( NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal ( CCT) domain, which recognizes a unique peptide ( Arg-Phe-Xaa-Val) motif present in OSR1-and SPAK-activating kinases ( with-no-lysine kinase 1 ( WNK1) and WNK4) as well as its substrates ( NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-PheXaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.

SPAK and OSR1 are two protein kinases that play important roles in regulating the function of numerous ion co-transporters. They are activated by two distinct mechanisms that involve initial phosphorylation at their T-loops by WNK kinases and subsequent binding to a scaffolding protein termed MO25. To understand this latter SPAK and OSR1 regulation mechanism, we herein show that MO25 binding to these two kinases is enhanced by serine phosphorylation in their highly conserved WEWS motif, which is located in their C-terminal domains. Furthermore, we show that this C-terminal phosphorylation is carried out by WNK kinases in vitro and involves WNK kinases in cells. Mutagenesis studies revealed key MO25 residues that are important for MO25 binding and activation of SPAK and OSR1 kinases. Collectively, this study provides new insights into the MO25-mediated activation of SPAK and OSR1 kinases, which are emerging as important players in regulating ion homeostasis. (C) 2018 Elsevier Inc. All rights reserved.