Functional Insights into the Activation Mechanism of Ste20-related Kinases

Abstract

Mammalian Ste20-related kinases modulate salt transport and ion homeostasis through physical interaction and phosphorylation of cation-chloride cotransporters. Identification of a sea urchin (Strongylocentrotus purpuratus) ortholog of the mouse Oxidative Stress Response 1 (OSR1) kinase prompted the cloning and testing of the functional effect of a non-mammalian kinase on a mammalian cotransporter. Heterologous expression of sea urchin OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevisoocytes activated the cotransporter indicating evolutionary conservation of the WNK4-OSR1-NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full-length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras by replacing specific portions of the suOSR1 gene with complimentary mouse OSR1 sequences. Co-expression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which possibly prevented the kinase from acquiring an active conformation. Interestingly, non-functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co-expressed in frog oocytes. Sea urchin/mouse OSR1 chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20-related kinases.

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

“…Indeed, proline-rich regions are believed to bring proteins together to increase the likelihood of subsequent interactions (39). The PAPA box, (AP) 6 , of the light chain myosin kinase was shown to target the kinase to actin, thereby allowing their direct interaction (40). Hence we can speculate that the presence of the PAPA box could facilitate the transport of SPAK to the apical membrane, where it interacts with the sodium chloride cotransporter NKCC2.…”

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

“…Recent studies with WNK4 KO mice indicate that physiologic activation of SPAK, OSR1, and NCC by a low sodium diet are all dependent on WNK4 (21). However, the requirement for regulatory co-factors, such as the calcium-binding protein 39 (Cab39, also called mouse protein-25, MO25), which activates SPAK/OSR1 in vitro by binding to and stabilizing the active conformation (22,23) is presently unknown. Most importantly, it still remains unclear whether SPAK, OSR1, or both kinases are required for physiological stimulation of NCC.…”

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

“…Recently, a scaffolding protein distantly related to armadillo proteins named Cab39 (Calcium-binding protein 39 or MO25 for mouse protein 25), has been demonstrated to enhance the WNK4/SPAK-mediated phosphorylation of NCC and NKCC1 (19,20). Cab39 was proposed to facilitate the structural changes in SPAK/OSR1 that lead to a closed or active conformation of the kinases upon phosphorylation of T-loop residue by WNK4.…”