Functional Insights into the Activation Mechanism of Ste20-related Kinases

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.