Cation Chloride Cotransporters Interact with the Stress-related Kinases Ste20-related Proline-Alanine-rich Kinase (SPAK) and Oxidative Stress Response 1 (OSR1)

Kerstin Piechotta, Jianming Lu, Eric Delpire
2002 Journal of Biological Chemistry  
Cells respond to stress stimuli by mounting specific responses. During osmotic and oxidative stress, cation chloride cotransporters, e.g. Na-K-2Cl and K-Cl cotransporters, are activated to maintain fluid/ion homeostasis. Here we report the interaction of the stress-related serine-threonine kinases Ste20-related proline-alaninerich kinase (SPAK) and oxidative stress response 1 (OSR1) with the cotransporters KCC3, NKCC1, and NKCC2 but not KCC1 and KCC4. The interaction was identified using yeast
more » ... tified using yeast two-hybrid assays and confirmed via glutathione S-transferase pull-down experiments. Evidence for in vivo interaction was established by coimmunoprecipitation of SPAK from mouse brain with anti-NKCC1 antibody. The interacting region of both kinases comprises the last 100 amino acids of the protein. The SPAK/OSR1 binding motif on the cotransporters consists of nine residues, starting with an (R/ K)FX(V/I) sequence followed by five additional residues that are essential for binding but for which no consensus was found. Immunohistochemical analysis of choroid plexus epithelium revealed co-expression of NKCC1 and SPAK on the apical membrane. In contrast, in choroid plexus epithelium from NKCC1 null mice, SPAK immunostaining was found in the cytoplasm. We conclude that several cation chloride co-transporters interact with SPAK and/or OSR1, and we hypothesize that this interaction might play a role during the initiation of the cellular stress response. Environmental change in a variety of physiological contexts elicits cellular responses via the activation of cell surface receptors. Depending on the nature of the stimulus, different protein families are involved. Osmotic and oxidative changes, for instance, have been shown to activate cation chloride cotransporters as well as stress signaling pathways. Cation chloride cotransporters are intrinsic membrane proteins that move Na ϩ , K ϩ , and Cl Ϫ ions across plasma membranes in a tightly coupled electroneutral fashion. They mediate secondary active transport and are driven by the gradients generated by the Na ϩ /K ϩ -ATPase. The cation chloride cotransporter family consists of at least seven members. A group of three Na ϩ -dependent inward cotransporters comprises one Na-Cl cotransporter
doi:10.1074/jbc.m208108200 pmid:12386165 fatcat:rel2gavfijh4xcyuikldpgvmia