Abstract
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-alanine-rich 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 two-hybrid assays and confirmed via glutathione S-transferase pull-down experiments. Evidence for in vivo interaction was established by co-immunoprecipitation 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.
Highlights
Environmental change in a variety of physiological contexts elicits cellular responses via the activation of cell surface receptors
Sequence analysis revealed that this clone represents the regulatory domain of the serine-threonine kinase Ste20-related proline-alaninerich kinase (SPAK), which was first cloned from rat brain by Ushiro et al [18]
On the basis of its structure, with a regulatory domain located at the carboxyl terminus and the catalytic domain located at the amino terminus, SPAK can be classified within the germinal center kinase (GCK) subgroup, the prototype of which is SPS1 (Fig. 1A)
Summary
Environmental change in a variety of physiological contexts elicits cellular responses via the activation of cell surface receptors. We performed a yeast two-hybrid screen to search for key proteins that interact with cation chloride cotransporters. We identified the serine-threonine kinases SPAK and OSR1 as direct interactors of several members of the cation chloride cotransporter family.
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