Cation Chloride Cotransporters Interact with the Stress-related Kinases Ste20-related Proline-Alanine-rich Kinase (SPAK) and Oxidative Stress Response 1 (OSR1)
Copyright policy )Cation Chloride Cotransporters Interact with the Stress-related Kinases Ste20-related Proline-Alanine-rich Kinase (SPAK) and Oxidative Stress Response 1 (OSR1)
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.
- Vanderbilt University United States
- Vanderbilt University Medical Center United States
Mice, Knockout, Binding Sites, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Sodium-Potassium-Chloride Symporters, Recombinant Fusion Proteins, Molecular Sequence Data, Intracellular Signaling Peptides and Proteins, Brain, Protein Serine-Threonine Kinases, MAP Kinase Kinase Kinases, Mice, Inbred C57BL, Mice, Oxidative Stress, Animals, Amino Acid Sequence, Sequence Alignment, DNA Primers, Gene Library, Glutathione Transferase
Mice, Knockout, Binding Sites, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Sodium-Potassium-Chloride Symporters, Recombinant Fusion Proteins, Molecular Sequence Data, Intracellular Signaling Peptides and Proteins, Brain, Protein Serine-Threonine Kinases, MAP Kinase Kinase Kinases, Mice, Inbred C57BL, Mice, Oxidative Stress, Animals, Amino Acid Sequence, Sequence Alignment, DNA Primers, Gene Library, Glutathione Transferase
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