Abstract
Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Biotinylation
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Blotting, Western
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Brain / metabolism
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Electrophoresis, Polyacrylamide Gel
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Immunohistochemistry
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Mice
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Mice, Inbred C57BL
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Microscopy, Fluorescence
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Mitogen-Activated Protein Kinases / metabolism
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Models, Anatomic
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Molecular Sequence Data
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Mutation
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Nervous System / metabolism
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Oocytes / metabolism
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Phosphorylation
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Precipitin Tests
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Protein Binding
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Protein Serine-Threonine Kinases / chemistry*
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Protein Serine-Threonine Kinases / metabolism
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Protein Structure, Tertiary
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Sciatic Nerve / metabolism
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Sequence Homology, Nucleic Acid
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Sodium-Potassium-Chloride Symporters / chemistry*
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Sodium-Potassium-Chloride Symporters / metabolism
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Sodium-Potassium-Chloride Symporters / physiology
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Solute Carrier Family 12, Member 2
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Spinal Cord / metabolism
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Stress, Physiological
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Tissue Distribution
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Two-Hybrid System Techniques
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Xenopus Proteins / chemistry*
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Xenopus Proteins / metabolism
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Xenopus laevis / metabolism
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p38 Mitogen-Activated Protein Kinases
Substances
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SLC12A2 protein, human
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Slc12a2 protein, mouse
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Sodium-Potassium-Chloride Symporters
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Solute Carrier Family 12, Member 2
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Xenopus Proteins
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OXSR1 protein, human
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Stk39 protein, mouse
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Protein Serine-Threonine Kinases
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STK39 protein, Xenopus
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases