Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel
| Autor: | Rebecca A. Falin, Rebecca Morrison, Kevin Strange, Amy-Joan L. Ham |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Cytoplasm
Voltage-dependent anion channel Physiology Molecular Sequence Data Gating Protein Serine-Threonine Kinases Mass Spectrometry Article Dephosphorylation 03 medical and health sciences 0302 clinical medicine Chloride Channels Genetic model Animals Humans Voltage-Dependent Anion Channels Amino Acid Sequence Phosphorylation Caenorhabditis elegans Caenorhabditis elegans Proteins Cell Size 030304 developmental biology 0303 health sciences Binding Sites Protein-Serine-Threonine Kinases biology Kinase Articles Cell biology Kinetics biology.protein Signal transduction Ion Channel Gating Protein Kinases 030217 neurology & neurosurgery Signal Transduction |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| DOI: | 10.1085/jgp.200810080 |
| Popis: | Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl− cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events. |
| Databáze: | OpenAIRE |
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