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GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel.

ABSTRACT: CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7alpha and GLC-7beta. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle- and cell volume-regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl- content, cell volume, and epithelial fluid secretion.

SUBMITTER: Denton J

PROVIDER: S-EPMC2217494 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel.

Denton Jerod J Nehrke Keith K Yin Xiaoyan X Morrison Rebecca R Strange Kevin K

The Journal of general physiology 20050201 2

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7alpha and GLC-7beta. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner ...[more]

PMID: 15684092

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Project description: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-7alpha and GLC-7beta. 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.

Project description:CLC anion channels are homodimeric proteins. Each subunit is comprised of 18 α-helices designated "A-R" and an intracellular carboxy-terminus containing two cystathionine-β-synthase (CBS1 and CBS2) domains. Conformational coupling between membrane and intracellular domains via poorly understood mechanisms is required for CLC regulation. The activity of the C. elegans CLC channel CLH-3b is reduced by phosphorylation of a carboxy-terminus "activation domain," which disrupts its interaction with CBS domains. CBS2 interfaces with a short intracellular loop, the H-I loop, connecting membrane helices H and I. Alanine mutation of a conserved H-I loop tyrosine residue, Y232, prevents regulation demonstrating that the loop functions to couple phosphorylation-dependent CBS domain conformational changes to channel membrane domains. To gain further insight into the mechanisms of this coupling, we mutated conserved amino acid residues in membrane helices H and I. Only mutation of the H-helix valine residue V228 to leucine prevented phosphorylation-dependent channel regulation. Structural and functional studies of other CLC proteins suggest that V228 may interact with Y529, a conserved R-helix tyrosine residue that forms part of the CLC ion conduction pathway. Mutation of Y529 to alanine also prevented CLH-3b regulation. Intracellular application of the sulfhydryl reactive reagent MTSET using CLH-3b channels engineered with single-cysteine residues in CBS2 indicate that V228L, Y529A, and Y232A disrupt putative regulatory intracellular conformational changes. Extracellular Zn2+ inhibits CLH-3b and alters the effects of intracellular MTSET on channel activity. The effects of Zn2+ are disrupted by V228L, Y529A, and Y232A. Collectively, our findings indicate that there is conformational coupling between CBS domains and the H and R membrane helices mediated by the H-I loop. We propose a simple model by which conformational changes in H and R helices mediate CLH-3b regulation by activation domain phosphorylation.

Dataset Information

GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel.

Publications

GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel.

Similar Datasets

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