Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

Abstract

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.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

“…As shown previously (5) and in Table 1, the S742E/S747E mutation reduces the current amplitude, hyperpolarizes the activation voltage, and increases the rise time. These changes fully mimic those induced by GCK-3 phosphorylation of S742 and S747 (5).…”

“…Although the biophysical mechanisms of CLC Cl À and H þ transport have been studied extensively, relatively little is known about the molecular mechanisms of CLC regulation. Numerous CLCs are regulated by phosphorylation (3)(4)(5)(6), intracellular adenosine ligands (7,8), accessory proteins (9,10), and extracellular Ca 2þ (11).…”

“…Channel activation requires dephosphorylation of two C-terminus serine residues, S742 and S747, by type 1 protein phosphatases. Phosphorylation of these residues is mediated by the conserved Ste20 kinase GCK-3 (5,6).…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…This however, is a requirement of the basophilic PAK subfamily [162,163]. Although some GCKs such as Mst1, still phosphorylate substrates containing a basic residue at P(-2), phosphorylation of the GCKs SPAK (Ste20/SPS1-related proline alanine-rich kinase) and OSR1 (oxidative stressresponse 1), further confirms that GCKs do not require basic residues in the consensus sequence of their substrates [161,162,164]. Those GCKs that maintain the preference for a basic residue in the substrate at P-2 however, have been shown to contain a basic residue at P(-2) with respect to their primary site of autophosphorylation [162].…”

“…Ste20 kinases comprise a superfamily of 27 identified mammalian kinases that are divided into p21-activating kinase (PAK) or germinal center kinase (GCK) subfamilies (Appendix G) [161,162]. Since SLK is classified as a GCK-V kinase, our analysis of current Ste20 phosphorylation literature will focus on the GCK subfamily.…”