Potassium-Chloride Cotransporter 3 Interacts with Vav2 to Synchronize the Cell Volume Decrease Response with Cell Protrusion Dynamics

Adèle Salin-Cantegrel,Rebecca Gaudet,Sarah Rasheed,François M Charron,Jean-Yves Lapointe,Patrick A Dion,Guy A Rouleau,Janet Laganière,Masoud Shekarabi

doi:10.1371/journal.pone.0065294

Abstract

Loss-of-function of the potassium-chloride cotransporter 3 (KCC3) causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD) response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF) Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC.

Highlights

Mutations in the potassium chloride cotransporter 3 (KCC3) gene cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disorder characterized by neuronal and axonal swelling [1,2,3]
In order to determine whether the poly-proline site was functionally required, we used a mutant form of the cotransporter lacking the vast majority of the C-terminus (KCC3DCterm) and performed the mutagenesis of two of the proline residues in the wild type KCC3 to generate another mutant form, KCC3mPro (Figure 1B)
Since KCC3 mutations can affect its intracellular transit in mammalian cells [5], we first determined whether the generated forms could reach the oocyte plasma membrane

Summary

Introduction

Mutations in the potassium chloride cotransporter 3 (KCC3) gene cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disorder characterized by neuronal and axonal swelling [1,2,3]. In response to cell swelling, most potassium chloride cotransporters (KCC1, KCC3 and KCC4) are activated and cause an electroneutral ion co-transport as part of the regulatory volume decrease (RVD) response [13]. Hypo-osmotic challenges cause dramatic re-organization of cortical actin while F-actin destabilizing agents slow down swelling recovery, providing evidence of a functional role of actin reorganization in the RVD response [14,15,16,17]. Vav is a member of the Vav family of protooncogenes and has been involved in a large array of basic cellular processes such as cell proliferation, cell adhesion, cell spreading and cell migration as well as in neuronal specific dynamics such as neurite outgrowth, dendrite remodeling and commissural axon migration in the central nervous system [20,21,22]

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Conclusion