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PLoS One
2013 May 15;85:e65294. doi: 10.1371/journal.pone.0065294.
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Potassium-chloride cotransporter 3 interacts with Vav2 to synchronize the cell volume decrease response with cell protrusion dynamics.
Salin-Cantegrel A
,
Shekarabi M
,
Rasheed S
,
Charron FM
,
Laganière J
,
Gaudet R
,
Dion PA
,
Lapointe JY
,
Rouleau GA
.
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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.
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Figure 2. Evaluation of wild-type and mutant KCC3ΔCterm, KCC3mPro functions in Xenopus oocyte flux assays.(A) Wild-type KCC3 and mutant KCC3ΔCterm, KCC3mPro transit to the plasma membrane in Xenopus Laevis oocytes. Immunofluorescence staining of KCC3 using a specific antibody shows all generated forms at the plasma membrane of the injected oocytes. Control oocytes were injected with water instead of the wild-type and mutant KCC3 cRNAs. (B) Directed mutagenesis of the proline motif leads to impaired function of KCC3 in Xenopus Laevis oocyte flux assay. 86Rb+ flux of wild-type and mutant KCC3 is measured under hypotonic conditions in presence or absence of KCCs inhibitor, furosemide.
Figure 3. KCC3 proline-rich segment mediates binding to Vav2 SH3-domain.The proline-rich sequence interacts with a collection of SH3 domains detected after incubation of a biotinylated wild-type peptide (LLNMPGPPRNPEGDE) with a commercially available SH3 domain array (Panomics). Wild-type peptide binds to the second SH3 domain (D2) of the Vav proteins Vav2 and Vav3 but not to the SH3-domain of cortactin. To control for binding specificity of the wild-type peptide on the array, a peptide mutated for two proline residues (LLNMPGQARNPEGDE) was used in parallel experiments.
Figure 4. Vav2 interacts with the C-terminal domain of KCC3.(A) Vav2 endogenously expressed by HeLa cells binds to the C-terminal domain of KCC3 in GST-pull down assays. Lysate = whole protein lysate extracted from HeLa cells; Lysate* = pre-cleared HeLa cell protein lysate incubated with sepharose beads; GST = pre-cleared protein lysate incubated with GST only; GST-WT = pre-cleared protein lysate incubated with chimeric protein where KCC3 C-teminus was fused to GST; GST-mPro = pre-cleared protein lysate incubated with chimeric protein where KCC3 C-terminus was mutated for two prolines (PP→QA) and fused to GST. (B) KCC3 and Vav2 are endogenously expressed in HeLa cells. Transient over-expression of KCC3 in HeLa cells is associated with abundant accumulation of the wild-type protein (but not the proline mutated form) in cellular protrusions. (C) Vav2 and KCC3 co-immunoprecipitate together in HeLa cells. Lysate = whole protein lysate extracted from HeLa cells; Mock = protein lysate extracted from HeLa cells transfected with the control empty vector; KCC3, KCC3mPro and KCC3ΔCterm = protein lysate from HeLa cells transfected with the indicated KCC3 form.
Figure 5. KCC3 colocalizes with the active form of Vav2 in actin-rich membrane protrusions induced by hypotonic conditions.(A) Distribution of wild-type and mutant KCC3 forms in transiently transfected HeLa cells. Note the aberrant distribution of the KCC3 mutant forms in the cytoplasm. (B) The active form of Vav2 accumulates with wild-type KCC3 but not with KCC3mPro or KCC3ΔCterm at the cell periphery. Wild-type KCC3 accumulates with pVav2 in actin-rich plasma membrane protrusion under hypotonic conditions (left panel). These results were obtained after a 10 min treatment either in an isotonic or in a hypotonic medium. In the merged images, KCC3 reactivity is indicated in red, pY-Vav2 is indicated in green and polymerized actin detected by phalloidin is indicated in blue. Arrows indicate co-localisation; the stars (*) indicate actin-rich membrane not showing KCC3 immuno-detection; the arrowheads indicate actin-rich membrane showing KCC3 immuno-reactivity but not pVav2 co-localisation.
Figure 6. KCC3 is involved in processes dependent of actin structural dynamic.(A) Involvement of KCC3 in the formation of in cell protrusions. KCC3 co-localizes with the active phosphorylated form of Vav2 at lamellipodia-rich migrating front of HeLa cells that stably overexpress KCC3 (top panel). HeLa cells stably expressing the wild-type form of KCC3 were subjected to a hypotonic choc and show intense co-localization of KCC3 and pY-Vav2 to hypotonicity-induced membrane protrusions (Low panel). Involvement of KCC3 in these processes was assessed after a 5 min treatment in this experiment. On the merged image, KCC3 is indicated in red and Vav2 is stained in green. Co-localization is observed by the overlapping of the two stainings seen as a yellow signal. (B) KCC3 role in cell migration. Stable overexpression of KCC3 in cervical cancer (HeLa) cells accelerates wound closure in scratch assays experiments. (C) Spreading assay determines novel role of KCC3 in cell spreading. HeLa cells were visualized for their polymerized actin with rodamin-phalloidin. Quantification of the spreading cells on poly-lysine coated and uncoated slides (n = 3).
Figure 1. Identification of a functional proline-rich motif in the C-terminal domain of KCC3.(A) Protein sequence of the KCC3 C-terminal domain. The C-terminus of KCC3 contains a distal poly-proline motif indicated in bold characters which is predicted to be a binding site for SH3 domains with a non-canonical class I recognition specificity (ELM motif data base). The C-terminus also contains a hydrophobic tetrad and a predicted Tyrosine residue (both underlined) involved in cation-chloride co-transporter trafficking. Predicted PDZ domains interacting motifs are shown in italic. Three HMSN/ACC non-sense mutations are indicated by a dot (.). The phosphorylated sites are indicated by a star (*). (B) KCC3 C-terminal domain is highly conserved in the proline-rich region. The motif is conserved at 100% between KCC3 homologs but also between KCC3 orthologs KCC1, KCC2 and KCC4. The proline stretch was mutated in order to generate mPro mutant forms of KCC3 polypeptide and protein.
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