Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA.
Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G(13)-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin, and sphingosine-1-phosphate. CLIC4 recruitment is strictly dependent on Galpha(13)-mediated RhoA activation and F-actin integrity, but not on Rho kinase activity; it is constitutively induced upon enforced RhoA-GTP accumulation. Membrane-targeted CLIC4 does not seem to enter the plasma membrane or modulate transmembrane chloride currents. Mutational analysis reveals that CLIC4 translocation depends on at least six conserved residues, including reactive Cys35, whose equivalents are critical for the enzymatic function of GSTs. We conclude that CLIC4 is regulated by RhoA to be targeted to the plasma membrane, where it may function not as an inducible chloride channel but rather by displaying Cys-dependent transferase activity toward a yet unknown substrate.
Division of Cell Biology and Center for Biomedical Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Bas
B
Ponsioen
Leonie
L
van Zeijl
Michiel
M
Langeslag
Mark
M
Berryman
Dene
D
Littler
Kees
K
Jalink
Wouter H
WH
Moolenaar
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004252:DNA Mutational Analysis; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; C464459:CLIC4 protein, human; D018118:Chloride Channels; D008246:Lysophospholipids; D011993:Recombinant Fusion Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein; D003545:Cysteine; C032881:lysophosphatidic acid