Kinectin is a key effector of RhoG microtubule-dependent cellular activity.
RhoG is a member of the Rho family of GTPases that activates Rac1 and Cdc42 through a microtubule-dependent pathway. To gain understanding of RhoG downstream signaling, we performed a yeast two-hybrid screen from which we identified kinectin, a 156-kDa protein that binds in vitro to conventional kinesin and enhances microtubule-dependent kinesin ATPase activity. We show that RhoG(GTP) specifically interacts with the central domain of kinectin, which also contains a RhoA binding domain in its C terminus. Interaction was confirmed by coprecipitation of kinectin with active RhoG(G12V) in COS-7 cells. RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, mainly in the endoplasmic reticulum but also in lysosomes. Kinectin distribution in REF-52 cells is modulated according to endogenous RhoG activity. In addition, by using injection of anti-kinectin antibodies that challenge RhoG-kinectin interaction or by blocking anti-kinesin antibodies, we show that RhoG morphogenic activity relies on kinectin interaction and kinesin activity. Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytoskeletal effects and switches cells to a RhoA phenotype when RhoG activity is inhibited or microtubules are disrupted. The functional links among RhoG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal transport is facilitated by RhoG activation or kinectin overexpression and is severely stemmed upon RhoG inhibition. These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kinectin also mediates RhoG- and RhoA-dependent antagonistic pathways.
Vignal
E
E
Centre de Recherche en Biochimie Macromoléculaire, CNRS-UPR1086, 34293 Montpellier cedex 5, France.
Blangy
A
A
Martin
M
M
Gauthier-Rouvière
C
C
Fort
P
P
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D001798:Blood Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins; D018715:Microscopy, Video; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D019138:Antibodies, Blocking; D001798:Blood Proteins; C088547:KTN1 protein, human; D008565:Membrane Proteins; C489578:Rhog protein, rat; C075397:RHOG protein, human; D020558:GTP Phosphohydrolases; D016547:Kinesins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein