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.
Mesh Terms:
Animals, Antibodies, Blocking, Biological Transport, Blood Proteins, COS Cells, Cytoskeleton, Endoplasmic Reticulum, Fibroblasts, GTP Phosphohydrolases, Gene Expression, Humans, Jurkat Cells, Kinesin, Lysosomes, Membrane Proteins, Microscopy, Video, Microtubules, Phenotype, Protein Binding, Protein Structure, Tertiary, Rats, Transfection, Two-Hybrid System Techniques, cdc42 GTP-Binding Protein, rac1 GTP-Binding Protein, rho GTP-Binding Proteins, rhoA GTP-Binding Protein
Animals, Antibodies, Blocking, Biological Transport, Blood Proteins, COS Cells, Cytoskeleton, Endoplasmic Reticulum, Fibroblasts, GTP Phosphohydrolases, Gene Expression, Humans, Jurkat Cells, Kinesin, Lysosomes, Membrane Proteins, Microscopy, Video, Microtubules, Phenotype, Protein Binding, Protein Structure, Tertiary, Rats, Transfection, Two-Hybrid System Techniques, cdc42 GTP-Binding Protein, rac1 GTP-Binding Protein, rho GTP-Binding Proteins, rhoA GTP-Binding Protein
Mol. Cell. Biol.
Date: Dec. 01, 2001
PubMed ID: 11689693
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