Kenectin 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 dow nstream signaling, we performed a yeast two-hybrid screen from which we ide ntified kinectin, a 156-kDa protein that binds in vitro to conventional kin esin and enhances microtubule-dependent kinesin ATPase activity. We show th at RhoG(GTP) specifically interacts with the central domain of kinectin, wh ich also contains a RhoA binding domain in its C terminus. Interaction was confirmed by coprecipitation of kinectin with active RhoG(G12V) in COS-7 ce lls. RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, main ly in the endoplasmic reticulum but also in lysosomes. Kinectin distributio n in REF-52 cells is modulated according to endogenous RhoG activity. In ad dition, by using injection of anti-kinectin antibodies that challenge RhoG- kinectin interaction or by blocking anti-kinesin antibodies, we show that R hoG morphogenic activity relies on kinectin interaction and kinesin activit y. Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytos keletal effects and switches cells to a RhoA phenotype when RhoG activity i s inhibited or microtubules are disrupted. The functional links among RboG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal trans port is facilitated by RhoG activation or kinectin overexpression and is se verely stemmed upon RhoG inhibition. These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kine ctin also mediates RhoG- and RhoA-dependent antagonistic pathways.