EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6 [Signal Transduction]

October 8th, 2014 by Kanamarlapudi, V.

ARF6 small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. GTP-exchange factors (GEFs) activate ARF6. The Exchange factor for ARF6 (EFA6)R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, PH and CC domains of the other EFA6 family GEFs. Here we report the functional characterisation of EFA6R. Endogenous EFA6R is present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R are targeted to the PM. In vitro, GFP-EFA6R associates weakly but preferentially with PI 4,5-P2 (PIP2) through the PH domain. EFA6R requires both its PH and CC domains localised at the C-terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6RΔCC) was released from the PM into the cytosol upon PIP2 depletion whereas EFA6R release from the PM requires both PIP2 depletion and actin destabilisation. These results suggest that the dual targeting via the PH and CC domains is important for the PM localisation of EFA6R. EFA6R specifically catalyses the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulates ARF6 localisation and thereby actin stress fibers loss. The GEF activity of EFA6R is dependent on the presence of the Sec7 domain. The PH and CC domains are also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R.
  • Posted in Journal of Biological Chemistry, Publications
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