ARL4D Recruits cytohesin-2/ARNO to modulate actin remodeling

Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
Molecular Biology of the Cell (Impact Factor: 4.47). 12/2007; 18(11):4420-37. DOI: 10.1091/mbc.E07-02-0149
Source: PubMed


ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.

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Available from: Fang-Jen Lee
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    • "We tested whether the coiled-coil domain might bind to this auto-inhibitory conformation to provide an additional layer of negative regulation by determining if disrupting the psuedosubstrate conformation prevents binding of the coiled-coil domain to the rest of the protein. The Arf family members, Arf6 and Arl4, can bind to cytohesin PH domains when they are in the GTP-bound state [20]–[22]. This interaction promotes recruitment of the cytohesin to membranes [20]–[22]. "
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    ABSTRACT: Cell migration is regulated by a number of small GTPases, including members of the Arf family. Cytohesins, a family of Arf-activating proteins, have been extensively implicated in the regulation of Arfs during migration and cell shape change. Membrane association of both the Arf and its activating protein is a prerequisite for Arf activation. Therefore regulating the extent of cytohesin membrane association is a mechanism for controlling the initiation of cell movement. We have discovered a novel intramolecular interaction that controls the association of cytohesins with membranes. The presence of the coiled-coil domain reduces the association of cytohesin 2 with membranes. We demonstrate that this domain interacts with more C-terminal regions of the protein. This interaction is independent of another previously identified autoinhibitory conformation. A threonine residue (T276) in the cytohesin 2 PH domain is a target for phosphorylation by Akt. Mutation of this threonine to aspartic acid, to mimic phosphorylation, disrupts the binding of the coiled-coil domain to c-terminal regions and promotes membrane association of cytohesin 2. The presence of a second autoinhibitory interaction in the cytohesins suggests that these proteins can act a signal integrators that stimulate migration only after receive multiple pro-migratory signals.
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    • "Members of the cytohesin family of GEFs are recruited to the plasma membrane based on the affinity of their PH domain for specific phosphatidylinositol phosphates. A further mechanism is through interaction with the GTP bound form of ARF6 and ARL4 which leads to cytohesin recruitment and activation of ARF6 and ARF1 [32-34]. The activation of ARF proteins stimulates signaling pathways that regulate membrane trafficking and cell motility. "
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    • "The ARL4D gene was silenced using synthetic 21-nt RNA duplexes according to previously described protocols [10]. The following pairs of oligonucleotides with hairpin, terminator, and overhanging sequences were annealed and inserted into the BglII–HindIII sites of the pSUPER or pSUPER.gfp/neo "
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