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Vol. 18, Issue 11, 4420-4437, November 2007

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ARL4D Recruits Cytohesin-2/ARNO to Modulate Actin Remodeling

Chun-Chun Li^*, Tsai-Chen Chiang^*, Tsung-Sheng Wu^*, Gustavo Pacheco-Rodriguez, Joel Moss, and Fang-Jen S. Lee^*

*Institute of Molecular Medicine, College of Medicine, National Taiwan University, and Department of Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan; and Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1434

Submitted February 20, 2007; Revised August 21, 2007; Accepted August 27, 2007
Monitoring Editor: Francis Barr

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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Fang-Jen S. Lee (fangjen{at}ha.mc.ntu.edu.tw)

Abbreviations used: AD, activation domain; ARF, ADP-ribosylation factor; ARL, ARF-like protein; ARNO, ARF nucleotide-binding-site opener; BD, binding domain; GAP, GTPase-activating protein; GEF, guanine nucleotide-exchange factor; GST, glutathione S-transferase; NLS, nuclear localization signal; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PIP₃, phosphatidylinositol 3,4,5-trisphosphate; TBS, Tris-buffered saline; GRP1, general receptor for phosphoinositides 1.