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. INTRODUCTIONADP-ribosylation factors (ARFs) are small GTPases involved in membrane transport, maintenance of organelle integrity, and activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase Chavrier and Goud, 1999;Takai et al., 2001;D'Souza-Schorey and Chavrier, 2006). ARF1 is mainly associated with the Golgi apparatus, and it regulates vesicle budding of transport events (Stearns et al., 1990;Balch et al., 1992). ARF6 can regulate peripheral membrane dynamics and actin rearrangements at the plasma membrane (Donaldson, 2003;Sabe, 2003) such as stress fibers disassembly (D'Souza-Schorey et al., 1997;Boshans et al., 2000), formation of plasma membrane protrusions and ruffles (Radhakrishna et al., 1996;D'Souza-Schorey et al., 1997;Franco et al., 1999), cell migration (Palacios et al., 2001;Santy and Casanova, 2001), cell adhesion (Palacios et al., 2001), and regulation of endosomal membrane traffic (D'Souza-Schorey et al., 1995;Radhakrishna and Donaldson, 1997). Similar to other guanosine triphosphate (GTP)-binding proteins, ARF function depends on the highly controlled binding and hydrolysis of GTP. The conformational changes that accompany the binding of GDP or GTP can lead directly to changes in the affinity of the GTPase for proteins, lipids, and membranes. Interconversion between the two states of ARFs is most likely achieved through guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) Donaldson and Jackson, 2000;Jackson and Casanova, 2000).ARF-GEFs are linked to vesicular trafficking and ...

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plusend-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in woundhealing assays were remarkably similar. Treatment of cells with BIG1-or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

ARF-like proteins (ARLs) are distinct group of members of the ARF family of Ras-related GTPases. Although ARLs are very similar in primary structure to ARFs, their functions remain unclear. We cloned mouse (m) and human (h) ARL5 cDNAs to characterize the protein products and their molecular properties. mARL5 mRNA was more abundant in liver than in other adult tissues tested. mARL5, similar to mARL4, was developmentally regulated and localized to nuclei. hARL5 interacted with importin-α through its C-terminal bipartite nuclear localization signal. When expressed in COS-7 cells, mutant hARL5(T35N), which is predicted to be GDP bound, was concentrated in nucleoli. The N-terminus of hARL5, like that of ARF, was myristoylated. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL5(Q80L),predicted to be GTP bound, interacted with heterochromatin protein 1α(HP1α), which is known to be associated with telomeres as well as with heterochromatin, and acted as a transcriptional suppressor in mammalian cells. The interaction was reproduced in COS cells, where hARL5(Q80L) was co-immunoprecipitated with HP1α. hARL5 interaction with HP1α was dependent on the nucleotide bound, and required the MIR-like motif. Moreover,hARL5(Q80L), but not hARL5 lacking the MIR-like motif, was partly co-localized with overexpressed HP1α. Our findings suggest that developmentally regulated ARL5, with its distinctive nuclear/nucleolar localization and interaction with HP1α, may play a role(s) in nuclear dynamics and/or signaling cascades during embryonic development.

Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells. C ell migration requires the coordinated spatiotemporal regulation of actomyosin function for alterations in cell shape and adhesion. Nonmuscle myosin II (NM II) is critical for regulation of structural remodeling and migration of nonmuscle cells. NM II comprises two heavy chains of 230 kDa, two 20-kDa regulatory light chains (RLCs), and two 17-kDa essential light chains that assemble into bipolar filaments with actin-stimulated ATPase activity. The resultant contractility and actin crosslinking drive assembly of actin filaments that form the actin cytoskeleton (1). In mammalian cells, NM II heavy chain proteins (NMHC) IIA, IIB, and IIC encoded, respectively, by three genes (Myh9, Myh10, and Myh14), are 60-80% identical. Three hexameric isoforms of NM II named NM IIA, IIB, and IIC, with both shared and unique properties, are designated by NMHC component, which accounts for their differences, and all function in events like cell polarization, migration, and adhesion that involve mechano-sensing and motility (2, 3).Reversible phosphorylation of specific amino acids in the pair of RLCs and/or the heavy chains alters NM II activity. Without phosphorylation, NM II folds into a compact structure, in which one head interacts with the second head of the same molecule. The tails interact also with the heads to prevent ATP hydrolysis and thereby filament assembly. Phosphorylation of RLC on T18 and/or S19 disrupts head-head and head-tail interactions and promotes the formation of contractile actin bundles by enhancing the actin-activated ATPase of NM II (1). Rho-associated protein kinase (ROCK) (4), myotonic dyst...