Identification of Cytohesin-2/ARNO as an ARL4D Interactor
To identify potential effectors of ARL4D, we used a yeast two-hybrid system and used the ARL4D(Q80L), a putative GTP-bound form of ARL4D, as bait to screen a mouse embryonic cDNA library. We identified several candidate genes, including eight independent clones for cytohesin-2/ARNO. We next assessed specificity among ARF/ARL family members by yeast two-hybrid assays (, A and B). Our data showed that ARNO interacted specifically with ARL4D and ARL4A, but not with ARF1 or other ARLs. Furthermore, ARNO interacted with ARL4D(WT), ARL4A(WT), ARL4D(Q80L), and ARL4A(Q79L), but not with ARL4DΔC, the GTP-binding-defective mutant ARL4D(T35N) or ARL4A(T34N). These results indicated that the interactions were specific, nucleotide dependent, and involved the C terminus of ARL4D.
Figure 1. ARNO interacts with ARL4D. (A) Schematic representation of ARL4D molecule and mutants. G1–G3 are conserved regions important for binding to phosphate/Mg2+ and the guanine base. ARL4D(Q80L) is a putative GTPase-defective mutant, and it is predicted (more ...)
To confirm the ARL4D and ARNO interaction, we performed in vitro GST pull-down assays. We tested whether purified, bacterially expressed GST-ARNO could bind to ARL4D or its mutants (C). GST-ARNO pulled down ARL4D, ARL4D(Q80L), and ARL4D(G2A), but not ARL4D(T35N) or ARL4DΔC. GST alone failed to pull down ARL4D constructs. Next, we used coimmunoprecipitation experiments to demonstrate that ARL4D interacts with ARNO. Proteins immunoprecipitated from lysates of 293T cells coexpressing ARL4D or mutants and FLAG-ARNO with anti-FLAG M2 affinity gel were immunoblotted with antibodies against ARL4D or FLAG. As shown in D, ARL4DWT, ARL4D(Q80L), or ARL4D(G2A), but not ARL4D(T35N) or ARL4DΔC, was coimmunoprecipitated with FLAG-ARNO. Thus, we demonstrated interactions of recombinant ARNO and ARL4D that were nucleotide dependent and involved in the C-terminal NLS domain by using proteins synthesized in bacteria or in 293T cells.
The PH Domain and the Polybasic C Domain of ARNO Are Both Necessary and Sufficient for Interaction with ARL4D
All ARNO clones isolated from the yeast two-hybrid screening contained an intact PH domain and partial Sec7 domain, suggesting that the C-terminal PH domain of ARNO may be involved in the interaction with ARL4D. To identify the specific domains of ARNO responsible for this interaction, a series of ARNO-deletion mutants was generated (A) and tested for the ability to interact with ARL4D in yeast two-hybrid assays (B). We found that the C-terminal 140 amino acids (ARNOCT) alone were sufficient for the interaction; the N-terminal coiled-coil and central Sec7 domains were not required. ARL4D interaction with the ARNO PH domain (ARNOPH) was much weaker than that of the C-terminal PH domain and polybasic c domain (ARNOCT) (B), indicating that, besides the PH domain, the C-terminal polybasic c domain is also important. The C-terminal polybasic stretch of cytohesin-1 and ARNO was reported to be important for its collaboration with the PH domain in membrane recruitment and phospholipids PIP3
binding (Nagel et al., 1998a
; Macia et al., 2000
; Dierks et al., 2001
). To determine whether the basic charge amino acids in the C-terminal polybasic stretch is important for the interaction between ARL4D and ARNO, we used site-directed mutagenesis to generate an ARNO mutant, ARNOC7A, in which the seven basic residues were replaced with alanine. Interestingly, ARNOC7A, like ARNO, showed similar interaction ability for ARL4D (B). This result indicates that the C-terminal basic amino acids are not involved in the interaction between ARL4D and ARNO. These constructs were expressed in relatively equal amounts in the transformed yeast (unpublished data).
Figure 2. The PH domain of ARNO interacts with ARL4D. (A) Schematic representation of ARNO and its deletion mutants. ARNO contains a coiled-coil domain (a.a. 10-63), Sec7 domain (a.a. 72-201), PH domain, and polybasic c domain (a.a.386-399). (B) ARL4D and its activated (more ...)
By coimmunoprecipitation, we further showed that ARL4D(Q80L) was coimmunoprecipitated with FLAG-ARNO and FLAG-ARNOCT, but not FLAG-ARNOΔCT (C). Thus, our results demonstrate that the PH domain and polybasic c domain of ARNO are necessary and sufficient for interaction with ARL4D.
ARNO Does Not Catalyze Nucleotide Exchange on ARL4D
ARNO has previously been shown to catalyze guanine nucleotide exchange on ARF6 in vitro (Frank et al., 1998a
). To determine whether ARL4D is a substrate of ARNO, we measured the ability of ARNO to catalyze the binding of GTPγS on ARF6 and ARL4D. Consistent with the previous observation (Frank et al., 1998a
), ARF6 undergoes spontaneous nucleotide exchange even in the absence of ARNO and addition of ARNO results in a stimulation of GTPγS binding (Supplemental Figure S1A and S1B). However, no stimulation of GTPγS binding was observed when using ARL4D as substrate. Our data indicated that ARNO could not catalyze nucleotide exchange on ARL4D in the GTPγS binding assay.
Localization of ARL4D at the Plasma Membrane Is GTP Dependent
Our previous studies showed that N-terminally enhanced green fluorescent protein (EGFP)-tagged ARL4D is located in nuclei and partially in nucleoli and can interact with importin-α through its C-terminal bipartite NLS (Lin et al., 2002
). However, ARL4D has a myristoylation site at its N terminus and a NLS in its C terminus; thus, epitope tags at either end might sterically hinder and change its conformation, localization, and function. To examine the physiological phenotype of ARL4D, we first used two unique peptides (peptide N corresponding to a.a. 2-18 and peptide B a.a. 139-155 of ARL4D) to generate peptide-specific antibodies (Supplemental Figure S2A). A Blast search using the peptide sequences of ARL4D-N and ARL4D-B did not reveal any homologous peptides. The antibody against peptide B was more sensitive in detecting purified recombinant human ARL4D in low nanogram amounts, whereas no reaction was detected with 100 ng of recombinant human ARL4A, ARF1, or ARF6 proteins (Supplemental Figure S2B). Using this antibody, endogenous ARL4D was detected in three cell lines (Supplemental Figure S2C). The detected ~25-kDa band was similar in size to recombinant ARL4D protein expressed in HeLa cells.
To determine the subcellular localization of ARL4D, we first analyzed overexpression of ARL4D and its mutants in transiently transfected COS-7 cells by indirect immunofluorescence microscopy (A). The stacked images were obtained by using a confocal microscope. The single plane images of transfected cells are shown in Supplemental Figure S3. Interestingly, ARL4D was detected at the plasma membrane, in addition to the nucleus and cytoplasm. Like ARL4D(WT), ARL4D(Q80L) was detected in the nucleus and diffusely throughout the cytoplasm but concentrated most intensely at the plasma membrane, where it associated with areas of membrane folding or ruffles along the periphery of the cell. Notably, cells transfected with ARL4D(Q80L) also demonstrated membranous protrusion structures from their dorsal surface, and ARL4D(Q80L) was also concentrated within these structures. Nuclear, perinuclear punctate, and diffuse cytoplasmic labeling, but much less plasma membrane-associated signals, were detected in cells expressing ARL4D(T35N). Our results indicated that subcellular localization of ARL4D was guanine nucleotide dependent. We further showed that ARL4D(G2A), containing Ala substituted for Gly at position 2, was diffusely distributed in the cytoplasm (A), indicating that N-terminal myristoylation is important for association with the plasma membrane.
Figure 3. Subcellular localization of ARL4D and its mutants. (A) Localization of overexpressed untagged ARL4D. COS-7 cells grown on coverslips were transiently transfected with plasmids encoding ARL4D or its mutants. Forty-eight hours after transfection, cells (more ...)
We next used the ARL4D antibody to detect endogenous ARL4D. B shows the result of an immunoblot antibody competition analysis of total HeLa cell lysate. The detection of a 25-kDa protein was abolished by preincubation of the antibody with the ARL4D-B peptide immunogen but not with ARL4D-N peptide. Moreover, endogenous ARL4D distributed mainly in the membrane fraction after cytosol-membrane fractionation of HeLa cells (C). We also observed that an ~42-kDa band on immunoblots, which was only seen with nuclear fractions, was abolished by the addition of a specific ARL4D-B antigen (, B and C). Whether the ~42-kDa band is the source of the nuclear staining due to endogenous proteins needs to be further investigated. We also examined the subcellular localization of endogenous ARL4D in interphase HeLa cells. Endogenous ARL4D, like overexpression of ARL4D, was detected at the plasma membrane, in addition to the nucleus and cytoplasm (D). PMCA was used as a control. The signals of ARL4D were abolished when the antibody was preincubated with the ARL4D-B peptide used for immunization, but not with DMSO or ARL4D-N peptide.
Both the PH Domain and Polybasic c Domain, but Not GEF Activity of ARNO, Are Required for ARL4D-Induced Recruitment of ARNO to the Plasma Membrane
The PH domain of ARNO is required for its membrane targeting or translocation (Venkateswarlu et al., 1998
). We examined whether ARL4D could affect the subcellular localization of ARNO in transiently transfected COS-7 cells. A showed that FLAG-ARNO was detected as a diffuse signal throughout the cytoplasm and was not clearly observed at the periphery plasma membrane in COS-7 cells. Coexpression of FLAG-ARNO and ARL4D(WT) or its mutants reveals that part of FLAG-ARNO was colocalized with ARL4D(WT) or ARL4D(Q80L) at plasma membrane ruffles and dorsal membranous protrusions. This translocation of ARNO was not observed in cells coexpressing ARL4D(G2A) or ARL4D(T35N) (B). We confirmed the effect of ARL4D(Q80L) on ARNO translocation to the membrane fraction by subcellular fractionation (Supplemental Figure S4). We also observed similar effects of ARL4D on ARNO in HeLa and Madin Darby canine kidney (MDCK) cells (unpublished data). The localization of ARL4D or its mutants was not altered when coexpressed with FLAG-ARNO (compare with and B). Quantitation of the ARNO fluorescence signal confirmed that ARL4D(WT) or ARL4D(Q80L) induced ARNO redistribution to the plasma membrane (, C and D). Moreover, overexpression of ARF1(Q71L) or ARL1(Q71L) did not affect the distribution of FLAG-ARNO (unpublished data). Collectively, these data indicate that ARL4D can induce ARNO redistribution to the plasma membrane and this effect is GTP dependent.
Figure 4. ARL4D induces ARNO redistribution to plasma membrane protrusions and ruffles. COS-7 cells were transfected with FLAG-ARNO alone (A) or cotransfected with FLAG-ARNO and ARL4D mutants (B). Cells were fixed, permeabilized, labeled with anti-FLAG M2 and anti-ARL4D (more ...)
We next examined whether the PH domain or polybasic c domain is required for inducing redistribution of ARNO by ARL4D. COS-7 cells were transfected with ARNOΔCT or ARNOCT and/or ARL4D(Q80L). Similar to that of wild-type ARNO, the localization of ARNOΔCT or ARNOCT is detected in the cytoplasm, and it did not seem to concentrate at the lateral margins (Supplemental Figure S5A and S5B). Although ARL4D(Q80L) could not induce redistribution of ARNOΔCT to the plasma membrane, ARNOCT and ARNOC7A were translocated to ARL4D(Q80L)-enriched plasma membrane in a manner similar to full-length FLAG-ARNO (Supplemental Figure S5B and S5C). Moreover, ARL4D(Q80L) can only recruit less amounts of ARNOPH than full-length ARNO to the plasma membrane (Supplemental Figure S5D). Our data demonstrate that ARL4D mediates redistribution of ARNO through its C-terminal PH domain and polybasic c domain.
ARNO is a GEF for ARF1 and ARF6 (Chardin et al., 1996
; Frank et al., 1998a
); thus, we were interested to learn whether ARL4D-regulated translocalization of ARNO is dependent on ARNO GEF activity. To test this, we constructed a catalytically inactive GEF form of ARNO, ARNO(E156K). When expressed in COS-7 cells, ARNO(E156K) exhibited a distribution similar to that of wild-type ARNO (Supplemental Figure S6A). ARNO(E156K) was coprecipitated by ARL4D(Q80L) (C), and, like the wild-type ARNO, ARNO(E156K) was also recruited to the ARL4D(Q80L)-enriched plasma membrane region (Supplemental Figure S6B). Moreover, coexpression of ARNO(E156K) or down-regulation of endogenous ARNO by siRNA in COS-7 cells did not affect the distribution of ARL4D(WT) (unpublished data). These data suggest that induced redistribution of ARNO by ARL4D does not require ARNO GEF activity.
ARL4D Promotes Activation of ARF6
Translocation of ARNO to the plasma membrane is a critical event for ARNO GEF activity and ARF6 activation. We examined whether ARL4D-induced ARNO membrane-targeting can promote the activation of ARF6. We showed in A that cells cotransfected with ARF6 and ARL4D(Q80L) exhibited colocalization along plasma membrane ruffles and protrusions. Conversely, ARF6 showed little or no colocalization with ARL4D(T35N). We carried out a pull-down assay to detect ARF6 states by using a GST fusion construct containing the VPS27, Hrs, and STAM- and ARF-binding domains of GGA3 (Santy and Casanova, 2001
). ARF6(Q67L) bound to GST-GGA3 was used as a control (B). In COS-7 cells, wild-type ARF6-myc was coexpressed with either hemagglutinin (HA)-ARNO, ARL4D(Q80L), or ARL4D(T35N). ARL4D(Q80L), but not ARL4D(T35N), stimulated ARF6 activation. Consistent with a previous report (Santy and Casanova, 2001
), ARF6 activation was stimulated by ARNO (B). Moreover, cells coexpressed with HA-ARNO and ARL4D(Q80L) increased the GTP-bound form of endogenous ARF6 (C). Together, these results indicate that ARL4D recruits ARNO to the plasma membrane and thus promotes activation of ARF6.
Figure 5. ARL4D promotes activation of ARF6. (A) ARL4D(Q80L) and ARF6 colocalize along plasma membrane ruffles and protrusions. COS-7 cells cotransfected with vectors encoding ARL4D(Q80L) or ARL4D(T35N) together with ARF6-myc were processed for immunofluorescence (more ...)
ARL4D(Q80L) Induces Disassembly of Actin Stress Fibers
Both ARNO and ARF6 have been demonstrated to modulate the assembly and organization of the actin cytoskeleton (Radhakrishna et al., 1996
; D'Souza-Schorey et al., 1997
; Frank et al., 1998b
; Boshans et al., 2000
). Next, we investigated whether the downstream effect of ARL4D-induced ARNO translocation and ARF6 activation might be involved in the organization of the actin cytoskeleton (). Consistent with previous reports (D'Souza-Schorey et al., 1997
; Frank et al., 1998b
; Boshans et al., 2000
), expression of ARNO, but not the catalytically inactive ARNO(E156K) mutant, resulted in the reduction of stress fibers, and a decrease in actin stress fibers was elicited by overexpression of ARF6(Q67L), but not ARF6(T27N) (, A and C). As expected, examination of actin organization in cells expressing ARL4D(Q80L) revealed a loss of stress fibers, and this phenotype was not observed in mock-transfected cells, indicating that the loss of stress fibers is a consequence of ARL4D(Q80L) expression (, A and C). A similar phenotype was seen with the expression of relatively higher levels of wild-type ARL4D (C; data not shown). Expression of ARL4D(T35N) or ARL4D(G2A) did not result in the reduction of stress fibers (, A and C). Quantitative fluorescence analyses for F-actin intensity are shown in C. Together, our data indicate that ARL4D(Q80L), similar to ARNO and ARF6(Q67L), induces disassembly of actin stress fibers.
Figure 6. ARL4D(Q80L) induces actin stress fiber disassembly. (A) COS-7 cells were transfected with an expression vector encoding FLAG-ARNO, FLAG-ARNO(E156K), ARL4D(Q80L), ARL4D(T35N), ARL4D(G2A), ARF6(Q67L)-myc, and ARF6(T27N)-myc, respectively. Forth-eight hours (more ...)
ARL4D Modulates Actin Remodeling through ARNO and ARF6
Having demonstrated that overproduction of ARL4D manipulates the downstream effect in a manner similar to that of ARNO and ARF6, we next examined whether ARL4D may act coordinately with ARNO and ARF6 to modulate actin organization. Two experiments for abolishing GEF activity and reducing the expression level of ARNO helped dissect this possibility. First, cells transfected with ARNO(E156K) showed no effect on stress fiber organization and inhibited the decrease in ARL4D(Q80L)-induced actin remodeling (, B and C). Second, we introduced ARNO or ARL4D siRNAs into COS-7 cells, and we showed that expression of endogenous ARNO and ARL4D were markedly reduced when cells were treated with ARNO-specific and ARL4D-specific siRNAs (D). These siRNAs were specific, and they did not interfere with expression of other proteins, such as calnexin, α-tubulin, or ARF6. We further used siRNA knockdown to ask whether reduction of ARNO could block ARL4D(Q80L)-mediated actin remodeling. The localization of ARL4D(WT) or ARL4D(Q80L) in ARNO knockdown cells remained unchanged; however, ARL4D(Q80L)-induced stress fiber reduction was significantly decreased (E; data not shown). In contrast, reduction of ARL4D had no affect on ARNO-mediated decrease in actin stress fibers (F). It clearly demonstrated that ARNO is the direct downstream effector of ARL4D on the actin remodeling. Consistent with the result from ARNO GEF inactivation, an inactive form of ARF6 (T27N), blocked ARL4D(Q80L)-induced actin remodeling. ARL4D(T35N) did not block ARF6(Q67L)-mediated cytoskeletal rearrangements (, B and C) or interfere with the ARNO-mediated reduction of actin stress fibers (, B and C). These data demonstrate that ARL4D effects on actin remodeling lie upstream of ARNO and ARF6.
Requirement for ARL4D in Cell Migration
Expression of ARNO or activation of ARF6 stimulates MDCK cell migration (Palacios et al., 2001
; Santy and Casanova, 2001
), and suppression of ARF6 blocks invasive and migration activities of breast cancer cells (Hashimoto et al., 2004
). To investigate the potential role of ARL4D in cell migration, we assessed whether knockdown of the endogenous ARL4D would impair cell motility. We used a Transwell migration assay with control siRNA, ARL4D siRNA, and ARNO siRNA in HeLa cells. The cells were plated in the upper chamber containing filters that had been coated with fibronectin on the underside, and they were allowed to migrate for 6 h. As shown in , B and C, HeLa cells transfected with either ARL4D siRNA or ARNO siRNA showed a significantly reduced motility compared with control siRNA. We also used a wound-healing assay to monitor cell migration and obtained similar results. Namely, knockdown of ARL4D or ARNO expression caused a delay in wound closure (D). Quantification of the wound area covered by the migrating monolayer cell is shown in E. Together, these results provide evidence that ARL4D play a physiological role in cell motility.
Figure 7. Requirement for ARL4D in cell migration. (A) HeLa cells transfected with ARL4D siRNA, ARNO siRNA, or a control siRNA were lysed 48 h after transfection, and then they were assayed for expression by immunoblotting. (B and C) HeLa cells transfected with (more ...)
ARL4D-induced Translocation of ARNO to the Plasma Membrane Is Independent of PI3-Kinase Signaling
It has been demonstrated that cytohesin-2/ARNO responds to the PI3-kinase signaling cascade through the selectivity of PH domains for binding PIP3
, and cells stimulated with agonists such as epidermal growth factor (EGF), nerve growth factor (NGF), or insulin showed translocation of cytohesin-2/ARNO from the cytosol to the plasma membrane; this redistribution was inhibited by PI3K inhibitors (wortmannin and LY294002) (Venkateswarlu et al., 1998
; Mansour et al., 2002
). To investigate whether the effects of ARL4D on inducing redistribution of ARNO to the plasma membrane were dependent on PI3K, we first examined the subcellular localization of ARL4D and ARNO in transfected cells when PI3K activity was stimulated by EGF or was blocked by PI3K inhibitors (, A and B). To confirm the inhibition of PI3K–Akt activation, the phosphorylation level of Akt (Ser473) was examined in each experiment (data not shown). As shown in a previous report (Mansour et al., 2002
), EGF stimulated plasma membrane ruffling and translocation of ARNO to membrane ruffles (A). This translocation of ARNO was abrogated by the addition of wortmannin or LY294002 (, A and C). However, the localization of ARL4D at the plasma membrane was not affected by wortmannin or LY294002, indicating that localization of ARL4D is not dependent on PI3K signaling (A). Consistent with this finding, ARL4D-mediated redistribution of ARNO to the plasma membrane was not blocked by wortmannin when cells were incubated with or without EGF (, B and C). Quantitative fluorescence analyses confirm that ARNO redistribution to the plasma membrane by ARL4D was independent on PI3K activation (C).
Figure 8. ARL4D-induced translocation of ARNO is not dependent on PI3K signaling. (A) Inhibitors of PI3K do not inhibit the plasma membrane localization of ARL4D. COS-7 cells transfected with ARL4D or FLAG-ARNO were serum starved, treated with EGF or wortmannin (more ...)
A previous report showed that a mutation (R279C) in the PH domain of ARNO abolishes PIP3
binding and does not show EGF-stimulated translocation of the R279C mutant to the plasma membrane (Venkateswarlu and Cullen, 2000
). Thus, we examined whether ARL4D could induce translocation of ARNO(R279C) to the plasma membrane (, E–G). ARNO(R279C) was able to interact with ARL4D by yeast two-hybrid analysis (D). Consistent with a previous report, ARNO(R279C) resides primarily in the cytosol (E, left; Venkateswarlu and Cullen, 2000
). When coexpressed with ARL4D(Q80L), we observed that ARNO(R279C) translocated to the plasma membrane (E, right). Moreover, ARL4D-mediated redistribution of ARNO(R279C) to the plasma membrane was not blocked by wortmannin when cells were incubated with or without EGF (, F and G). Collectively, these data suggested that ARL4D-mediated association of ARNO to the plasma membrane is dependent on interaction with plasma membrane-associated ARL4D, but not in a PIP3
ARL4D Recruits Other Members of Cytohesin Family, but Not Akt PH Domain, to the Plasma Membrane
Because of highly structural conservation of the PH domain in the cytohesin family (Ogasawara et al., 2000
), we next examined whether ARL4D can induce redistribution of other cytohesins, including cytohesin-1, cytohesin-3, and cytohesin-4, to the plasma membrane. All FLAG-tagged cytohesins were diffusely localized throughout the cytoplasm (Supplemental Figure S7). When coexpressed with ARL4D(Q80L), the redistribution of FLAG-cytohesin-1, FLAG-cytohesin-3, and FLAG-cytohesin-4 to plasma membrane ruffles and protrusions was detected (Supplemental Figure S7). Together, these results illustrate that the effect of ARL4D on the subcellular localization of all members of the cytohesin family is similar.
Despite high primary sequence variability, PH domains retain a conserved three-dimensional organization consisting of seven-stranded β-sandwich structure, with one corner capped off by a C-terminal α-helix and another by three interstrand loops. However, different PH domains have different affinities to several kinds of phospholipids (Lemmon, 2004
; Balla, 2005
). There has been speculation about the key regulator(s) of their specificity (Lemmon, 2004
; Balla, 2005
; Varnai et al., 2005
), especially concerning the protein–protein interaction. Similar to the PH domain of ARNO, the PH domain of Akt showed growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane (Gray et al., 1999
). Thus, we next examined whether ARL4D can induce translocation of Akt-PH-GFP to the plasma membrane. However, a yeast two-hybrid assay showed that ARL4D could not interact with Akt-PH (unpublished data). In control, Akt-PH-GFP is localized to the cytoplasm and the nucleus (H, top). In EGF-stimulated cells with or without coexpressing ARL4D, we found that Akt-PH-GFP localized to the plasma membrane (, H and I). However, unlike the effect on ARNO, ARL4D did not induce redistribution of Akt-PH-GFP to the plasma membrane in the presence of wortmannin (, H and I). This suggests that the interaction between ARL4D and cytohesin family proteins does not extend to another PIP3 membrane-associated PH domain-containing protein, and it reflects a novel and specific relationship between ARL4D and cytohesin family proteins.