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(Journal of Leukocyte Biology. 2002;71:718-728.)
© 2002 by Society for Leukocyte Biology

ARNO but not cytohesin-1 translocation is phosphatidylinositol 3-kinase-dependent in HL-60 cells

Sylvain G. Bourgoin*, Martin G. Houle*, Indrapal N. Singh{dagger}, Danielle Harbour*, Steve Gagnon*, Andrew J. Morris{ddagger} and David N. Brindley{dagger}

* Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Pavillon CHUL et Département d’Anatomie-Physiologie, Faculté de Médecine, Université Laval, Québec, Canada;
{ddagger} Department of Pharmacological Sciences and the Institute for Cell and Developmental Biology, Stony Brook Health Science Center, Stony Brook, New York; and
{dagger} Signal Transduction Laboratories, Department of Biochemistry and Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada

Correspondence: Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Pavillon CHUL, Local T1-49, 2705 Boulevard Laurier, Ste-Foy, Québec, G1V 4G2, Canada. E-mail: sylvain.bourgoin{at}crchul.ulaval.ca


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ABSTRACT
 
Cytohesin-1 and ARNO are guanine nucleotide-exchange factors (GEFs) for ADP-ribosylation factor (Arf). Here, we show that ARNO is expressed in HL-60 cells and established that granulocytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils. Me2SO-differentiated HL-60 cells expressed ARNO and cytohesin-1 isoforms with a diglycine and a triglycine motif in their PH domains, respectively. In vitro, ARNO diglycine and cytohesin-1 triglycine enhanced phospholipase D1 (PLD1) activation by Arf1 with near-maximal effects at 250 nM. These effects were marked particularly at low Mg2+ concentrations. PLD activation was well-correlated with GTP binding to Arf1, and cytohesin-1 was always more potent than ARNO in the PLD- and GTP-binding assays. Increasing Mg2+ concentrations reduced PLD and Arf1 activation by Arf-GEFs. fMetLeuPhe and phorbol 12-myristate 13-acetate stimulated ARNO and cytohesin-1 as well as Arf1 translocation to HL-60 cell membranes. fMetLeuPhe-mediated ARNO recruitment, but not cytohesin-1 and Arf1 translocation, was blocked by phosphatidylinositol 3-kinase inhibitors. The combined results demonstrate that cytohesin-1 triglycine participates in a major phosphatidylinositol 3-kinase-independent pathway linking cell-surface receptors to Arf1 activation and translocation in human granulocytes.

Key Words: Sec7 • ADP-ribosylation factor • pleckstrin homology domain • RhoA • guanine nucleotide-exchange factors • inflammation


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INTRODUCTION
 
Activation of neutrophils is a key component of the inflammatory response. Indeed, fMet-Leu-Phe (fMLP) stimulates several neutrophil responses following its binding to specific cell-surface receptors and the production of lipid second messengers [1 , 2 ]. Some fMLP-induced responses are mediated by phosphatidate (PA), a product of the hydrolysis of phosphatidylcholine (PtdCho) by phospholipase D (PLD). PA is the precursor of other lipid second messengers such as diacylglycerol or lyso-PA through its hydrolysis by lipid phosphate phosphatases or phospholipase A1 or A2 [3 ], respectively. Studies in human granulocytes showed that PA regulates the oxidative burst [4 ], phagocytosis [5 ], and secretion [6 ] in response to fMLP stimulation.

Two related genes encoding for differently regulated PLD enzymes, PLD1 [7 ] and PLD2 [8 ], have been characterized. Splice variants of PLD1 (PLD1a and PLD1b) and PLD2 (PLD2a and PLD2b) have been shown [9 , 10 ]. The relative levels of expression of PLD1 and PLD2 mRNA in cell lines vary considerably [10 , 11 ]. Undifferentiated HL-60 cells predominantly express PLD1 but not PLD2 mRNA [11 ]. However, the expression of PLD1 and PLD2 is enhanced during granulocytic differentiation [12 , 13 ]. PLD1a is the major PLD isoform found in granulocyte membranes [14 ].

Whereas PLD isoforms displayed an absolute requirement for phosphatidylinositol 4,5-bisphosphate (PIP2) in vitro [15 , 16 ], PLD1 is strongly activated by the small GTPases, ADP-ribosylation factor (Arf) and Rho, and the protein kinase C (PKC) isofoms, -{alpha} and -ß. Using granulocyte membranes or cytosol-depleted cells, cytosolic Arf1 [17 ] and RhoA [18 ] reconstitute guanosine 5'-O-thiotrisphosphate (GTP{gamma}S)-induced PLD activity. The contribution of Arf and Rho GTPAses in receptor-mediated PLD activities has been explored but remains ill-defined [19 20 21 22 23 ]. RhoA, Arf1, and Arf6 are recruited to granulocyte membranes in response to fMLP [22 , 24 25 26 ]. Several studies suggest a role for Arf6 but not Arf1 in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase regulation [26 ] and Fc receptor for immunoglobulin G (IgG; Fc{gamma}) receptor-mediated phagocytosis [27 ]. These physiological functions of Arf6 appear to depend on PLD activation [4 , 5 , 26 , 27 ]. Moreover, although phosphoinositide 3-kinase (PI 3-kinase) inhibitors inhibited fMLP-mediated PLD activation, they do not suppress fMLP-mediated recruitment of Arf1 to membranes [22 ]. This result implies that a PI 3-kinase-dependent component of PLD activation distinct from Arf1 remains unidentified in granulocytes and that the product of the kinase activity, phosphatidylinositol 3,4,5-trisphosphate (PIP3), is not required to recruit Arf guanine nucleotide-exchange factors (Arf-GEFs) to membrane domains where they will activate Arf1.

Arf-GEFs are broadly divided into two classes: those that are sensitive to inhibition by brefeldin A (BFA) and those that are not [28 , 29 ]. BFA has no inhibitory effect on fMLP-stimulated PLD activity in granulocytes [30 ]. Therefore, it is likely that a BFA-insensitive Arf-GEF, e.g., cytohesin-1 [31 ], ARNO [32 ], or GRP-1 [33 ], contributes to PLD activation by Arf(s) in granulocytes. Cytohesin-1 [31 ], ARNO [32 ], mouse GRP-1 [34 ], and its human homolog ARNO3 [35 ] are GEFs for Arf class I (Arf1 and Arf3) and class III (Arf6) as well. These GEFs have in common a C-terminal, coiled-coil domain; a Sec7 domain-stimulating nucleotide exchange; and an N-terminal pleckstrin homology (PH) domain, which binds PIP3 with high affinity [34 , 36 , 37 ]. This domain mediates PIP3- and PIP2-dependent stimulation of nucleotide exchange on myristoylated Arf1 (myrArf1) and/or Arf6 by GRP-1 [34 ] and ARNO [23 , 32 ], respectively. It is interesting that receptor-mediated cell activation causes a rapid translocation of ARNO [37 ], GRP-1 [38 ], and cytohesin-1 [39 ] to plasma membranes in response to PI 3-kinase activation. Cytohesin-1 is expressed abundantly but not exclusively in hematopoietic tissues [40 , 41 ], and it interacts with the cytoplasmic tail of lymphocyte function-associated antigen-1 (LFA-1) integrin ß chain (CD18), regulating the adherence of lymphocytes [41 ] and monocytes [42 ].

In this study, we show that cytohesin-1 but not ARNO expression increases with granulocytic differentiation of HL-60 cells. Furthermore, in vitro, we found that cytohesin-1 was a better stimulator of Arf1-induced PLD1 activity compared with ARNO. In granulocytes, ARNO and cytohesin-1 were mainly cytosolic and were recruited rapidly to the membrane fraction following stimulation with fMLP or phorbol 12-myristate-13 acetate (PMA). The fMLP-induced translocation of ARNO but not of cytohesin-1 required the activation of PI 3-kinase. The latter results identify ARNO with a diglycine motif in its PH domain as a PI 3-kinase-sensitive component of HL-60 cells activation.


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MATERIALS AND METHODS
 
Materials
HL-60 cells were obtained from America Type Culture Collection (Manassas, VA). The prokaryote expression vector pET30b was from Novagen (Madison, WI). ARNO cDNA (IMAGE clone ID, 180774) was obtained from Research Genetics (Huntsville, AL) and was subcloned in pET30b between the NcoI-HindIII restriction sites. Wild-type bovine Arf1 cDNA was kindly provided by Dr. P. Chardin (Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis). Saccharomyces cerevisiae N-myristoyltransferase (NMT) expression vector (pBB131) was a generous gift from Dr. J. I. Gordon (Washington University School of Medicine, St. Louis, MO). Cytohesin-1 cDNA inserted in the pQE30 expression vector [31 ] was kindly provided by Dr. J. Moss (National Institutes of Health, Bethesda, MD). [35S]GTP{gamma}S, L-{alpha}-dipalmitoyl phosphatidyl[N-methyl-3H]choline ([3H]PtdCho), enhanced chemiluminescence (ECL) reagents, DEAE Sepharose CL-6B, protein A-Sepharose, Sephadex G-75 superfine, and the HiTrap column for affinity purification of (His)6-tagged proteins were purchased from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada). Phospholipids were from Sigma Aldrich Canada (Oakville, Ontario). RhoA and PKC{alpha} antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The Arf1 polyclonal antibody was described in previous studies [43 ]. It recognizes Arf1 (Arf class I) and Arf5 (Arf class II) but not Arf6 (Arf class III). ARNO (serum 18) and cytohesin-1 (serum 139) polyclonal antibodies, as well as mAb 6G11 and 2E11, were generated and characterized in previous studies [44 ].

Production and preparation of recombinant proteins
To produce myrArf1, the BL21(DE3)pLysS host strain was cotransfected with the pET11d/Arf1 and pBB131/NMT expression vectors. Transformed bacteria were grown at 37°C, and myristate (50 µM) bound to serum albumin was added to exponentially growing bacteria 10 min before induction with 1 mM isopropyl-1-thio-ß-Dgalacto-pyranoside (IPTG). Induction was performed at 24°C for 3 h to increase the yield of myristoylated protein [45 ]. Arf1 was purified as described previously [43 ] and was estimated to be >80% pure by Coomassie blue staining of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.

Cytohesin-1/pQE30 and ARNO/pET30b constructs were transfected in the M15 and BL21(DE3)pLysS host strains, respectively. Protein expression was induced with 1 mM IPTG for 3 h at 37°C. Proteins were purified using a HiTrap chelating column, according to the instructions of the manufacturer. Recombinant proteins were dialyzed against buffer A containing 50 mM Hepes, pH 7.2, 100 mM KCl, 5 mM NaCl, 1 mM MgCl2, 0.5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 0.25 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 µg/ml aprotinin, and 2.5 µg/ml leupeptin and were stored at -80°C. The purity of (His)6-tagged cytohesin-1 and ARNO was estimated to be >90%.

Neutrophil purification and HL-60 cell cultures
Neutrophils were purified as described previously [14 ]. HL-60 cells were grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Differentiation was induced by the addition of 1.25% (v/v) Me2SO for 7 days [46 ].

Cell stimulation
Cell suspensions were treated with 1 mM di-isopropylfluorophosphate (DFP) for 30 min at 24°C. Samples (107 cells/ml) were incubated for 5 min at 37°C and stimulated with 100 nM PMA for 2 min or incubated 5 min with 10 µM cytochalasin B (CB) before stimulation with 100 nM fMLP for 2 min. Incubations were stopped by diluting the cells 1:5 with ice-cold RPMI (or Hanks’ balanced salt solution). Membrane and cytosolic fractions were prepared as described previously [24 ].

Immunoprecipitation of cytohesin-1 and ARNO
Cell suspensions (4x107 cells/ml), membranes (6x107 cell eq/ml), and cytosolic proteins (6x107 cell eq/ml) were mixed with an equal volume of nondenaturing lysis buffer (2x) containing 50 mM Tris-HCl, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM NaVO4, 1 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin (final concentrations), adjusted to pH 7.4. Lysates were incubated on ice for 15 min before centrifugation at 13,000 rpm for 15 min. Nonidet P-40 (0.12%), 0.005% bovine serum albumin, 20 µg/ml aprotinin, and 20 µg/ml leupeptin (final concentrations) were added to the supernatants, which were precleared with protein-A Sepharose and used subsequently for precipitation with ARNO or cytohesin-1 antibodies noncovalently bound to protein-A Sepharose. The beads were washed three times with ice-cold lysis buffer containing 1% Nonidet P-40 and incubated for 1 h at 37°C in Laemmli’s sample buffer. Immune complexes were then analyzed by Western blotting with ARNO or cytohesin-1 antibodies [44 ]. Proteins were revealed using the ECL reagent. Autoradiographs were obtained by exposing Kodak X-omat film to membranes. RhoA, Arf, and PKC{alpha} blots were performed as described previously [21 ].

Measurements of PLD activity in vitro
Human PLD1 was overexpressed in Sf9 cells infected with recombinant baculoviruses at a multiplicity of 10 [7 ]. After 48 h, the Sf9 cells were washed twice with ice-cold phosphate-buffered saline and resuspended in ice-cold buffer B: 162 mM NaCl, 8.1 mM Na2HPO4, 27 mM KCl, 1.5 mM KH2PO4, 2.5 mM ethylenediaminetetraacetate (EDTA), 1 mM dithiothreitol (DTT), and 0.1 mM PMSF, adjusted to pH 7.4. Samples were sonicated and centrifuged at 100,000 g for 60 min at 4°C. Membrane pellets were resuspended in buffer B at a protein concentration of 2–3 mg/ml and stored at -80°C. The PLD assay measures the release of [3H]choline from [3H]PtdCho according to Brown et al. [47 ], with slight modifications. The substrate was presented in the form of phospholipid vesicles composed of phosphatidylethanolamine/PIP2/PtdCho in a 16:1.4:1 molar ratio. Vesicles were prepared by mixing phospholipids in 50 mM Hepes, pH 7.5, 2 mM EDTA, 100 mM KCl, 1 mM MgCl2, and 1 mM DTT (buffer C). The final concentration of [3H]PtdCho was 8.3 µM (about 120,000 dpm per assay). The PLD assay was performed by mixing, on ice, phospholipid vesicles (30 µl), Sf9 membranes (10 µg protein), and Arf1 with buffer C to a final volume of 100 µl. The final free Mg2+ concentration was estimated to be 1 µM [48 ]. Samples were incubated for 2 min at 37°C, and the reaction was started by the addition of 30 µM GTP{gamma}S. Reactions were terminated by the addition of 1.35 ml chloroform:methanol:water (1:1:0.7, by vol), followed by mixing and centrifugation to separate water-soluble metabolites from [3H]PtdCho. [3H]Choline was extracted from the aqueous phase and quantitated by scintillation counting, essentially as described by Fonnum [49 ].

Nucleotide binding assay
Arf1 (2 µM), alone or in combination with Arf-GEFs, was incubated at 37°C with [35S]GTP{gamma}S (10 µM, about 1000 cpm/pmol) in buffer C, and the phospholipid vesicles were used for the PLD assay. Samples (100 µl) were diluted with 2 ml ice-cold 20 mM Hepes, pH 7.5, 100 mM NaCl, and 10 mM MgCl2 and were filtered on 25 mm GN-6 membrane filters (Gelman Laboratory, Ann Arbor, MI). Filters were washed twice, dried, and assayed by liquid scintillation counting.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Sec7 PH domains
cDNA from Me2SO-differentiated HL-60 cells was the template for PCR analysis. The PH domains of cytohesin-1 and ARNO were amplified using the forward (5'-GGAATGACCTCACTCACTTTCT-3', 5'-GGAATGACCTGACCCACCTT-3') and reverse (5'-GTCTAAGCTTCAGTGTCGCTTCGTGGAGG-3', 5'-GCATAAGCTTTCAGGGCTGCTCCTGCTTCTT-3') primers, respectively. Pwo DNA polymerase (Roche Diagnostics, Laval, Quebec, Canada) was used for all PCR reactions. Products were gel-purified and recovered using the QIAquick Gel Extraction kit. The sequences were analyzed by automated sequencing.


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RESULTS
 
Expression of Sec7 proteins in human granulocytes
We generated antibodies against cytohesin-1 and ARNO to examine their presence in human granulocytes. The ARNO antiserum 18 detects as little as 2 ng (His)6-tagged ARNO and is 50-fold less sensitive in detecting cytohesin-1 (Fig. 1A ). Furthermore, antiserum 18 immunoprecipitated ARNO selectively (Fig. 2B ; and ref. [44 ]). In contrast, cytohesin-1 antibodies (antiserum 139) detected 25 ng (His)6-tagged cytohesin-1 (Fig. 2B) and 100 ng (His)6-tagged ARNO (not shown). The expression of ARNO and cytohesin-1 in neutrophils, undifferentiated, and Me2SO-differentiated HL-60 cells was analyzed using the antisera 18 and 139, respectively. However, we detected no specific signal in the 48-kDa region by immunoblotting, indicating that the level of protein expression was below the detection limit of the antibodies. Therefore, we immunoprecipitated ARNO and cytohesin-1 from cells with the appropriate antibodies, and the immune complexes were probed with the same antibody. Endogenous ARNO was detected at 48 kDa, just below the heavy IgG chain in undifferentiated HL-60 cells (Fig. 1B , lane 2), and there was a decrease (44%, n=2) in the levels of ARNO following granulocytic differentiation induced with Me2SO for 7 days (lane 3). No ARNO was immunoprecipitated from HL-60 cells using the preimmune antiserum (lane 1), and the levels of endogenous ARNO were low in human neutrophils (lane 4). Similarly, no cytohesin-1 was immunoprecipitated from human granulocytes using the preimmune antiserum (lane 5). However, a 48-kDa protein could be immunoprecipitated with the cytohesin-1 antiserum 139. The levels of endogenous cytohesin-1 were very low in undifferentiated HL-60 cells (lane 6), but the expression of the protein increased 34-fold (n=2) following granulocytic differentiation induced with Me2SO. The levels of cytohesin-1 in HL-60 granulocytes (lane 7) were very similar to those detected in neutrophils (lane 8). Figure 2 shows that cytohesin-1 (50.2±12.8 ng/2x107 cells, n=3) is fivefold more abundant in Me2SO-differentiated HL-60 cells than ARNO (9.8±3.8 ng/2x107 cells, n=3), as estimated by densitometry scanning. Although ARNO could be immunopreciptated by cytohesin-1 antibodies [44 ], the amounts recovered in immune complexes (2.4±0.88 ng/2x107 cells, n=3) were negligible as compared with cytohesin-1 (Fig. 2A and 2B) .



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  		Figure 1. Differential expression of ARNO and cytohesin-1 in human granulocytes. (A) The selectivity of the polyclonal ARNO antibodies (antiserum 18) was tested on purified (His)6-tagged ARNO and cytohesin-1. (B) ARNO and cytohesin-1 were immunoprecipitated from cell lysates obtained from 2 x 107 HL-60 cells, Me2SO-differentiated HL-60 cells (d), and neutrophils (PMN) using the ARNO antiserum 18 (lanes 2–4), the cytohesin-1 antiserum 139 (lanes 6–8), or the preimmune antiserum (lanes 1 and 5). The immune complexes were subjected to Western blotting with ARNO (left panel) or cytohesin-1 (right panel) polyclonal antibodies. ARNO and cytohesin-1 are detected at 48 kDa as indicated by the arrow. One experiment representative of two with similar results is shown.



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  		Figure 2. Cytohesin-1 is expressed more abundantly than ARNO in human granulocytes. (A) ARNO was immunoprecipitated from cell lysates obtained from 2 x 107 differentiated HL-60 cells using the ARNO antiserum 18 (lane 1) or the cytohesin-1 antiserum 139 (lane 2). Samples were blotted with the ARNO antiserum 18. The arrow shows presence of ARNO just below the heavy IgG chain. (B) Cytohesin-1 was immunoprecipitated using the ARNO antiserum 18 (lane 1) or the cytohesin-1 antiserum 139 (lane 2). Samples were blotted with the cytohesin-1 antiserum 139. The arrow shows presence of cytohesin-1 just below the heavy IgG chain. For quantification of endogenous ARNO and cytohesin-1 by densitometry, increasing amounts of recombinant ARNO (rec-ARNO) and cytohesin-1 (rec-Cyto-1) were electrophoresed on the same gels, respectively. One experiment representative of three with similar results is shown.

Cytohesin-1 and ARNO are translocated to membranes upon fMLP and PMA stimulation
Previously, we have shown that Arf1 is translocated to granulocyte membrane fractions upon stimulation with fMLP and PMA [24 ]. This observation prompted us to examine whether the translocation of Arf1 induced by agonists (Fig. 3A ) could be a result of the activation and/or the recruitment of Arf-GEFs to granulocyte membranes. Therefore, to determine the subcellular distribution of these Arf-GEFs in HL-60 granulocytes, we immunoprecipitated ARNO and cytohesin-1 from the membrane and cytosolic fractions (Fig. 3B) . Although the majority of ARNO was recovered in the cytosol (lane 1), about 38% (n=2) of the protein was recovered in membranes (lane 4). Stimulation of HL-60 granulocytes with fMLP or PMA for 2 min or more (5 min; not shown) altered the subcellular distribution of ARNO significantly. Figure 3B shows that the majority of endogenous ARNO (60%, n=2) shifted to membranes in response to fMLP (lane 5), and this is reflected by a decrease in the cytosol (lane 2). Significantly more ARNO was recruited to membranes (75%, n=2) in response to PMA (lane 6) as compared with fMLP (lane 3). The bulk of cytohesin-1 (82%, n=2) was soluble (lanes 7 and 10). Upon stimulation with fMLP (lane 11) or PMA (lane 12), the levels of membrane-associated cytohesin-1 increased 2.1- and 4.9-fold, respectively, and this was reflected by a decrease (40–50%, n=2) in the levels of cytosolic cytohesin-1. Similarly, fMLP and PMA promoted the recruitment of cytohesin-1 to neutrophil membranes (not shown). The data indicate that the translocation of ARNO and cytohesin-1 to granulocyte membranes in response to PMA or fMLP stimulation accompanies the recruitment of Arf1 (Fig. 3A) .



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  		Figure 3. fMLP- and PMA-mediated translocation of ARNO and cytohesin-1. (A) Membrane proteins from control (C) and PMA- and fMLP-stimulated cells were resolved on a 12% SDS-PAGE and probed with a polyclonal Arf1 antibody. (B) Me2SO-differentiated HL-60 cells were pretreated for 5 min with 10 µM CB or the vehicle (Me2SO) and stimulated with 100 nM fMLP or 100 nM PMA for 2 min, respectively. ARNO and cytohesin-1 were immunoprecipitated from lysates of 3 x 107 cells, and membranes and cytosols in equivalent proportions were analyzed by Western blotting with ARNO (left panel) or cytohesin-1 (right panel) polyclonal antibodies. ARNO and cytohesin-1 are detected at 48 kDa as indicated by the arrow. One experiment representative of two with similar results is shown.

Role of PI 3-kinase in Arf1 and Arf-GEFs translocation
ARNO and cytohesin-1 have PH domains that can bind phosphoinositides, and PIP3, the product of PI 3-kinase, could potentially promote plasma-membrane targeting of Arf-GEFs [37 , 39 ]. Because PI 3-kinase-{gamma} is activated by fMLP in neutrophils [50 ], we examined the effect of PI 3-kinase inhibitors, wortmannin [51 ] and LY294002 [52 ], on fMLP-induced cytohesin-1 and ARNO translocation. fMLP-induced translocation of cytohesin-1 to membranes was not inhibited by the PI 3-kinase inhibitors (Fig. 4A and 4B ). In contrast, wortmannin and LY294002 inhibited the translocation of ARNO in a dose-dependent manner. Nearly complete inhibition was achieved with 300 nM wortmannin or 30 µM LY294002 (Fig. 4C and 4D) . The results establish that the recruitment of ARNO but not of cytohesin-1 is dependent on the activation of PI 3-kinase by fMLP. Next, we determined whether the translocation of Arf1 was also dependent on PI 3-kinase activation. Figure 4E shows that concentrations of LY294002, found to inhibit ARNO translocation, have no inhibitory effect on fMLP-induced Arf1 recruitment to membranes. The highest concentration of LY294002 used marginally inhibited the translocation of RhoA (Fig. 4F) . Surprisingly, PKC{alpha} translocation induced by fMLP was blocked by LY294002 in a dose-dependent manner, with almost complete inhibition at 30 µM (Fig. 4E) . Thus, the inhibition of ARNO translocation by PI 3-kinase inhibitors does not impact Arf1 recruitment to membranes, suggesting that cytohesin-1 plays a major role in the activation of Arf1 in human granulocytes.



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  		Figure 4. PI 3-kinase participates in ARNO but not cytohesin-1 translocation elicited by fMLP. Me2SO-differentiated HL-60 cells were pretreated for 5 min with 10 µM CB and stimulated with 100 nM fMLP for 2 min. Where indicated, the cell suspensions were pretreated with the indicated concentrations of wortmannin (A and C) or LY294002 (B and D) for 10 min. Cytohesin-1 (A and B) and ARNO (C and D) were immunoprecipitated from membranes (3x107 cell/eq) and analyzed by Western blotting for ARNO or cytohesin-1 with the polyclonal antibodies or mAb 2E11 and 6G11. Results were normalized to the fMLP-mediated response and are means ± SE of three independent experiments. (E and F) Effect of LY249003 on Arf1, RhoA, and PKC{alpha} translocation elicited by fMLP. Cell suspensions were stimulated as above, and the membrane fractions were probed for Arf1, RhoA, and PKC{alpha}. One experiment representative of three with similar results is shown.

Analysis of ARNO and cytohesin-1 PH domains
cDNA samples of human tissues have been shown to contain native sequences coding for a diglycine or a triglycine motif in the ARNO and cytohesin-1 N-terminal PH domains [53 ]. It is interesting that ARNO and cytohesin-1 PH domains with a diglycine motif show high affinity for PIP3 over PIP2, and PH domains with a triglycine motif have very similar binding affinity for PIP3 and PIP2 [54 ]. Taken together with our results, the physiological and biochemical behavior of cytohesin-1 and ARNO in HL-60 cells may relate to the presence of a triglycine and a diglycine motif in their PH domains. Therefore, we analyzed the nucleic acid sequences of native ARNO and cytohesin-1 PH domains from the same sample of cDNA obtained from Me2SO-differentiated HL-60 cells. The results of two independent PCR analyses revealed the presence of a diglycine motif in the region encoding the ARNO PH domain (5'-GGCTGGCTCCTGAAGCTGGGGGGCCGGGTGAAGACGTGG-3', GWLLKLGGRVKTW), whereas the PH domain of cytohesin-1 had a triglycine motif (5'-GGCTGGCTATTGAAACTCGGAGGTGGCAGGGTAAAGACTTGG-3', GWLLKLGGGRVKTW). The high-binding specificity of the ARNO diglycine to PI 3-kinase products, as opposed to ARNO or cytohesin-1 containing triglycine [54 ], explains the sensitivity of ARNO translocation to inhibition by the PI 3-kinase inhibitors in HL-60 cells.

Cytohesin-1 and ARNO augment GTP{gamma}S-dependent PLD1 activity in the presence of Arf1
Cytohesin-1 triglycine and ARNO diglycine were overexpressed as (His)6-tagged proteins in Escherichia coli to analyze, in vitro, their effect on the activation of PLD by Arf1. We measured PLD1 activity using Sf9 membranes and lipid vesicles containing PIP2 and PtdCho, which allow for the specific detection of PLD1 activity in the presence of Arf1 and GTP{gamma}S [9 ]. There was little [3H]choline release when 30 µM GTP{gamma}S was added to Sf9 membranes (not shown). Arf1, 0.1–5 µM, induced a dose-dependent activation of PLD1 (not shown), which was stimulated markedly by PIP2 [9 ]. Unless otherwise stated, the PLD assay was performed at 37°C for 20 min in the presence of 2 µM Arf1 and 12 µM PIP2. ARNO or cytohesin-1 alone increased basal PLD1 activity slightly, but this effect remained marginal as compared with the levels of PtdCho hydrolysis in the presence of 2 µM Arf1 (1776±206 pmoles/mg protein, n=5). Because the levels of PLD activity varied from one experiment to another, the effects of Arf-GEFs were expressed as fold-increase of [3H]choline release stimulated by Arf1 to allow comparison between the two Arf-GEFs (Fig. 5A and 4B ). [3H]Choline release was increased by 50%, 176%, and 265% with 0.05, 0.25, and 1.25 µM ARNO as compared with Arf1 alone, respectively (Fig. 5A) . Although ARNO and cytohesin-1 have similar stimulatory effects, cytohesin-1 was more potent than ARNO (compare Fig. 5A and 5B , with 5C and FD ). This was evident particularly at low Mg2+ and Arf-GEF concentrations. Indeed, the levels of [3H]choline released by PLD1 activated with Arf1 were enhanced markedly by 50 nM cytohesin-1 (285%) and reached a plateau at 250 nM. Increasing the free Mg2+ concentration to 1 mM reduced the stimulatory effect of Arf-GEFs considerably.



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  		Figure 5. Dependence of Arf1-stimulated PLD activity on ARNO and cytohesin-1. Sf9 membranes (10 µg protein) were resuspended in buffer C and incubated under standard conditions (30 µM GTP{gamma}S) with varying concentrations of ARNO (A) or cytohesin-1 (B) in the presence of 2 µM Arf1 for 20 min at 37°C. Where indicated, the composition of the buffer was changed by adding 1 mM free Mg2+. For comparison among the different assay conditions, the results were expressed as fold-increase in PLD1 activity stimulated by Arf1 in the absence of Arf-GEF and the indicated Mg2+ concentration. Results are means of two independent experiments performed in duplicate. (C and D) Effect of ARNO and cytohesin-1 on the kinetic of [3H]PtdCho hydrolysis. Sf9 membranes resuspended in buffer C containing 1 µM free Mg2+ (C) or 1 mM free Mg2+ (D) were incubated with 2 µM Arf1 and 30 µM GTP{gamma}S at 37°C with or without 500 nM ARNO or cytohesin-1. At selected times, reactions were terminated, and the release of [3H]choline was measured. Results are means of two independent experiments, each performed in duplicate.

Arf1 and time-dependence of PLD activation
We monitored PLD1 activity with various concentrations of Arf1 in the presence or the absence of Arf-GEFs. In the range of Arf1 concentrations tested (0.1–3 µM), both Arf-GEFs accelerated PLD1 activity, with cytohesin-1 being twofold more potent than ARNO (not shown). The kinetics of PLD1 activation by Arf1 in the presence of ARNO and cytohesin-1 were also examined. Figure 5C shows a linear increase in the levels of [3H]PtdCho hydrolysis up to 40 min and shows that [3H]choline release reached a plateau between 40 and 60 min. At all time points tested, ARNO and cytohesin-1 (500 nM) accelerated the hydrolysis of [3H]PtdCho two- and threefold, respectively. The addition of 1 mM Mg2+ to the PLD assay buffer reduced PLD1 activation by Arf1 significantly (Fig. 5D) .

Effect of cytohesin-1 triglycine and ARNO diglycine on GTP{gamma}S binding to Arf1
Together, our results demonstrate that the presence of a diglycine or a triglycine motif in the PH domain of Arf-GEFs impacts their biochemical properties in vitro and in vivo. Because cytohesin-1 triglycine was more potent than ARNO diglycine in the PLD assay, we examined the possibility that this difference may relate to their intrinsic capacity of catalyzing GTP{gamma}S binding to Arf1 in the presence PIP2. Therefore, we compared the ability of ARNO diglycine and cytohesin-1 triglycine to stimulate GTP-binding on Arf1 (Fig. 6 ) using conditions similar to those used for the PLD assay to allow comparison between [3H]PtdCho hydrolysis by PLD1 and the activation of Arf1. As shown in Figure 6A 6B 6C 6D , spontaneous GTP{gamma}S binding on Arf1 was optimal at 1 µM free Mg2+. The addition of 0.5 µM ARNO or cytohesin-1 stimulated GTP{gamma}S binding markedly on Arf1, with optimal loading observed in the presence of 1 µM free Mg2+ (Fig. 6A) . Inclusion of 1 mM Mg2+ in the assay reduced the activation of Arf1 by cytohesin-1 and ARNO fivefold (Fig. 6B) . Consistently, cytohesin-1 was more potent than ARNO in promoting GTP-binding on Arf1 (Fig. 6A and 6B) . This was evident particularly at a suboptimal concentration (50 nM) of ARNO and cytohesin-1 (Fig. 6C and 6D) . Thus, the observation that [3H]PtdCho hydrolysis is reduced by Mg2+ is consistent with data demonstrating that physiologic Mg2+ concentrations can decrease spontaneous and stimulated nucleotide exchange on Arf1. Furthermore, our data indicate that the ability of cytohesin-1 and ARNO to increase Arf1-mediated PLD activation can be correlated directly to the activity of the exchange factors in controlling the levels of Arf1 in the GTP{gamma}S-bound form. GTP-binding on recombinant Arf6 was also enhanced strongly by cytohesin-1, whereas the stimulatory effect of ARNO was weak (unpublished results).



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  		Figure 6. ARNO and cytohesin-1-mediated nucleotide exchange on Arf1. Arf1 (2 µM) was incubated with 10 µM GTP{gamma}S and the lipid vesicles used for the PLD assay. Nucleotide-exchange activity was assayed in the presence of 1 µM (A and C) or 1 mM free Mg2+ (B and D). (A and B) GTP{gamma}S binding on Arf1 was performed in the absence ({square}) or presence of 500 nM ARNO ({triangleup}) or cytohesin-1 ({blacktriangleup}). (C and D) GTP{gamma}S binding on Arf1 was performed in the absence ({square}) or presence of 50 nM ARNO ({triangleup}) or cytohesin-1 ({blacktriangleup}). One experiment representative of three with similar results is shown. Results are means of triplicate.


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DISCUSSION
 
The characterization of Arf-GEFs with selectivity for Arf class I (Arf1 and 3) and class III (Arf6) is an important issue for elucidating the function and the mechanism of PLD activation by physiological agonists in human granulocytes. The rapidly growing family of Arf-GEFs containing a Sec7 domain includes the BFA-sensitive GEFs—yeast Gea1 and Gea2 [55 ] and bovine BIG1 and BIG2 [56 ]—and the BFA-insensitive—ARNO [32 ], cytohesin-1 [31 ], ARNO3/GRP-1 [33 , 35 ], and cytohesin-4 [53 ]. The amino acids that confer sensitivity to BFA have been mapped in the Sec7 domain. Several studies have shown that PLD activation by agonists is sensitive to inhibition by BFA [20 , 57 ]. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines [58 ], and this activity was stimulated greatly by Arf1 and GTP analogs. This stimulation could be inhibited by BFA. However, BFA does not interfere with the ability of Arf1 to stimulate PLD activity that had been purified partially from Golgi. These data indicate that a BFA-sensitive Arf-GEF resides in the Golgi of these cells [59 ]. Two different PLD isoforms, PLD1 [7 ] and PLD2 [8 , 10 ], have been cloned recently, and both enzymes are expressed in Me2SO-differentiated HL-60 cells [12 , 46 ].

Several studies clearly indicate that granulocyte PLD, mainly PLD1a [14 ], is activated by Arf1 [17 , 22 , 24 ] or Arf6 [26 ]. However, in granulocytes, the activation of PLD induced by fMLP and PMA [22 , 60 ] or the stimulation by Arf1 of PLD activity in Golgi-enriched membranes [30 ] is not sensitive to inhibition by BFA. These studies in human granulocytes suggest that the activation of PLD by Arf1 and/or Arf6 involves BFA-insensitive Arf-GEFs. In this study, we demonstrate the presence of ARNO and cytohesin-1 in human granulocytes and show that cytohesin-1 expression is enhanced markedly during granulocytic differentiation of HL-60 cells induced by Me2SO. We suggest that cytohesin-1 could be the BFA-insensitive Arf-GEF, which promotes the activation of PLD1 by Arf1 in granulocytes. This conclusion is based on the following observations: Activation of PLD by various agonists is not blocked by BFA (ref. [30 ]; and unpublished results); stimulation of HL-60 granulocytes with fMLP and PMA induces concomitant translocation of Arf1, cytohesin-1, and ARNO to membranes; cytohesin-1 is expressed more abundantly as compared with ARNO, based on an average volume of 400 µm3/cell, the endogenous concentrations of ARNO and cytohesin-1 in HL-60 granulocytes were estimated to be >=25 and 250 nM, respectively; and cytohesin-1 is more potent than ARNO in promoting the activation of PLD1 by Arf1 in vitro. However, because ARNO and cytohesin-1 also induce Arf6 activation in vitro, the activation of other Arf isoforms by these BFA-insensitive Arf-GEFs in intact HL-60 cells cannot be excluded.

fMLP stimulates the activation of PI 3-kinase-{gamma} [50 ]. It is interesting that PI 3-kinase inhibitors reduce fMLP-induced PLD activation in human granulocytes [22 ]. However, although PLD activation is decreased, PI 3-kinase inhibitors do not interfere with the ability of fMLP to recruit Arf1 to membranes [22 ]. As described previously for Arf1 [22 , 24 ] and Arf6 [26 ], this study provides evidence that cytohesin-1 and ARNO were translocated rapidly to the membrane fraction within a minute of stimulation with fMLP and remained associated with membranes after 5 min (not shown). ARNO and cytohesin-1 have PH domains that bind PIP2 or PIP3 with high affinity [34 , 36 , 37 ]. Therefore, it is possible that the driving force for targeting ARNO and cytohesin-1 to membranes is the activation of PI 3-kinase, which increases the levels of membrane PIP3. Indeed, the recruitment of ARNO [37 ], GRP-1 [38 ], and cytohesin-1 [39 ] to plasma membranes has been shown to require the activation of PI 3-kinase. However, it is not clear in the studies mentioned above whether the isoform of ARNO or GRP-1 used has a triglycine or a diglycine motif in its PH domain. This is particularly important because ARNO, GRP-1, and cytohesin-1 with a diglycine motif in their PH domain display high selectivity in their binding to PIP3 versus PIP2, and the presence of a third glycine increases the affinity of these domains for PIP2 considerably [54 ]. To our knowledge, there is no study about the mechanisms regulating the translocation of endogenous ARNO and cytohesin-1 to membranes of HL-60 cells. Here, we show that the ability of fMLP to recruit ARNO to the membrane fraction requires the activation of PI 3-kinase. In contrast, the translocation of cytohesin-1 is insensitive to wortmannin and LY294002. It is noteworthy that human ARNO, GRP-1, and cytohesin-1 have been shown to contain the diglycine or the triglycine motif in their PH domain. It is interesting that RT-PCR analyses using mRNA isolated from Me2SO-differentiated HL-60 cells have established that ARNO diglycine and cytohesin-1 triglycine are the isoforms expressed in HL-60 granulocytes. Based on these data, it can be predicted that ARNO can be recruited selectively by the products of PI 3-kinase, whereas PIP2 and PIP3 will induce membrane localization of cytohesin-1 in granulocytes. Consistent with this prediction, ARNO but not cytohesin-1 was sensitive to inhibition by PI 3-kinase inhibitors. Therefore, the lack of inhibition of Arf1 and cytohesin-1 translocation by PI 3-kinase inhibitors strengthens our conclusion that cytohesin-1 but not ARNO is the Arf-GEF involved in Arf1 activation in HL-60 cells and neutrophils [44 ]. A possibility is that cytohesin-1 and ARNO are recruited in different membrane domains and activate distinct Arf isoforms. In this scenario, cytohesin-1 and ARNO would activate Arf1 in the Golgi [30 ] and Arf6 in the plasma membrane [23 , 26 ], respectively. Although we cannot totally exclude a role for GRP-1 in the recruitment of Arf1 to membrane microdomains, cytohesin-4 is unlikely to be involved because it is not expressed in granulocytes [53 ].

Clearly, there are pathways other than PI 3-kinase involved in Arf-GEF membrane-targeting. One possibility is the PKC pathway because ARNO has been shown to regulate cytoskeletal reorganization in Hela cells, and cortical actin rearrangement induced by PMA is not observed in cells expressing the inactive mutant E156K or a truncated ARNO lacking the PH domain [61 ]. A role for PKC in human granulocytes is suggested by the ability of PMA to recruit ARNO and cytohesin-1 to HL-60 cell membranes. ARNO and cytohesin-1 have PKC-phosphorylation sites in their basic C-terminal domain. However, phosphorylation of ARNO by PKC does not stimulate enzyme activity but rather decreases its nucleotide-exchange activity and its interaction with cell membranes [36 , 62 ]. We cannot ignore the possible activation of phosphoinositol kinases by PKC isoforms, such as PKCµ (PKD), which acts as a scaffold for type II phosphoinositol 4-kinase and type I phosphatidylinositol 4-phosphate 5-kinase [63 ]. Increased synthesis of PIP2 or activation of PI 3-kinase [64 ] by PMA may potentially provide the link to ARNO and cytohesin-1 translocation in granulocytes.

In previous work [60 ], it was established that a 50-kDa fraction isolated from cytosol of HL-60 granulocytes stimulated PLD activity. This fraction contained cytohesin-1 but not ARNO. We had hoped to be able to isolate cytohesin-1 from the stimulating fraction and establish its role in PLD activation. However, this proved not to be practical because of the low amounts of cytohesin-1. Therefore, an alternative approach was adopted. Cytohesin-1 triglycine and ARNO diglycine, which are both found in HL-60 granulocytes, were overexpressed in E. coli to analyze, in vitro, their effect on the activation of PLD by Arf1 and GTP{gamma}S binding to Arf1. As shown previously for ARNO [32 , 48 ], maximal activation of Arf1 by cytohesin-1 triglycine requires low Mg2+ concentration and PIP2. Cytohesin-1 was always more potent than ARNO in the GTP-binding to Arf1 and Arf6 (unpublished results) or the PLD assay. The difference in effects of the two GEFs on GTP-binding to Arf1 is unlikely to reflect the quality of the recombinant proteins, because similar results were obtained with three different preparations of Arf-GEFs. The in vitro data suggest that differences in the binding affinities of ARNO diglycine and cytohesin-1 triglycine for PIP2 impact nucleotide-exchange activities, thereby modulating the recruitment and concentration of active Arf(s) in the proximity of its target PLD1 in cells. Because PLD requires PIP2 and/or PIP3 for activity [65 ], the impact of their concentrations on the Arf-GEF-mediated increase in the levels of PtdCho hydrolysis by Arf1 could not be tested in vitro. ARNO and cytohesin-1 share 83% homology, and the Sec7 domain is the most conserved [66 67 68 ]. The Sec7 domains of ARNO and cytohesin-1 differ by only five residues. Whether these residues in the Sec7 domain or the more divergent N- and C-terminal domains could impact on interaction with Arf1 has not been examined.

We do not know the relative functions of ARNO and cytohesin-1 in granulocyte physiology because there is, at least in these cells, no other available data. Arf-GEFs are likely to be involved in the activation of PLD by Arf1 or Arf6 depending on cell models. Indeed, cytohesin-1 and ARNO have been shown to increase PLD activation by Arf(s) in membranes of HEK-293 cells [69 , 70 ]. However, the endogenous Arf isoforms stimulating PLD activity in HEK-293 cells were not characterized. Several studies indicate that ARNO is the membrane-associated Arf-GEF involved in the Arf6-dependent PLD activation in chromaffin cells [23 ] and kidney epithelial cells [71 ]. Furthermore, in PLB-985 granulocyte-like cells, Arf6 but not Arf1 regulates fMLP-mediated activation of the NADPH oxidase, and for this physiological function, Arf6 requires activation of PLD [26 ]. The fact that PI 3-kinase inhibitors inhibit PLD activation [22 ] and the translocation of endogenous ARNO but not Arf1 and cytohesin-1 to granulocyte membranes does not support a role for cytohesin-1 and Arf1 in fMLP-induced PLD activation in intact HL-60 cells. Whether the inhibitory effects of PI 3-kinase inhibitors on PLD activity are linked to inhibition of Arf6 activation by ARNO remains to be determined. However, our observations suggest that cytohesin-1 triglycine and ARNO diglycine have different functions in stimulated granulocytes, and future work should also address the physiological function of cytohesin-1 and its role in the activation of Arf1. Cytohesin-1 has been shown to bind to the cytoplasmic tail of the integrin ß2 chain CD18 and to promote the LFA-1 ({alpha}Lß2 integrin)-dependent binding of Jurkat cells [41 ] and monocytes [42 ] to intracellular adhesion molecule 1. ß2 Integrin cross-linking increases PLD activity, and PLD-derived PA could increase the affinity of ß2 integrin for their ligands [72 ]. Whether cytohesin-1 binds to the cytoplasmic tail of the integrin ß2 chain is not known in granulocytes. Currently, we are determining the subcellular distribution of cytohesin-1 and its colocalization with PLD, small GTPases, and LFA-1 in human neutrophils.

In conclusion, cytohesin-1 triglycine is expressed more abundantly than ARNO diglycine in human granulocytes. Cytohesin-1 and ARNO are translocated to membranes following stimulation with fMLP. However, fMLP-induced translocation of ARNO to membranes was dependent strictly on PI 3-kinase activation, whereas that of cytohesin-1 was not. Cytohesin-1 was more potent than ARNO in enhancing GTP binding to Arf1 and PLD activation by Arf1. Taken together, our results demonstrate that the presence of a diglycine motif in the PH domain of ARNO and a triglycine motif in the PH domain cytohesin-1 is sufficient to confer different mechanisms of recruitment to granulocyte membranes and modify the biochemical properties of these Arf-GEFs in vivo or in vitro. The divergent properties of endogenous ARNO and cytohesin-1 provide novel information concerning the regulation of Arf1 activation in human granulocytes.


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ACKNOWLEDGEMENTS
 
This work was supported by a Senior Scholarship and research grants from the Arthritis Society (S. G. B.) and the Medical Research Council of Canada (D. N. B.). S. G. B. was the recipient of a Visiting Scientist Award, and D. N. B. holds a Medical Scientist Award from the Alberta Heritage Foundation for Medical Research.

Received July 26, 2001; revised November 9, 2001; accepted December 13, 2001.


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K. Venkateswarlu
Interaction Protein for Cytohesin Exchange Factors 1 (IPCEF1) Binds Cytohesin 2 and Modifies Its Activity
J. Biol. Chem., October 31, 2003; 278(44): 43460 - 43469.
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