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Originally published In Press as doi:10.1074/jbc.M304078200 on August 14, 2003

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

Kanamarlapudi Venkateswarlu{ddagger}

From the Department of Pharmacology, The University of Bristol, Bristol BS8 1TD, United Kingdom

Received for publication, April 17, 2003 , and in revised form, August 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ADP-ribosylation factor (ARF)1 family of small GTPases regulate membrane trafficking at multiple sites within the cell (for reviews, see Refs. 1 and 2). Like other small GTPases, ARFs also function as molecular on/off switches by shuttling between an inactive GDP- and an active GTP-bound form. In mammalian cells, the ARF family consists of six ARF isoforms (ARF1–6). ARF1 and ARF6 are the best characterized and the most distantly related members of the ARF family. ARF1 localizes to the cytosol in the GDP-bound form and attaches in the GTP-bound form to the Golgi complex where it regulates vesicle formation by recruiting cytosolic coat proteins (COPI and AP1) onto the Golgi membranes (3). ARF1 has been implicated in cellular events such as protein secretion (2). ARF6, by contrast, associates with a tubular endosomal compartment in its inactive GDP-bound state and localizes to the plasma membrane in its active GTP-bound state. It primarily regulates vesicle trafficking between these two compartments by acting as a GDP/GTP-regulated switch (4). ARF6 plays an important role in cellular events such as receptor-mediated endocytosis, regulated exocytosis, and cell spreading by regulating cortical actin rearrangement beneath the plasma membrane and membrane ruffling (2, 4). ARFs are regulated by two kinds of proteins: ARF guanine nucleotide exchange factors (GEFs), which promote exchange of bound GDP with GTP, and ARF GTPase-activating proteins, which activate the hydrolysis of bound GTP to GDP (5, 6).

ARF GEFs are divided into two major groups, high molecular weight and low molecular weight, on the basis of sequence similarity, functional differences, and sensitivity to brefeldin A, a fungal metabolite that inhibits activation of ARF1 but not ARF6 (7). The high molecular weight mammalian ARF GEFs (>100 kDa in size) consist of BIG1, BIG2, and GBF1 (Golgi-specific brefeldin A resistance factor 1). They localize to and regulate membrane trafficking in the Golgi region (4, 8). BIG1 and BIG2 have been reported to be sensitive to brefeldin A. The low molecular weight mammalian ARF GEFs include the cytohesin family, the EFA6 (exchange factor for ARF6) family, and ARF-GEP100 (8, 9). These ARF GEFs are insensitive to brefeldin A and appear to be involved in ARF6-mediated membrane trafficking and cytoskeletal reorganization. The cytohesin family of ARF GEFs (cytohesin 1, cytohesin 2/ARNO (ARF nucleotide binding site opener), cytohesin 3/GRP1 (general receptor for phosphoinositides 1), and cytohesin 4) consist of a central catalytic Sec7 domain flanked by a C-terminal pleckstrin homology (PH) domain and an N-terminal coiled-coil (CC) domain (5). The CC domain of cytohesin 2 has been shown to mediate homodimerization, whereas the PH domain of cytohesins 1–3 have been shown to bind the inositol lipid second messenger PtdIns(3,4,5)P3 in vitro (10–13). We and others have demonstrated that cytohesins 1–3 also bind PtdIns(3,4,5)P3 in vivo by showing translocation of the GFP-tagged proteins in a PH domain-dependent manner from the cytosol to the plasma membrane in response to PtdIns(3,4,5)P3 production in agonist-stimulated cells (11–17). It was reported recently that the splice variants of cytohesins 2 and 3 that contain a triglycine motif instead of diglycine motif in the loop connecting the first and second {beta} strands in the PH domain bind PtdIns(3,4,5)P3 and PtdIns(4,5)P2, indicating possible diversity in the regulation of cytohesins by PtdIns lipids (18, 19). Although cytohesins appear to regulate most of the ARFs in vitro by facilitating GDP/GTP exchange, in vivo cytohesins 1–3 mainly regulate activation of ARF6 in a PtdIns(3,4,5)P3-dependent manner by localizing at the plasma membrane (14, 20, 21).

A few studies, however, have reported that cytohesins 1–3 localize to the Golgi through the interaction of the CC domain with an adaptor and inhibit cell secretion and Golgi disassembly (22–24). Binding partners for the CC domain of cytohesin 1 (Cybr/CASP) and cytohesin 3 (GRASP and GRSP1) have recently been identified (25–28). However, the functional consequences of these interactions have not been fully determined, although Cybr has been shown to enhance cytohesin 1 ARF GEF activity in vitro (25). We report here the characterization of a novel protein termed IPCEF1 (interaction protein for cytohesin exchange factors 1) that has been identified by a yeast two-hybrid screen of a rat brain cDNA library using cytohesin 2 as bait. IPCEF1 associates with cytohesin 2 and localizes to the cytosol in intact cells and translocates with cytohesin 2 to the plasma membrane of cells stimulated with epidermal growth factor (EGF) where it modulates the ARF6 GEF activity of cytohesin 2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—All chemicals were obtained from Sigma unless otherwise specified. DNA restriction enzymes were from Roche Applied Science.

Plasmids—Full-length human cytohesin 2 and its deletion mutant cDNA sequences were amplified by PCR using cytohesin 2/pEGFPC1 plasmid as a template (11), Pfu DNA polymerase (Stratagene), and the following sets of primers containing EcoRI (sense, underlined) and SalI (antisense, underlined) restriction sites: FL (the full-length human cytohesin 2, 1–399 aa), sense primer 5'-CGCGAATTCATGGCCAAGGAGCGGCGCAGG-3' and antisense primer 5'-CGCGTCGACTCAGGGCTGCTCCTGCTTCTT-3'; {Delta}PH (cytohesin 2 deletion mutant without the PH domain, 1–252 aa), FL sense primer and antisense primer 5'-CGCGTCGACTCAGTCATTCCCGTCATCCTCAGG-3'; Sec7 (cytohesin 2 Sec7 domain, 61–252 aa), sense primer 5'-CGCGAATTCATGCGGAACCGGAAGATGGCAATG-3' and {Delta}PH antisense primer; and CC (the CC domain of cytohesin 2, 1–67 aa), FL sense primer and antisense primer 5'-CGCGTCGACTCACATTGCCATCTTCCGGTTCCG-3'. The resulting cDNAs were digested with EcoRI and SalI and cloned into the same sites of bait plasmid pBTM116 (29) for expression as LexA DNA binding domain (BD) fusion proteins and pGEX 4T1 vector (Amersham Biosciences) for expression as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli. The pGEX constructs for full-length cytohesins 1 and 3 and centaurin-{alpha}1 were described previously (12, 13, 30). The full-length cytohesin 4 cDNA was amplified from mammalian gene collection clone 48780 by PCR using sense primer 5'-CGCGAATTCATGGACCTGTGCCACCCAGAGCCCG-3' and antisense primer 5'-CGCGTCGACTCACTGCTTGCTGGCAATCTTC-3' containing EcoRI (underlined) and SalI (underlined) sites, respectively. The cDNA was then digested with EcoRI and SalI and ligated to pGEX-4T1. Bait constructs encoding human cytohesins 1, 3, and 4 and centaurin-{alpha}1 were prepared by subcloning the full-length sequences from the respective pGEX constructs into pBTM116 vector. IPCEF1 and its deletion mutant sequences were isolated by PCR using IPCEF1/pGEMT (see below) and the following sets of primers containing EcoRI (sense, underlined) and SalI (antisense, underlined) sites, digested with EcoRI-SalI, and cloned into the same sites of pGAD424 (Clontech) for expression as GAL4 activation domain (AD) fusion proteins in yeast cells and pCMV-tag2 (Stratagene) for expression as FLAG-tagged proteins in mammalian cells: IPCEF1 FL (IPCEF1 full-length, 1–406 aa), sense primer 5'-CGCGAATTCATGAGTCGGAGAAGGATATCCTG-3' and antisense primer 5'-CGCGTCGACAAGAGAATTTTCCACACAGTCAG-3'; {Delta}N101 (IPCEF1, 102–406 aa), sense primer 5'-CGCGAATTCATGTGGCTAAACAAACTTGGATTTG-3' and IPCEF1 FL antisense primer; {Delta}N188 (IPCEF1, 189–406 aa), sense primer 5'-CGCGAATTCATGAAAGAAAGACAGTCGTGGCTTG-3' and IPCEF1 FL antisense primer; {Delta}N285 (IPCEF1, 286–406 aa), sense primer 5'-CGCGAATTCATGGAGAAACTGTACAAGTCATTG-3' and IPCEF1 FL antisense primer; and {Delta}C121 (IPCEF1, 1–285 aa), IPCEF1 FL sense primer and antisense primer 5'-CGCGTCGACCTCATCGTCTTCGGCGATTTTAT-3'. ARF6-HA/pXS construct, kindly provided by Dr. J. Donaldson (National Institutes of Health), was used to express ARF6 with a C-terminal hemagglutinin (HA) epitope tag in mammalian cells. ARF6-HA/pXS was also used as a template to isolate full-length ARF6 by PCR using forward (5'-CTAGCCATGGGGAAGGTGCTATCCAAAATC-3') and reverse (5'-CGGAATTCGAGATTTGTAGTTAGAGGTTAACC-3') primers containing NcoI (underlined) and EcoRI (underlined) restriction sites, respectively. The PCR product was digested with NcoI-EcoRI and cloned into the same sites of pTric-His2b (Novagen) for expression of protein with a C-terminal His6 epitope tag in E. coli. Similarly the full-length metallothionein 2A (MT2A) cDNA was amplified from mammalian gene collection clone 12397 by PCR using sense (5'-CGCGAATTCATGGACCCCAACTGCTCCTGCGCCGC-3') and antisense (5'-CGCGTCGACGGCGCAGCAGCTGCACTTGTCCG-3') primers having EcoRI (underlined) and SalI (underlined) sites, respectively. The cDNA was digested with EcoRI-SalI and ligated to pGEX-4T1 for expression as GST fusion protein in E. coli. pBB131 plasmid encoding yeast N-myristoyltransferase (obtained from Prof. J. I. Gordon, Washington University) was used to myristoylate ARF6 in E. coli. The authenticity of all constructs was verified by nucleotide sequencing using an ABI Prism 211 automated sequencer (PerkinElmer Life Sciences). All bait constructs tested negative for autoactivation of reporter gene activity in the yeast two-hybrid reporter strain L40 (contains HIS3 and {beta}-galactosidase reporters) (29).

Yeast Two-hybrid Screening—Yeast two-hybrid screening was performed as described previously (31). Briefly, yeast strain L40 was first transformed with cytohesin 2/pBTM116 construct and then with a rat brain pGAD10 cDNA library (Clontech) using the lithium chloride method (31). The transformation mixture was grown overnight at 30 °C in synthetic medium lacking tryptophan and leucine to select the transformants carrying the plasmids. A total of 10 million transformants were assayed for growth on synthetic medium in the absence of histidine, leucine, and tryptophan. Positive colonies were then reassayed for growth on the histidine-lacking medium and for {beta}-galactosidase activity. {beta}-Galactosidase filter assays were carried out as described previously (46). The pGAD plasmids were recovered from the transformed yeast colonies using E. coli strain HB101 as a recipient strain and selecting on M9 minimal medium. The cDNA inserts of the recovered plasmids were sequenced as described above. The 5' coding sequence of IPCEF1 was obtained by 5'-end rapid amplification of cDNA ends (5'-RACE) from the Marathon-Ready cDNA library of rat brain (Clontech) using antisense primer 5'-GTCCCACATCTTCGGTAGCAGGCGA-3' (741–765 base pairs of IPCEF1) and an adapter sense primer provided with the library. The 5'-RACE was carried out as described in the manufacturer's manual. The RACE product was sequenced and then ligated to the truncated IPCEF1 cDNA to obtain a full-length cDNA by PCR. The PCR product was cloned into pGEMT vector (Promega) and sequenced as described.

Gene Expression Analysis—Expression of IPCEF1 gene in various rat tissues was analyzed by amplifying a 375-base pair 3'-translated region of IPCEF1 from the multiple tissue cDNA panel (Clontech) by PCR (94 °C for 1 min; 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 1 min; and 68 °C for 5 min) using the IPCEF1{Delta}N285 sense and IPCEF1 FL antisense primers (described above) and DNA polymerase (Clontech). Glyceraldehyde-3-phosphate dehydrogenase expression in multiple tissue cDNA was also analyzed by PCR using sequence-specific primers provided by the manufacturers.

Cell Culture and Transfection—COS and normal rat kidney (NRK) cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and 2 mM glutamine in a humidified incubator with 5% CO2 at 37 °C. COS and NRK cells were transiently transfected with plasmid DNA using the liposomal transfection reagent FuGENE 6 (Roche Applied Science) at a ratio of 4 µl of reagent/1 µg of DNA according to the manufacturer's instructions.

GST Fusion Protein Pull-down Assay—COS cells were plated into 100-mm Petri dishes and allowed to grow to 70–80% confluency. Then they were transfected with FLAG-tagged full-length IPCEF1 or its deletion mutant-encoding plasmids (5 µg of DNA). After 48 h of transfection, cells were washed twice with phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 1% protease inhibitor mixture, Sigma). The lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. 90% of the cell extract was incubated with 20 µg of GST or GST-fused protein coupled to glutathione beads (Amersham Biosciences) for 1 h at 4 °C. The resin was washed three times with the lysis buffer and boiled in SDS-PAGE sample buffer. The samples were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Whatman) and probed with anti-FLAG M2 monoclonal antibody (Sigma). GST fusion proteins were expressed in E. coli strain BL21(DE3) and purified by glutathione-Sepharose chromatography (Amersham Biosciences) according to the method described previously (12).

Co-immunoprecipitation—COS cells were co-transfected with GFP or GFP-cytohesin 2 and FLAG or FLAG-tagged full-length IPCEF1 plasmids (1:1 ratio, 10 µg of total DNA). After 2 days, COS cells were lysed as described above, and the cell extracts were incubated with 5 µg of anti-GFP polyclonal antibody (Clontech) for 30 min at 4 °C. The immunocomplexes were incubated with 30 µl of protein A-Sepharose (Sigma) for 2 h at 4 °C and washed five times with lysis buffer. The bound protein was detected by immunoblotting using anti-FLAG M2 monoclonal antibody. To examine the interaction of FLAG-IPCEF1 with endogenous cytohesin 2, NRK cells were transfected with FLAG-tagged IPCEF1 plasmid, and cell extracts were prepared as described above. The cell extracts of NRK cells were incubated with 10 µl of preimmune serum or anti-cytohesin 2 polyclonal antibody (Santa Cruz Biotechnology, Inc.) for 30 min at 4 °C. The cells extracts of NRK cells were also incubated with GST or GST-cytohesin 2 CC for 60 min and then with 10 µl of anti-FLAG monoclonal M2 antibody (Sigma) for 30 min at 4 °C. The immunocomplexes were incubated with 30 µl of protein A- or protein G-Sepharose for 2 h at 4 °C and washed five times with lysis buffer. The bound protein was detected as described above with an anti-cytohesin 2 polyclonal antibody.

In Vitro ARF6 Activation Assay—Myristoylated ARF6 with His6 tag at the C terminus was expressed in E. coli BL21(DE3) strain by cotransforming with yeast N-myristoyltransferase plasmid (pBB131) and purified using a Ni2+ affinity column (Novagen) according to the procedure described previously by Glenn et al. (32). GST-cytohesin 2 and GST-IPCEF1 recombinant proteins were prepared as described previously (12). [35S]GTP{gamma}S binding to recombinant myristoylated ARF6 was performed in triplicate using a rapid filtration procedure (33). Briefly, 50 µl of the reaction buffer (50 mM HEPES, pH 7.5, 1 mM MgCl2, 0.1 M KCl, 1 mM dithiothreitol) containing 1 µM ARF6, 4 µM [35S]GTP{gamma}S (0.2 µCi), and 50 µg of liposomes (70% phosphatidylcholine, 30% phosphatidylglycerol, and 5% PtdIns(3,4,5)P3) was incubated with either GST (200 nM), GST-cytohesin 2 (50 nM), GST-IPCEF1 (10–200 nM), or GST-cytohesin 2 (50 nM) + GST-IPCEF1 (10–200 nM) at 37 °C. Reactions were stopped at the indicated times by adding 1.5 ml of ice-cold washing buffer (20 mM HEPES, pH 7.5, 0.1 M NaCl, and 10 mM MgCl2) to each reaction tube. Samples were filtered through nitrocellulose membranes in a vacuum manifold (Millipore). The filters were washed three times with 2 ml of ice-cold washing buffer and dried. Scintillation fluid was added to the filters, and they were counted in a Beckman LS 5000 {beta}-counter to quantify the amount of [35S]GTP{gamma}S-bound protein.

In Vivo ARF6 Activation Assay—This assay was performed as described previously (34). COS cells transfected with ARF6-HA, GFP, or GFP-cytohesin 2 and FLAG or FLAG-tagged full-length IPCEF1 plasmids (3:1:1 ratio, 10 µg of total DNA) were serum-starved for 2 h and incubated for 5 min with or without 200 ng/ml EGF. In some experiments 8 µg of ARF6-HA, 2 µg of GFP-cytohesin 2, 5–0 µg of FLAG, and 0–5 µg of FLAG-IPCEF1 vectors were used for transfection. The cells were then lysed in 0.5 ml of lysis buffer B (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 0.1% protease inhibitor mixture (Sigma)). The lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. 90% of the cell extract was incubated with GST-MT2A coupled to glutathione beads in the presence of 2 mM ZnCl2. After1hof mixing at 4 °C, the beads were washed three times with wash buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM MgCl2, 1% Triton X-100, 2 mM ZnCl2, and 0.1% protease inhibitors), boiled in SDS-PAGE sample buffer, and analyzed by immunoblotting using a monoclonal anti-HA antibody (Covance). Immunoblots were scanned and the GTP-bound ARF6 precipitated with GST-MT2A was normalized to total ARF levels in the lysates to compare ARF6GTP levels in cells transformed with the indicated constructs. The GST-MT2A fusion protein was expressed in BL21(DE3) strain of E. coli and coupled to glutathione beads as described previously (12).

Immunofluorescence—NRK cells were seeded onto 13-mm coverslips in a 24-well plate (50–60% confluency) and were transfected with GFP or GFP-cytohesin 2 and FLAG-tagged IPCEF1 or its deletion mutant-encoding plasmids (1:1, 0.5 µg of total DNA). After 2 days, cells were serum-starved for 2 h. The cells were then incubated with 200 ng/ml EGF (Sigma) for 5 min and fixed immediately with 4% paraformaldehyde in phosphate-buffered saline for 15 min. The cells were washed three times with phosphate-buffered saline, permeabilized with 0.2% Triton X-100 for 10 min, blocked with blocking buffer (1% bovine serum albumin in wash buffer (phosphate-buffered saline + 0.1% Triton X-100)) for 30 min and incubated with anti-FLAG M2 antibody (10 µg/ml) in blocking buffer for 1 h. The cells were washed three times with wash buffer, then incubated with a 1:500 dilution of goat TRITC-conjugated anti-mouse antibody (Jackson Immunoresearch Laboratories, Inc.) in blocking buffer for 1 h, and mounted on slides with mounting solution (0.1 M Tris-HCl, pH 8.5, 10% Mowiol (Calbiochem), and 50% glycerol) containing 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma). Immunofluorescence staining was visualized using a Leica TCS-NT confocal microscope equipped with a krypton/argon laser. All images presented are single sections in the z-plane.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Novel Protein That Interacts with Cytohesin 2 by Yeast Two-hybrid Screening—To identify proteins that may interact with cytohesin 2, we screened a yeast two-hybrid cDNA library derived from rat brain using full-length human cytohesin 2 as bait. One of the positive clones was shown to encode part of a novel protein that we referred to as IPCEF1. Retransformation experiments confirmed that IPCEF1 specifically interacts with cytohesin 2 but not with unrelated bait proteins such as centaurin-{alpha}1 (data not shown, see Fig. 7). Since the PH domain of cytohesin 2 shares significant homology with the C-terminal PH domain of centaurin-{alpha}1, the inability of IPCEF1 to interact with centaurin-{alpha}1 not only confirms the specificity of interaction between IPCEF1 and cytohesin 2 but also suggests that the cytohesin 2 interacts with IPCEF1 through a region other than the PH domain.



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  		FIG. 7.
Interaction with IPCEF1 is highly conserved among cytohesin family ARF GEFs. IPCEF1 interacts with other members of cytohesin family in yeast two-hybrid (A) and GST pull-down assays (B). A, cytohesins 1–4 and centaurin-{alpha}1 fused to the DNA BD of LexA (pBTM) were co-transformed with IPCEF1 fused to the AD of GAL4 (pGAD) into L40 yeast strain, and the transformants were tested for their ability to grow in the absence of histidine and to express {beta}-galactosidase, which is analyzed by assaying the conversion of X-gal into a blue-colored product. A, left to right, shows three clones of each transformant grown on solid medium with (+His) and without (–His) histidine and a filter {beta}-galactosidase assay ({beta}-gal). B, the lysates of COS cells expressing FLAG-IPCEF1 were incubated with GST-tagged cytohesins 1–4 and centaurin-{alpha}1 coupled to glutathione beads. After washing the beads, the bound proteins were analyzed by immunoblotting using an anti-FLAG antibody (Blot). These findings were reproduced three times with identical results.

 

By using rat brain 5'-RACE cDNA, we isolated the missing 5' sequence and ligated this by PCR to the truncated IPCEF1 sequence that was isolated by the two-hybrid screening to obtain a full-length IPCEF1 (deposited into EMBL/GenBankTM/DDBJ under accession number AJ536192 [GenBank] ). The entire nucleotide sequence of rat IPCEF1 along with the deduced amino acid sequence is shown in Fig. 1. The cDNA sequence contained an open reading frame of 1221 base pairs, extending from nucleotides 141 to 1361, encoding for a protein of 406 amino acids with a calculated molecular mass of 45.7 kDa and a theoretical isoelectric point (pI) of 7.53. The 140-nucleotide 5'-untranslated region includes a Kozak consensus sequence upstream of the ATG codon, and the 3'-untranslated region extends for 196 nucleotides following the TAG stop codon. IPCEF1 contains no signal sequence or potential transmembrane domain.



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  		FIG. 1.
Sequence analysis of IPCEF1. The nucleotide sequence and the deduced amino acid sequence, in single letters, of IPCEF1. Amino acid sequences of the PH domain and the partial cDNA isolated by the yeast two-hybrid screening are shown in bold and underlined letters, respectively. The Kozak sequence (CAAC) is indicated by bold italic letters. The sequence has been deposited in the EMBL/GenBankTM/DDBJ data base with accession number AJ536192 [GenBank] . *, the stop codon.

 

Analysis of the IPCEF1 deduced amino acid sequence reveals the presence of a PH domain at the N-terminal end (Fig. 1), indicating that IPCEF1 is a new member of the PH domain-containing protein family. Data base searches revealed the presence of rat IPCEF1 homologues as uncharacterized cDNA and also as open reading frames obtained from genome projects in a number of organisms including human, mouse, and Drosophila. IPCFE1 displays 79% amino acid identity to human KIAA0403 (Fig. 2), which was identified as a protein of unknown function in a brain gene-cloning project. The high homology between IPCEF1 and KIAA0403 suggests that these proteins are species orthologues. Because it contains the PH domain that mediates protein-PtdIns lipid interactions, we analyzed whether IPCEF1 binds PtdIns lipids using an in vitro protein-lipid overlay assay described previously (35). To this end, a nitrocellulose blot spotted with various concentrations of PtdIns lipids was incubated with purified GST-IPCEF1, and any bound protein was detected by immunoblotting with an anti-GST antibody. Using this assay, we found no interaction between IPCEF1 and PtdIns lipids. However, cytohesin 2, used as a positive control in the binding assays, bound specifically to PtdIns(3,4,5)P3 lipid as expected (data not shown).



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  		FIG. 2.
Alignment of the amino acid sequence of rat IPCEF1 and the human protein KIAA0403. The deduced amino acid sequence of rat IPCEF1 is aligned with that deduced for the human homologue KIAA0403 using the ClustalW program. Identical amino acids are indicated by * in the consensus sequence. The percentage identity is 79%, and percentage similarity is 85% between the two proteins.

 

Tissue Distribution of IPCEF1 mRNA—To gain an insight into the possible functional roles of IPCEF1, we analyzed the expression of IPCEF1 mRNA using a PCR-based assay with cDNA prepared from a number of rat tissues. Sequence-specific primers were used to obtain a 375-base pair fragment corresponding to rat IPCEF1 (nucleotides 989–1364). As shown in Fig. 3, rat IPCEF1 is widely distributed. IPCEF1 expression was most abundant in brain, spleen, lung, and testes. A relatively low, but significant, expression of IPCEF1 was also detected in kidney. There was very low expression of IPCEF1 in liver, and no expression of IPCEF1 was detected in either heart or skeletal muscle.



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  		FIG. 3.
IPCEF1 mRNA expression pattern in rat tissues. An IPCEF1{Delta}N285 (375-bp) cDNA fragment was amplified from rat multiple tissue cDNA panel by PCR. Lane 1, positive control (FLAG-IPCEF1 plasmid DNA); lane 2, negative control (no DNA); lanes 3–10, heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testes. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression in multiple tissue cDNA was also analyzed by PCR to ensure that an equal amount of cDNA from each tissue was used. The bottom panel shows glyceraldehyde-3-phosphate dehydrogenase (1 kb) expression in different tissues. The expression analysis was repeated twice with identical results.

 

Identification of the Binding Domains of Cytohesin 2 and IPCEF1—To understand the structural requirements for the interaction between cytohesin 2 and IPCEF1, we used a deletion mutant approach to map the specific domains of cytohesin 2 and IPCEF1 required for their association. A series of cytohesin 2 deletion mutants were generated as LexA DNA BD fusion constructs, and we tested their ability to bind IPCEF1 fused to the AD of GAL4 in the yeast two-hybrid system (Fig. 4A). The results demonstrated that only the fusion constructs containing the N-terminal CC domain of cytohesin 2 were capable of interacting with IPCEF1, whereas the C-terminal PH domain and the central Sec7 domain were not required for the interaction. To define the region of IPCEF1 involved in binding cytohesin 2, we generated a series of AD fusion constructs containing various truncations of IPCEF1 and analyzed their interaction with BD-cytohesin 2 in a yeast two-hybrid assay (Fig. 4B). The C-terminal 121 amino acids were found to be required for binding cytohesin 2, whereas the rest of the protein was not required for the interaction. Together these results demonstrate that the association of IPCEF1 with cytohesin 2 is mediated by the CC domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1.



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  		FIG. 4.
Identification of the interacting domains of IPCEF1 and cytohesin 2 by yeast two-hybrid assay. A, mapping of the IPCEF1 binding domain of cytohesin 2. Schematic representation of cytohesin 2 and its deletion mutants encoded by the BD fusion cDNA constructs is shown in the top panel. B, mapping of the cytohesin 2 binding region within IPCEF1. Schematic representation of IPCEF1 and its deletion mutants encoded by the AD fusion cDNA constructs is shown in the top panel. *, the insert of the clone isolated by yeast two-hybrid screening using cytohesin 2 as bait. The BD fusion (pBTM) constructs were co-transformed with the AD fusion (pGAD) constructs into L40 yeast strain and tested for their ability to grow in the absence of histidine and to express {beta}-galactosidase, which is analyzed by assaying the conversion of X-gal into a blue-colored product. The bottom panel in both A and B, left to right, shows three clones of each transformant grown on solid medium with (+His) and without (–His) histidine as well as filter {beta}-galactosidase assays ({beta}-gal). These findings were reproduced three times with three different yeast transformants.

 

We next used a GST pull-down assay to determine whether the interaction between IPCEF1 and cytohesin 2 detected in yeast also takes place in vitro. For this purpose, the cytohesin 2 and its deletion mutants were expressed as GST fusion proteins and analyzed for their ability to interact with FLAG-tagged IPCEF1 and its deletion mutants expressed in COS cells. As shown in Fig. 5A, IPCEF1 interacted with GST fusion proteins containing the CC domain of cytohesin 2 but not with the fusion proteins lacking the CC domain or with GST alone. Similarly GST-cytohesin 2 showed strong interaction with either IPCEF1 or the IPCEF1 deletion mutant containing the C-terminal 121 amino acids ({Delta}N285) but not with the deletion mutant lacking the C-terminal 121 amino acids ({Delta}C121) (Fig. 5B). In agreement with the yeast two-hybrid data, these results reveal that the CC domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1 are sufficient and necessary for the IPCEF1-cytohesin 2 interaction.



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  		FIG. 5.
Determination of IPCEF1 and cytohesin 2 binding sites by in vitro GST pull-down assay. A, the lysate of COS cells expressing FLAG-IPCEF1 was incubated with either GST alone, GST-cytohesin 2, or GST-cytohesin 2 deletion mutants, all coupled to glutathione beads. B, lysates of COS cells expressing FLAG-tagged IPCEF1 or its deletion mutants were incubated with either GST or GST-cytohesin 2 coupled to glutathione beads. After washing the beads, the bound proteins were separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with a monoclonal anti-FLAG antibody (Blot). Positions of molecular mass standards, Mr (kDa), are shown. One-twentieth of the cell lysates (Input) is also shown for each of the sets of experiments. These experiments were repeated three times with similar results.

 

Although IPCEF1 and cytohesin 2 are expressed ubiquitously (Fig. 3 and Ref. 36), we were unable to analyze either the relative amounts of endogenous cytohesin 2 and IPCEF1 or the in vivo interaction between the endogenous proteins due to the lack of specific antibodies for IPCEF1. Alternatively we have determined whether the IPCEF1-cytohesin 2 interaction occurs in vivo by co-immunoprecipitating the epitope-tagged proteins expressed transiently in mammalian cells. Co-immunoprecipitation analysis was performed using extracts of COS cells transfected with vectors encoding both GFP-tagged cytohesin 2 and FLAG-tagged IPCEF1. The cell extracts were immunoprecipitated with an anti-GFP polyclonal antibody, and the immunoprecipitates were then analyzed by immunoblotting using an anti-FLAG monoclonal antibody. As shown in Fig. 6A, FLAG-IPCEF1 co-immunoprecipitated with GFP-cytohesin 2 but not with GFP alone. This result clearly demonstrates that cytohesin 2 and IPCEF1 interact with each other in vivo. We have also immunoprecipitated endogenous cytohesin 2 from extracts of NRK cells transiently expressing FLAG-tagged IPCEF1 using an anti-cytohesin 2 polyclonal antibody and detected IPCEF1 in the immunoprecipitates by immunoblotting using an anti-FLAG monoclonal antibody (Fig. 6B, I, lane 3). IPCEF1 was not detected in the immunoprecipitates when control preimmune serum was used for immunoprecipitation (Fig. 6B, I, lane 2). Conversely we immunoprecipitated IPCEF1 from the extracts of NRK cells expressing FLAG-IPCEF1 using the anti-FLAG monoclonal antibody and detected endogenous cytohesin 2 in the immunoprecipitates by immunoblotting using the anti-cytohesin 2 polyclonal antibody (Fig. 6B, II, lane 3). The endogenous cytohesin 2 was not detected in immunoprecipitates of the cell lysates that were preincubated with the purified GST-tagged CC domain of cytohesin 2 (Fig. 6B, II, lane 2). These data confirm the specificity of interaction between cytohesin 2 and IPCEF1.



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  		FIG. 6.
In vivo interaction between cytohesin 2 and IPCEF1. A, co-immunoprecipitation of FLAG-IPCEF1 with GFP-cytohesin 2. COS cells were transfected with the indicated expression vectors. After 2 days of transfection, cells were lysed and immunoprecipitated (IP) with an anti-GFP antibody. After washing, immunoprecipitates were resolved by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with a monoclonal anti-FLAG antibody (Blot) to detect FLAG-tagged IPCEF1. 5% of the cell lysates (Input) was also immunoblotted with anti-GFP and anti-FLAG antibodies to ensure that GFP-cytohesin 2 and FLAG-IPCEF1, respectively, were expressed. B, co-immunoprecipitation of FLAG-tagged IPCEF1 with endogenous cytohesin 2. NRK cells transfected with FLAG-tagged IPCEF1 were lysed after 48 h and immunoprecipitated with control preimmune serum (lane 2) or an anti-cytohesin 2 antibody (lane 3), and FLAG-tagged IPCEF1 in the precipitate and cell lysate (lane 1) was visualized by immunoblot using an anti-FLAG antibody (I). The cell lysates were also incubated with GST-CC (lane 2) or GST (lane 3) prior to immunoprecipitation with an anti-FLAG antibody, and endogenous cytohesin 2 in the precipitates and cell lysate (lane 1) was detected by immunoblot using an anti-cytohesin 2 antibody (II). The experiment was repeated an additional two times with similar results.

 

The Interaction with IPCEF1 Is Highly Conserved among Cytohesin Family ARF GEFs—Since all members of the cytohesin family contain the N-terminal CC domain, we examined whether the other members of this family also interact with IPCEF1 using both the yeast two-hybrid and GST pull-down assays. Cytohesin 2 and centaurin-{alpha}1 were used as positive and negative controls, respectively, in these assays. First we generated cytohesins 1, 3, and 4 as BD fusion constructs and tested their ability to interact with the AD-IPCEF1 in the yeast two-hybrid assay. As shown in Fig. 7, all cytohesin family members were able to interact with IPCEF1. Similarly FLAG-IPCEF1 was precipitated from COS cell extracts by GST-tagged cytohesins 1, 3, and 4. Taken together, these results illustrate that IPCEF1 interacts with all members of the cytohesin family both in yeast and in vitro.

FLAG-IPCEF1 Translocates to the Plasma Membrane with GFP-Cytohesin 2 upon EGF Stimulation—We next analyzed the interaction between IPCEF1 and cytohesin 2 in intact cells by co-localization studies. NRK cells were transfected with vectors encoding FLAG-IPCEF1 and GFP-cytohesin 2 and immunostained with an anti-FLAG antibody to detect FLAG-IPCEF1, and the subcellular localization of proteins was analyzed using a laser scanning confocal microscope. FLAG-tagged IPCEF1 was diffusely localized throughout the cytoplasm when expressed on its own (data not shown), and its localization was unaltered when co-expressed with either GFP or GFP-cytohesin 2 (Fig. 8). It has been reported previously that exogenously expressed GFP-tagged cytohesins 1–3 as well as endogenous cytohesins 1–3 translocate in a PtdIns 3-kinase-dependent manner from cytosol to the plasma membrane in cells stimulated with agonists such as EGF, insulin, and formyl-methionyl-leucyl-phenylalanine (11–17, 20, 37). The specific interaction between IPCEF1 and cytohesin 2 suggests that PtdIns 3-kinase activation might also induce a change in the subcellular localization of IPCEF1. To explore this possibility, we analyzed the subcellular localization of IPCEF1 in cells stimulated with EGF, which activates PtdIns 3-kinase. EGF stimulation of NRK cells transfected with only FLAG-IPCEF1 had no affect on the intracellular localization of IPCEF1 (data not shown). However, when co-expressed with GFP-cytohesin 2, EGF stimulation promoted the redistribution of FLAG-IPCEF1 and GFP-cytohesin 2 from the cytosol to the plasma membrane (Fig. 8). This redistribution was inhibited by wortmannin, a PtdIns 3-kinase inhibitor (data not shown). The deletion mutant of IPCEF1 containing the C-terminal 121-amino acid cytohesin 2 binding domain ({Delta}N285) also co-migrated with GFP-cytohesin 2 to the plasma membrane after EGF stimulation, but the deletion mutant without the cytohesin 2 binding domain ({Delta}C121) failed to relocate under the same conditions. These results clearly demonstrate that the redistribution of IPCEF1 to the plasma membrane upon EGF stimulation requires cytohesin 2.



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  		FIG. 8.
IPCEF1 co-localizes with cytohesin 2 to the cytosol and upon EGF stimulation migrates to the plasma membrane by binding to cytohesin 2. NRK cells were transiently transfected with FLAG-tagged IPCEF1 or its deletion mutants along with GFP or GFP-tagged cytohesin 2. After 48 h, the cells were serum-starved for 2 h, stimulated with EGF (200 ng/ml), and fixed with paraformaldehyde. The fixed cells were then immunostained using an anti-FLAG primary antibody and a TRITC-labeled secondary antibody, mounted onto glass slides, and imaged using a confocal microscope. Similar results were obtained with five different cell preparations.

 

Effects of IPCEF1 on GEF Activity of Cytohesin 2—We finally examined whether the IPCEF1-cytohesin 2 interaction is functionally relevant for the ARF GEF activity of cytohesin 2. Since cytohesin 2 has been shown to activate ARF6 both in vitro and in vivo (20, 38), we analyzed the effect of IPCEF1 on the ARF6 GEF activity of cytohesin 2 using both in vitro and in vivo binding assays. For in vitro studies, purified myristoylated ARF6-His6 was incubated with [35S]GTP{gamma}S in the presence and absence of cytohesin 2 and/or IPCEF1, and the amount of [35S]GTP{gamma}S bound to ARF6 was analyzed. Cytohesin 2 increased the binding of GTP to ARF6 in a time-dependent manner, and the rate of binding was further increased by IPCEF1, which was ineffective when added in the absence of cytohesin 2 (Fig. 9A). Similar results were also obtained using the deletion mutant of IPCEF1 that contains the cytohesin 2 binding site ({Delta}N285) but not the deletion mutant that lacks the cytohesin 2 binding site ({Delta}C121) in place of IPCEF1 (data not shown). Furthermore IPCEF1 amplified cytohesin 2-mediated ARF6 activation in a concentration-dependent manner (Fig. 9B), reaching saturation when the amount of IPCEF1 added was equal to that of cytohesin 2 (2.5 pmol). ARF6 has been shown to interact specifically with an effector, MT2A, when it is in the active GTP-bound form (34). Recently Schweitzer and D'Souza-Schorey (34) have made use of this observation and developed a GST-effector pull-down assay to study ARF activation in vivo. To analyze the effect of IPCEF1 on the in vivo GEF activity of cytohesin 2, HA-tagged ARF6 was co-expressed with either GFP-cytohesin 2, FLAG-IPCEF1, or both in COS cells. Following serum starvation, cells were stimulated with EGF, the cells were lysed, and the activated ARF6 was purified from the cell lysates using GST-MT2A coupled to a glutathione resin. ARF6 activation in EGF-treated cells was stimulated by cytohesin 2, and the activation was further increased by IPCEF1 (Fig. 10). However, IPCEF1 was ineffective as a modulator for ARF6 activation in the absence of cytohesin 2. Although we used an equal amount of FLAG-IPCEF1 and GFP-cytohesin 2 plasmid DNA to transfect COS cells, the effect of IPCEF1 on the in vivo activation of ARF6 by cytohesin 2 was relatively small compared with its effect on the in vitro cytohesin 2 activity when the amount of protein added to the assay was equal to that of cytohesin 2. This may be due to the low expression of IPCEF1 compared with that of cytohesin 2 (data not shown, see Fig. 11, lane 3). To examine whether IPCEF1 affects the in vivo activation of ARF6 by cytohesin 2 in a concentration-dependent manner, we co-transfected COS cells with fixed amounts of DNA of HA-ARF6 and GFP-cytohesin 2 and varying amounts of FLAG-IPCEF1 DNA and analyzed ARF6 activation in EGF-stimulated cells using GST-effector pull-down assay. Consistent with the in vitro data, IPCEF1 modulated in vivo cytohesin 2 ARF6 GEF activity in a concentration-dependent manner, and its effect became saturated when its expression levels were comparable to that of cytohesin 2 (Fig. 11). Together these results suggest strongly that IPCEF1 potentiates ARF6 GEF activity of cytohesin 2 through direct interaction.



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  		FIG. 9.
Effect of IPCEF1 on the in vitro ARF6 GEF activity of cytohesin 2. A, ARF6 (50 pmol) was incubated alone or with GST (10 pmol), GST-cytohesin 2 (2.5 pmol), GST-IPCEF1 (1.25 pmol), or both GST-cytohesin 2 (2.5 pmol) and GST-IPCEF1 (1.25 pmol) for the indicated times at 37 °C. B, ARF6 (50 pmol) was incubated with GST-IPCEF1 (0.5–10 pmol) or GST-cytohesin 2 (2.5 pmol) and GST-IPCEF1 (0.5–10 pmol) for 10 min at 37 °C. The bound GTP was then measured. Values are shown as the means of three independent assays performed in triplicate and expressed as the amount of GTP{gamma}S bound to ARF6. Error bars indicate S.E.

 


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  		FIG. 10.
Effect of IPCEF1 on the in vivo ARF6 GEF activity of cytohesin 2. A, COS cells were transfected with the indicated expression plasmids. Two days later cells were serum-starved for 2 h, incubated in the presence or absence of EGF (200 ng/ml), and lysed, and ARF6GTP was precipitated using GST-MT2A coupled to glutathione beads. The precipitates were then immunoblotted (Blot) with anti-HA antibody. Before ARF6GTP precipitation, cell lysates were also immunoblotted using an anti-HA antibody to determine total ARF6. B, quantification of data obtained from three similar experiments. The mean of three experiments is represented. Error bars indicate S.E.

 


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  		FIG. 11.
Concentration-dependent potentiation of in vivo ARF6 GEF activity of cytohesin 2 by IPCEF1. A, COS cells were transfected with HA-ARF6, GFP-cytohesin 2, FLAG (5–0 µg), or FLAG-IPCEF1 (0–5 µg) vectors. After 2 days, cells were serum-starved for 2 h and stimulated with EGF (200 ng/ml). The cells were immediately lysed, ARF6GTP was precipitated using GST-MT2A coupled to glutathione beads, and the precipitates then immunoblotted (Blot) with anti-HA antibody. The cell lysates were also immunoblotted with anti-HA, anti-FLAG, and anti-GFP antibodies to determine HA-ARF6, FLAG-IPCEF1, and GFP-cytohesin 2 expression levels, respectively. B, quantification of data obtained from three similar experiments. The activation of ARF6 by cytohesin 2 in the absence of IPCEF1 is used as a control. The mean of three experiments is represented. Error bars indicate S.E.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using yeast two-hybrid screening, we have identified IPCEF1 from a rat cDNA library as a novel 406-amino acid protein that interacts with members of the cytohesin family of ARF GEFs. IPCEF1 mRNA has a wide distribution; it is detected in all tissues except heart and skeletal muscle. We confirm that IPCEF1 specifically binds cytohesins in vitro by GST pull-down assays and in vivo by immunoprecipitation assays. The cytohesin 2-IPCEF1 interaction is mediated by the CC domain of cytohesin 2 and the IPCEF1 C-terminal domain. IPCEF1 co-localizes with cytohesin 2 to the cytosol in intact cells and co-migrates with cytohesin 2 to the plasma membrane of cells stimulated with EGF. Moreover IPCEF1 potentiated ARF6 activation by cytohesin 2 in vitro as well as in EGF-stimulated cells, suggesting that it modulates the ARF6 GEF activity of cytohesin 2. This implies a conformational change in the catalytic Sec7 domain upon IPCEF1 binding to the CC domain of cytohesin 2 that leads to an alteration in the catalytic activity of the Sec7 domain.

In recent years, several groups have attempted to isolate cytohesin-interacting proteins that could influence the catalytic activity and localization of cytohesins using approaches such as yeast two-hybrid and cDNA expression library screening. These efforts resulted in the identification of a number of cytohesin-interacting proteins including GRSP1, GRASP, and Cybr/CASP (25–28). GRSP1 is a one FERM (band 4.1, ezrin, radixin, moesin homology domain) and two CC domain-containing protein and was shown to form a complex with cytohesin 3 that localized to the cytoplasm of Chinese hamster ovary cells, and this complex was recruited to plasma membrane ruffles upon stimulation with insulin (28). The gene expression of GRASP and Cybr/CASP, which contain PDZ and CC domains, is inducible by retinoic acid and cytokines, respectively (25–27). GRASP interacts with cytohesins 2 and 3, whereas CASP/Cybr interacts with cytohesins 1–3. GRASP has been shown to recruit cytohesin 3 to the plasma membrane in a constitutive manner. In COS cells, the Cybr/CASP complex appears to localize to the cytosol and translocates to the plasma membrane following EGF stimulation. It has been shown that the interaction between cytohesins and their interactors occurs through the CC domains in these proteins. However, the functional significance of the interactions between cytohesins and their interactors has not been determined except for Cybr/CASP, which was shown to enhance cytohesin 1 catalytic activity in vitro. Interestingly none of these proteins has any obvious homology with the currently described protein, suggesting that IPCEF1 is a novel protein that interacts specifically with the cytohesin family of ARF GEFs. Moreover we have demonstrated the functional relevance of the interaction between cytohesin 2 and IPCEF1 by showing an increase in ARF6 GEF activity of cytohesin 2 by IPCEF1 both in vitro and in vivo.

The Sec7 domain of cytohesins has also been implicated in protein-protein interactions (39, 40). Cytohesin 1 was originally identified by its ability to interact with the cytoplasmic tail of {beta}2 integrin, which is expressed exclusively in cells of the immune system and plays an essential role in the attachment of white blood cells to the endothelium (39, 41). Cytohesin 3 has also been demonstrated to mediate regulation of cell attachment via binding to {beta}2 integrin (40). Cytohesins 1 and 3 interact with {beta}2 integrin through the Sec7 domain. The interaction between cytohesin 1 and {beta}2 integrin results in an increase in the avidity of integrin-substrate binding, which is an important event in the stimulation of white blood cell adherence by the PtdIns 3-kinase pathway (42). However, the GEF activity of cytohesin 1 is not required for this effect, suggesting that the protein-protein interaction alone can regulate this signaling event. Cytohesin 1 was also shown to mediate F-actin-associated shape changes and transformation events triggered by the human herpes virus 8 protein kaposin A by directly interacting with that protein (33). These findings, together with the observation that some of the cytohesins exist in forms with different PtdIns binding specificities (18, 19), suggest that cytohesins participate in several cellular functions by becoming part of distinct complexes through association with their interactors.

In summary, we identified IPCEF1 as a novel cytohesin-interacting protein by yeast two-hybrid screening. IPCEF1 shows no noticeable similarity to known cytohesin interactors. IPCEF1 interacts with the CC domain of cytohesins through its C terminus. In NRK cells, IPCEF1 co-localizes with cytohesin 2 to the cytosol and recruits to the plasma membrane via binding to cytohesin 2 following EGF stimulation. We have also shown that IPCEF1 enhances cytohesin 2 ARF6 GEF activity in vitro and in vivo. Future studies will be aimed at determining the signaling events resulting from the interaction of IPCEF1 with cytohesins.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AJ536192 [GenBank] .

* This work was funded by the Biotechnology and Biological Science Research Council (BBSRC) and the Royal Society UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a David Phillips Research Fellowship from the BBSRC. To whom correspondence should be addressed: Dept. of Pharmacology, School of Medical Sciences, The University of Bristol, University Walk, Bristol BS8 1TD, UK. Tel.: 44-117-9289702; Fax: 44-117-9250168; E-mail: k.venkateswarlu{at}bristol.ac.uk.

1 The abbreviations used are: ARF, ADP-ribosylation factor; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; PtdIns, phosphatidylinositol; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; EGF, epidermal growth factor; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; RACE, rapid amplification of cDNA ends; BD, binding domain; AD, activation domain; NRK, normal rat kidney; BIG, brefeldin A-inhibitable GEF; GRSP1, GRP1 signaling partner 1; GRASP, GRP1-associated scaffold protein; CASP, cytohesin-associated scaffold protein; Cybr, cytohesin binder and regulator; IPCEF1, interaction protein for cytohesin exchange factors 1; aa, amino acids; X-gal, 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside; TRITC, tetramethylrhodamine isothiocyanate; CC, coiled-coil; FL, full-length; MT2A, metallothionein 2A; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back


   ACKNOWLEDGMENTS
 
I am grateful to Kevin Brandom for technical assistance. I thank Drs. Eamonn Kelly, Alastair Poole, and Matthew Jones for careful review of the manuscript and valuable suggestions. I also thank the Medical Research Council UK for providing an Infrastructure Award to establish the School of Medical Sciences Cell Imaging Facility and Dr. Mark Jepson and Alan Leard for assistance. I am also extremely grateful to Professor Jeremy Henley for sharing yeast two-hybrid screening reagents.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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