Isolation of the Novel Human Guanine Nucleotide Exchange Factor ...

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Endocrinology 144(5):1742–1752 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220984

Isolation of the Novel Human Guanine Nucleotide Exchange Factor Src Homology 3 Domain-Containing Guanine Nucleotide Exchange Factor (SGEF) and of C-Terminal SGEF, an N-Terminally Truncated Form of SGEF, the Expression of Which Is Regulated by Androgen in Prostate Cancer Cells ´ A FOURNIER, JOSE ´ E GRENIER, CATHERINE FILLION, YVAN LABRIE, HENG QI, ANDRE CLAUDE LABRIE

AND

Oncology and Molecular Endocrinology Research Center, CHUL Research Center, and Laval University, Quebe´c, Canada G1V 4G2 In searching for androgen-responsive genes in human prostate cancer cells, we have isolated two cDNAs that encode alternate forms of a novel Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF). The SGEF mRNA is widely expressed in human tissues, and the predicted 871amino acid SGEF protein contains Dbl homology and pleckstrin homology domains as well as an N-terminal proline-rich domain, a C-terminal Src homology 3 domain, and two nuclear localization signals. The second cDNA encodes a 139-amino acid N-terminally truncated form of SGEF designated Cterminal SGEF (CSGEF). In contrast to SGEF, CSGEF mRNA expression is restricted to prostate and liver. Moreover, CSGEF expression is up-regulated by androgens in LNCaP

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HO GUANOSINE triphosphatases (GTPases) form a subgroup of the Ras superfamily of guanine nucleotidebinding proteins. Guanine nucleotide-binding proteins act as molecular switches that cycle between a GDP-bound state, which is inactive, and an active GTP-bound state. The activation of Rho GTPases is catalyzed by guanine nucleotide exchange factors (GEFs) that displace GDP from GDP-bound Rho GTPases, thereby allowing Rho GTPases to bind to GTP, which is present at higher concentrations than GDP in the cell (1). The structural conformation of activated (GTP-bound) Rho GTPases specifies their interactions with effector molecules that are implicated in diverse signaling pathways. Rho GTPases play a key role in the regulation of several aspects of cellular function, including membrane trafficking, cytoskeletal organization, gene expression, and cell cycle progression (2). Approximately 60 different Rho GEFs have been identified in humans to date (3). The prototype of Rho GEFs is the dbl oncogene product, which was originally isolated from a diffuse B cell lymphoma. The region of the Dbl protein that is

Abbreviations: aa, Amino acids; CMV, cytomegalovirus; CSGEF, Cterminal Src homology 3 domain-containing guanine nucleotide exchange factor; DH, Dbl homology; GEF, guanine nucleotide exchange factor; GTPase, guanosine triphosphatase; HA, hemagglutinin; PH, pleckstrin homology; SGEF, Src homology 3 domain-containing guanine nucleotide exchange factor; SH3, Src homology 3.

cells, whereas that of SGEF is not. Up-regulation of CSGEF was sensitive to actinomycin D but did not require new protein synthesis. The SGEF gene is located on chromosome 3q25.2 and consists of at least 15 exons. Based on the structure of the SGEF and CSGEF cDNAs, we deduced that CSGEF expression is controlled by an alternate androgen-responsive promoter of the SGEF gene. We hypothesize that SGEF is a ubiquitous regulator of Rho guanosine triphosphatases, whereas CSGEF may function as an androgen-induced regulator of Rho guanosine triphosphatase activity in epithelial cells of the human prostate. (Endocrinology 144: 1742–1752, 2003)

responsible for its catalytic activity is referred to as the Dbl homology (DH) domain (4), a domain that is conserved in all Rho GEFs. Most Rho GEFs also contain a pleckstrin homology (PH) domain that is located immediately C terminal to the DH domain (3). The PH domain is involved in targeting Rho GEFs to specific subcellular sites, such as the plasma membrane. Interestingly, the Dbl oncogene product and a majority of other Rho GEFs function as oncogenes in transfection assays, and the DH-PH module is responsible for the oncogenic property of Rho GEFs. Rho GEFs also contain other functional domains, such as Src homology domains, which are likely to dictate the specific interactions and functions of individual Rho GEFs. Rho GTPases are activated by intracellular signals that are triggered by the stimulation of several growth factor receptors, cytokine receptors, adhesion receptors, or serpentine receptors. One of the possible mechanisms by which such signals can lead to Rho GTPase activation is likely to involve the activation of Rho GEFs. The current model for Rho GEF activation is that Rho GEFs exist in an inactive or partially activated state in the absence of stimulation. Although the specific mechanism(s) by which Rho GEFs are activated in response to external stimuli has not been completely resolved, it is generally thought that Rho GEF activation results from the relief of inhibitory intra- or intermolecular interactions and alterations in the subcellular

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Qi et al. • Androgen Regulation of CSGEF Expression in LNCaP Cells

localization of Rho GEFs, some of which could be related to changes in the phosphorylation state of Rho GEFs (5). Whereas Rho GEFs directly activate Rho GTPases by enhancing the formation of GTP-bound Rho GTPases, Rho GTPase activity can also be controlled at other levels. For instance, GTPase-activating proteins inactivate GTP-bound Rho GTPases by increasing the rate of hydrolysis of bound GTP. Another class of proteins, guanine nucleotide dissociation inhibitors, maintain Rho GEFs in an inactive state by inhibiting the exchange of GDP for GTP. Finally, other proteins can inhibit Rho GEFs directly, possibly by targeting them for degradation via the ubiquitin-proteosome pathway. Examples of these regulatory mechanisms are described in greater detail in some recent review articles (2, 3, 5). Another mechanism that could possibly be implicated in the regulation of Rho GEF and/or Rho GTPase activity could involve changes in the intracellular concentrations of Rho GEFs themselves or of proteins that modulate Rho GEF/Rho GTPase activity. To our knowledge, such a mechanism has not been described to date. In this report we present the identification of a novel putative human Rho GEF that we designated Src homology 3 (SH3) domain-containing guanine nucleotide exchange factor (SGEF). A particularly interesting characteristic of the SGEF gene is that it produces two transcripts, one of which encodes a protein with the structural features typical of other Rho GEFs. The second transcript encodes a much shorter protein that could function as a modulator of Rho GEF activity. Moreover, the two transcripts are differentially expressed in human tissues, and they are differentially regulated by androgen in LNCaP human prostate cancer cells. This is an interesting discovery because SGEF provides a direct link for cross-talk between the androgen receptor and Rho GTPase signaling pathways in prostate cells. Materials and Methods Cloning of C-terminal SGEF (CSGEF) and SGEF cDNAs The 240-bp cDNA fragment referred to as B45 was isolated using PCR-Select cDNA subtraction as previously described (6) and was subcloned into pCRII (Invitrogen, Carlsbad, CA) to yield plasmid pCRIISSH2-B45/S. The 299-bp DNA fragment containing B45 that was used to screen a cDNA library was excised from pCRII-SSH2-B45/S by digestion with EcoRI and radiolabeled with [␣-32P]deoxy-CTP using the DECAprime II Random Priming DNA Labeling Kit (Ambion, Inc., Austin, TX). A LNCaP cDNA expression library in pBK-CMV (provided by E. Le´vesque and D. Turgeon) was screened using standard procedures as described in the ZAP Express cDNA Synthesis Kit manual (Stratagene, La Jolla, CA). Positive clones were in vivo excised directly into phagemid pBK-CMV (Stratagene), and one of these clones (pBK-CMVB45–24) was subsequently used to generate a 586-bp HindIII/SacI fragment that was radiolabeled and served as probe to isolate full-length CSGEF cDNAs. The AK probe used to isolate SGEF cDNAs was generated by RT-PCR amplification of nucleotides 369 –719 of AK022884. The PCR product was subcloned into the SmaI site of pBluescript II KS⫹/⫺, and the fragment that served as probe for cDNA library screening was excised by digestion with EcoRI and BamHI.

Cell lines and culture conditions Cell lines were obtained from American Type Culture Collection (Manassas, VA). Routine as well as experimental cell culture conditions were described in detail previously (6). In brief, LNCaP cells used for R1881 dose response (see Fig. 5A), time course (see Fig. 5B), and cycloheximide/actinomycin D experiments (see Fig. 5C) were cultured in

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RPMI medium supplemented with hormone-depleted 0.25% fetal calf serum for 6 d before initiation of the indicated experiments (see figure legend). Each experiment was performed at least three times with LNCaP cells between passages 30 and 37.

Northern blot analysis Human multiple tissue Northern blot H (heart . . . pancreas) and H4 (spleen . . . PBL) containing 2 ␮g polyadenylated RNA/lane were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). Nylon membranes containing immobilized total RNA isolated from human breast and prostate cancer cell lines were prepared as previously described (6). The blots were hybridized to the radiolabeled B45 cDNA probe (described previously) using standard techniques (6). The human ␤-actin and glyceraldehyde-3-phosphate dehydrogenase cDNA probes used as loading controls were described previously (7).

Transient expression of epitope-tagged SGEF and CSGEF The full-length SGEF and CSGEF cDNAs were used as templates for PCR amplification of the open reading frames of SGEF and CSGEF. The forward (sense) oligonucleotides contained a BamHI restriction site, a Kozak consensus sequence (5⬘-GAGGCAGC-3⬘), and the first 24 coding nucleotides of SGEF or CSGEF. The reverse primer contained an XbaI restriction site and nucleotides corresponding to the reverse complement of the last 24 nucleotides of the SGEF/CSGEF-coding sequence (excluding the stop codon). The PCR products were cloned into the BamHI and XbaI sites of pcDNA3-HA in-frame with sequences encoding a C-terminal hemagglutinin (HA) epitope (Tyr-Pro-Tyr-Asp-Val-ProAsp-Tyr-Ala-Ser-Leu). The expressed fusion proteins contain amino acids (aa) 1– 871 of SGEF or 1–139 of CSGEF, followed by Ser-Arg-GlyPro (encoded by the XbaI and ApaI sites of the vector) and the HA epitope. The plasmids were sequenced to confirm that the open reading frames correspond exactly to the sequences presented in Figs. 2 and 3. For practical reasons, the expression plasmids were transfected into LNCaP (pcDNA3-CSGEF-HA) or T-47D (pcDNA3-SGEF-HA) cells. Forty-eight hours after transfection using standard lipofection techniques (7, 8), the cells were washed twice with PBS buffer, lysed at 37 C for 5 min, and sonicated in 200 ␮l lysis buffer [6 m urea, 20 mm Tris (pH 6.8), 10% (wt/vol) sodium dodecyl sulfate, 1 mm dithiothreitol, 0.7 ␮g/ml pepstatin, and 1⫻ Complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN)]. Insoluble materials were removed by centrifugation. SGEF-HA and CSGEF-HA were detected by immunoblotting using a commercially available polyclonal antibody and a chemiluminescent detection system.

Ribonuclease protection assays Ribonuclease protection assays were performed as previously described (6, 7) using the Riboprobe System-T7 (Promega Corp., Madison, WI) for cRNA probe synthesis and the RPA III Ribonuclease Protection Assay Kit (Ambion, Inc.) for RNA hybridization and digestion. The B45 and AK cRNA probes were generated from the same plasmids that were used to produce cDNA probes (see above). The B45 cRNA probe contains nucleotides 2294 –2533 of CSGEF, whereas the AK cRNA probe contains nucleotides 369 –719 of AK022884 (SGEF 1737–2087). The AL DNA fragment was produced by RT-PCR amplification of AL117429 nucleotides 476 – 820 and subcloned into the SmaI site of pBluescript II KS. The AL probe corresponds to SGEF nucleotides 2620 –2964 and CSGEF nucleotides 590 –934. Ten micrograms of total RNA were used for all ribonuclease protection assays, and ␤-actin mRNA levels were determined as an internal control. SGEF, CSGEF, and ␤-actin mRNA levels were quantitated by scanning densitometry, and SGEF or CSGEF to ␤-actin ratios were calculated. For each experiment, the CSGEF or SGEF/␤-actin ratio in the appropriate control cells was arbitrarily assigned a value of 1, and the CSGEF or SGEF/␤-actin ratios of treated cells were adjusted accordingly.

Nucleotide sequence accession numbers The GenBank accession numbers for SGEF and CSGEF are AF415175 and AF415176, respectively.

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Results Cloning of CSGEF and SGEF cDNAs

We used PCR-based cDNA subtraction to identify transcripts whose levels were up-regulated by androgens in LNCaP human prostate cancer cells that were exposed to the synthetic androgen R1881 (0.1 nm) for 24 h. As described previously (6, 7), the cDNA fragments isolated in this manner were sequenced to determine their identities, and novel cDNAs were used as probes in ribonuclease protection assays to confirm that the corresponding transcripts were indeed androgen responsive. The sequence of a 240-bp cDNA fragment we isolated (no. B45) corresponded exactly to nucleotides 24,789 –25,028 of human chromosome 3 BAC RP1123D24 of the Roswell Park Cancer Institute Human BAC Library (GenBank accession no. AC018452). Although B45 did share partial sequence identity with a few established sequence tags (AA195770, AV708562, AV724614, and N79370), we could not identify any matching full-length cDNAs, which suggested that B45 could correspond to the product of an as yet unidentified gene. Based on the novelty of B45, we performed confirmatory ribonuclease protection assays of LNCaP cells (these data are presented in Fig. 5A) and then proceeded to identify and characterize the corresponding gene. We used the radiolabeled B45 fragment as a probe to screen a LNCaP cDNA library and obtained five cDNAs that contained the B45 sequence. The B45 fragment was found to reside in the 3⬘-untranslated region of the cDNA. The longest


cDNA (clone 24) was 3050 bp in length and contained a 139-codon open reading frame. However, clone 24 lacked translation termination codons upstream of and in-frame with the putative initiating methionine, which indicated that it might not contain the complete open reading frame. A second screen was therefore performed using a 586-bp SacIHindIII fragment derived from the 5⬘ end of clone 24 to isolate cDNAs containing longer 5⬘-untranslated regions. The longest of the 24 cDNAs we isolated in the second screen (clones 15 and 36) overlapped significantly (⬎1850 bp) with clone 24 and contained 5⬘-untranslated regions with the required translation termination codons. The sequences of the longest overlapping cDNAs were merged to yield a 3105-bp cDNA that we designated CSGEF for reasons explained below. We then compared the CSGEF sequence to that of other GenBank cDNAs and found a related cDNA, clone DKFZp434D146 (GenBank accession no. AL117429) that had been isolated from human testis at the German Cancer Research Center. The first 1740 nucleotides of AL117429 are identical to nucleotides 115-1854 of CSGEF, but AL117429 lacks the B45 sequence and the more extensive 5⬘-untranslated region of CSGEF (Fig. 1). The identification of another related cDNA (GenBank accession no. AK022884) that was isolated from a teratocarcinoma by the New Energy and Industrial Technology Development Organization human cDNA sequencing project provided more interesting and significant clues to the structure of the gene that encodes

FIG. 1. Genomic organization of the human SGEF gene. The SGEF gene (top) is comprised of 15 exons (vertical lines) contained within approximately 136 kb of chromosome 3q25.2. A composite cDNA was drawn by merging exons 1–14 of the SGEF cDNA with exon 15 of the CSGEF cDNA. The position of a stretch of 27 adenosine nucleotides in exon 15 is shown. The portions of exons that code for the P-rich (proline-rich), DH, PH, and SH3 domains are shown as solid bars below the composite cDNA. The SGEF (AF415175) and CSGEF (AF415176) cDNAs are represented by rectangles. The longest open reading frames of SGEF and CSGEF are shown in gray, whereas noncoding portions are shown in white. The black rectangle at the 5⬘ end of CSGEF represents sequences derived from the 3⬘ end of intron 10. The vertical dashed lines demarcate the sequences that are identical in SGEF and CSGEF (exons 11–15). The nucleotide positions of the open reading frames and the common portions of SGEF and CSGEF are indicated. GenBank cDNAs AK022884 and AL117429 are represented by thick black lines. The solid portions of these lines are shared with SGEF and CSGEF, whereas the dashed portion of AK022884 is shared with SGEF only. The positions of the B45 cDNA fragment and the AK and AL probes used in Northern and/or ribonuclease protection assays are shown at the bottom of the diagram. The dashed horizontal line at the 3⬘ extremity of the SGEF cDNA indicates that the clone is probably incomplete at its 3⬘ end.


CSGEF. Nucleotides 70 –1827 of CSGEF are identical to the last 1758 nucleotides of AK022884, but the first 69 nucleotides of CSGEF diverge entirely from the first 731 nucleotides of AK022884. Although the deduced protein sequence of AK022884 contained recognizable functional domains (see below), the longest open reading frame of AK022884 was undefined. This suggested that AK022884 and CSGEF could correspond to alternative transcripts of a single gene. We therefore used a fragment of the AK022884 cDNA (nucleotides 369 –719) as a probe to screen the LNCaP cDNA library. This screen yielded a number of cDNAs, the longest of which was 3857 bp in length. This cDNA, which we hereafter refer to as SGEF, included AK022884 as well as an additional 1368 bp of upstream sequences. Genomic structure of SGEF

The exon-intron structure of the SGEF gene was deduced using the Genome Browser at University of California (Santa Cruz, CA; http://genome.ucsc.edu). SGEF is located on chromosome 3q25.2, and it is composed of 15 exons spanning more than 130 kb (Fig. 1). The sequences of the SGEF and CSGEF cDNAs were compared with the genomic sequence to determine exon/intron boundaries (Table 1), and these were found to agree with consensus splice donor and acceptor site sequences (9). The SGEF transcript is derived from exons 1–15, whereas the CSGEF transcript contains exons 11–15. In addition, exon 11 of CSGEF is preceded by sequences derived from the 3⬘ end of intron 10 that is contiguous to exon 11. The fact that the 5⬘-untranslated regions of SGEF and CSGEF are derived from distinct portions of the SGEF gene suggests that the expression of SGEF and CSGEF might be controlled by different promoters. The SGEF cDNA that we isolated contains a shorter portion of exon 15-derived sequences than the CSGEF cDNA (Figs. 1–3). However, in Northern blot assays (Fig. 6) the exon 15-specific B45 probe detected two transcripts, one whose


length is consistent with that of the CSGEF cDNA and a longer transcript whose length would be consistent with that of an SGEF transcript containing all of exon 15 (ⱖ5135 bp). It is probably relevant to note that exon 15 contains a stretch of 27 adenosine nucleotides (Fig. 1) that coincides with the 3⬘ end of the SGEF cDNA. We therefore hypothesize that the oligo(deoxythymidine) primer used in the construction of the LNCaP cDNA library hybridized to the polyadenylase sequence, thereby producing a 3⬘-truncated SGEF cDNA. For the purposes of the gene expression studies presented in this report, we have assumed that SGEF and CSGEF have identical 3⬘-untranslated regions. There are a few minor differences between the sequence of the open reading frame of SGEF and the genomic sequence of exons 2 and 12. Codon 29 of the SGEF cDNA encodes a leucine (CTT), whereas the genomic sequence encodes valine (GTT). Codon 60 of the SGEF cDNA encodes a serine (TCC), whereas the genomic sequence encodes leucine (CTC). Finally, genomic exon 12 contains an additional four bases (CAGT) that alter the open reading frame after codon 742 and result in a translation termination after codon 751. These differences are probably due to errors in the genomic sequence, especially since the 4-bp insertion in the genomic sequence of exon 12 is not present in the AK022884 cDNA. SGEF encodes a novel GEF

The longest open reading frame of the SGEF cDNA is derived from exons 2–15, and it is preceded by two inframe translation termination codons (Fig. 2). The sequence of the predicted 871-aa polypeptide was analyzed using the ProfileScan Server (http://hits.isb-sib.ch/cgibin/PFSCAN) of the ISREC (Swiss Institute for Experimental Cancer Research) to identify functional motifs. The SGEF protein contains two functional domains that are found in almost all GEFs for Rho GTPases. These are a DH

TABLE 1. Exon-intron structure of the SGEF gene Exons

Introns

No.

Size (bp)

Position in cDNA

Exon boundaries (5⬘. . .3⬘)

No.

Intron boundaries (5⬘. . .3⬘)

1 2 3 4 5 6 7 8 9 10 11a 11b 12 13 14 15a 15b

115 1134 40 146 57 161 153 130 75 90 155 226 210 68 105 1218 2496

1–115 116 –1249 1250 –1289 1290 –1435 1436 –1492 1493–1653 1654 –1806 1807–1936 1937–2011 2012–2101 2102–2256 1–226 2257–2466 2467–2534 2535–2639 2640 –3857 610 –3105

CCGCGGT. . .CCAGAAG GCAAGAC. . .GATAAAG AAAAAAA. . .GGGGAAG AAAATGC. . .CTCTGCG GTGAAAA. . .ACAAGAG GCTATCT. . .GCAAAAA GTTCTTT. . .AATTGTT AGCTACC. . .GATGGAT ACTATCT. . .TAGCAAG TTGGTTC. . .AATTAAG CCTTTTC. . .AGAAGAG AGCTTTT. . .AGAAGAG TGAAGAA. . .AGACGCA GAGCGAG. . .CGAACCT CACTGAC. . .AGCGATG GCTGGTA. . .ATTAATTpA GCTGGTA. . .TACTTGTpA

1 2 3 4 5 6 7 8 9 10 11

gtaaatt. gtaaaag. gtaagca. gtgagtg. gtatgtt. gtaagtg. gtaagca. gtaagac. gtaactg. gtattct. gtaagtc.

.tctttag .ctcttag .cttgcag .ctcctag .ttaccag .ttttcag .ccctcag .attacag .ttcccag .tttgtag .tttacag

422 1334 5124 19669 3326 34752 3451 3225 23150 7897 14359

12 13 14 — —

gtaagta. . .gttttag gtaagtt. . .gcctcag gtgagtg. . .ttcacag

12536 1536 506

. . . . . . . . . . .

Size (bp)

The exon-intron structure of the SGEF gene was deduced by comparing the cDNA sequences of SGEF and CSGEF to the genomic sequence of chromosome 3 retrieved from the UCSC Genome Browser. Exons are numbered from 1–15 where exons 11b and 15b correspond to the first and last exons of CSGEF. The first 71 bp of exon 11b correspond to the last 71 nucleotides of intron 10. The size of each exon and its position in the SGEF (CSGEF for exons 11b and 15b) cDNA are indicated. The stretches of adenosine residues at the end of exons 15a and 15b are represented by pA. Introns are numbered from 1–14.

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FIG. 2. Nucleotide and deduced aa sequences of human SGEF. DNA, Nucleotides are numbered on the left, and the first nucleotide of exons 1–15 is shown in white type on a black background. The two translation termination codons upstream of and in-frame with the putative initiating methionine are double-underlined, and a putative polyadenylation signal is underlined. Nucleotides 2100 –3857 of the SGEF cDNA are identical to nucleotides 70 –1827 of the CSGEF cDNA. The SGEF-specific AK probe used for ribonuclease protection assays contained nucleotides 1737–2087 of SGEF. The AL probe used for ribonuclease protection assays contained nucleotides 2620 –2964. Protein, The aa (in single-letter code) are numbered on the right, and the aa of putative functional domains are shown in bold. The two nuclear localization signals (aa 20 –37 and 425– 442) are underlined with a dashed line, and the proline-rich region (aa 106 –173) is double-underlined. The DH domain (aa 446 – 628) is underlined, the PH domain (aa 655–782) is highlighted in gray, and the SH3 domain (aa 789 – 850) is boxed. The termination codon is indicated by an asterisk.

domain (aa 446 – 628) adjacent to a PH domain (aa 655– 782). As is typical of a smaller number of Rho GEFs, SGEF also possesses a single SH3 domain that is located at the C-terminal end of the protein (aa 789 – 850). Three other functional domains were also identified. Like PDZ-RhoGEF (10), SGEF contains an N-terminal proline-rich region (aa 106 –173). Two bipartite nuclear localization signals situated at the N terminus (aa 20 –37, RRSIPQPHQLLGRSKPRP) and immediately N-terminal to the DH domain (aa 425– 442, KRKGLSQTVSQEERKRQE) were identified in SGEF. The longest open reading frame of the CSGEF cDNA is

derived from exons 12–15, and it is preceded by a single upstream translation termination codon that is encoded by intron 10 sequences (Fig. 3). The different exon content of CSGEF does not alter the reading frame of the coding sequence, except that the predicted first methionine of CSGEF corresponds to methionine 733 of SGEF. In contrast to SGEF, the longest open reading frame of CSGEF is only 139 codons long, and the predicted polypeptide lacks many of the features found in SGEF, hence the name CSGEF (C-terminal SGEF). CSGEF contains the last 50 aa of the PH domain and the complete SH3 domain. The calculated molecular masses of SGEF and CSGEF are



FIG. 3. Nucleotide and deduced aa sequences of human CSGEF. DNA, Nucleotides are numbered on the left, and the first nucleotide of exons 11–15 is shown in white type on a black background. The first 71 nucleotides of the cDNA are derived from the 3⬘ end of intron 10. The translation termination codon upstream of and in-frame with the putative initiating methionine is double-underlined. The polyadenylation signal is underlined, and the portion of exon 15 (nucleotides 1612–1664) that is absent in GenBank entry AK022655 is underlined with a dashed line. The B45 cDNA fragment isolated by cDNA subtraction (nucleotides 2294 –2533) is in bold and underlined. The AL probe used for ribonuclease protection assays contained nucleotides 590 –934. Protein, The aa (in singleletter code) are numbered on the right, and putative functional domains are shown in bold. The PH domain (aa 1–50) is highlighted in gray, and the SH3 domain (aa 57–118) is boxed. The termination codon is indicated by an asterisk. The longest open reading frame of CSGEF corresponds to codons 733– 871 of SGEF.

97 and 15 kDa, respectively. To estimate the molecular masses of SGEF and CSGEF, the proteins were transiently expressed in human cells as fusion proteins with C-terminal HA epitopes (Fig. 4). HA-tagged CSGEF migrated at an ap-

parent molecular mass of approximately 17 kDa, which is consistent with the calculated weight of CSGEF and the presence of 15 heterologous residues (SRGPYPYDVPDYASL) at its C terminus. Although multiple HA-tagged SGEF

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FIG. 4. Transient expression of SGEF and CSGEF. A, Schematic diagram of SGEF and CSGEF. The positions of the prolinerich domain, nuclear localization signals, and DH, PH, and SH3 domains are shown. B, Immunoblotting was performed on whole cell extracts of LNCaP (lanes 1 and 2) and T-47D (lanes 3 and 4) cells that were transiently transfected with control pcDNA3HA expression plasmid (lanes 1 and 3) or pcDNA3HA plasmids expressing CSGEF (lane 2) or SGEF (lane 4) fused to a C-terminal HA epitope. The expressed proteins were detected using a polyclonal antibody against HA. Molecular mass markers (in kilodaltons) are shown on the left. The multiple polypeptides detected in cells expressing SGEF-HA were attributed to translation initiation at internal start codons.

polypeptides were detected, the size of the largest polypeptide (100 kDa) was in agreement with the molecular mass of SGEF-HA. The shorter polypeptides, ranging from 33– 64 kDa in size, were attributed to internal initiation of translation at methionines located approximately between aa 281 and 571. SGEF and CSGEF mRNA levels are differentially regulated by androgen in LNCaP cells

After the isolation and sequencing of the B45 cDNA fragment, ribonuclease protection assays were performed to confirm that B45 corresponded to an androgen-regulated transcript. RNA samples were prepared from LNCaP cells that had been exposed to increasing concentrations of R1881 (0.1–10 nm) for 24 h and hybridized to the B45 cRNA probe. Relatively low levels of the B45 transcript were detected in control LNCaP cells, but the levels of the mRNA increased 4.4 –5.4 times in cells exposed to R1881, thereby confirming that B45 corresponds to an androgen-regulated transcript (Fig. 5A). Additional ribonuclease protection assays were subsequently performed after the isolation of CSGEF and SGEF cDNAs to determine whether both SGEF and CSGEF mRNAs were up-regulated by androgens. To this end we designed two cRNA probes, the AL probe (derived from AL117429) that detects portions of exons 14 and 15, which are present in both CSGEF and SGEF transcripts, and the AK probe (derived from AK022884) that detects exons 7–10, which are only present in the SGEF mRNA (Fig. 1). The AK probe detected a transcript for which the levels were not up-regulated in response to R1881 (Fig. 5A). On the other hand, the AL probe detected a transcript for which the levels increased 3.0 – 4.3 times in LNCaP cells exposed to R1881. Based on these results we concluded that CSGEF expression (i.e. mRNA levels) is regulated by androgens, whereas SGEF expression is not. These results were also confirmed using a probe specific to the 5⬘ end of the CSGEF mRNA (data not shown). To more precisely define the time course of CSGEF mRNA up-regulation, LNCaP cells were exposed to 0.1 nm R1881 and harvested 1–72 h later. CSGEF mRNA levels were de-

termined by ribonuclease protection assay using the AL cRNA probe (although this probe also detects SGEF, the low levels of SGEF mRNA were not expected to interfere with the assay). A modest (⬃2-fold compared with baseline), but reproducible, increase in CSGEF mRNA levels was detected as early as 3 h after the addition of R1881 to the culture medium (Fig. 5B). CSGEF mRNA levels increased steadily over time and peaked at 24 – 48 h after the addition of R1881. The kinetics of R1881-induced CSGEF mRNA up-regulation were similar to those of NKX3.1, a gene whose expression is directly regulated by androgen receptor at the transcriptional level (11). We therefore examined the effect of RNA and protein synthesis inhibitors on R1881-induced upregulation of CSGEF mRNA levels to determine whether CSGEF up-regulation occurs at the transcriptional level and whether protein synthesis is required. LNCaP cells were treated with R1881 (0.1 nm) in combination with the protein synthesis inhibitor cycloheximide (10 ␮g/ml), the RNA synthesis inhibitor actinomycin D (1 ␮g/ml), or the androgen receptor antagonist Casodex (3 ⫻ 10⫺6 m). CSGEF mRNA levels were determined by ribonuclease protection assay using the AL cRNA probe. R1881 alone caused a 4.2-fold increase in CSGEF mRNA levels (Fig. 5C). The effect of R1881 was completely blocked by actinomycin D, but not by cycloheximide, indicating that the effect of R1881 is transcriptional and does not require protein synthesis. Predictably, Casodex also blocked the effect of R1881, thereby confirming that R1881-induced up-regulation of CSGEF is mediated by the androgen receptor. SGEF is expressed ubiquitously, whereas CSGEF expression is restricted to prostate and liver

Northern blot analyses were performed to determine the tissue expression profile and size of SGEF transcripts. We used the B45 cDNA fragment as probe because we anticipated that it would detect both SGEF and CSGEF transcripts, as explained previously. We first performed a qualitative analysis of SGEF mRNA levels in total RNA samples prepared from control and androgen-treated LNCaP cells. A major 3.5-kb transcript was detected in control cells, and the



FIG. 5. Androgen regulation of CSGEF expression. A, Effects of increasing doses of R1881 on CSGEF and SGEF mRNA levels in LNCaP cells. LNCaP cells were exposed to 0.1–10 nM R1881 for 24 h, and ribonuclease protection assays were performed using the B45, AK (SGEF-specific), and AL cRNA probes. B, Time course of R1881-induced CSGEF mRNA up-regulation in LNCaP cells. Control and androgen-treated (0.1 nM R1881) cultures of LNCaP cells were harvested at the indicated intervals and processed for ribonuclease protection assays. CSGEF mRNA levels were detected using the AL riboprobe. C, Effect of the RNA synthesis inhibitor actinomycin D (ActD; 1 ␮g/ml), the protein synthesis inhibitor cycloheximide (CHX; 10 ␮g/ml), and the androgen receptor antagonist Casodex (CAS; 3 ⫻ 10⫺6 M) on R1881-induced up-regulation of CSGEF mRNA levels in LNCaP cells. CSGEF mRNA levels were detected using the AL riboprobe. mRNA (CSGEF and/or SGEF) to ␤-actin ratios are shown below each respective panel.

abundance of this transcript increased markedly after a 24-h exposure to 0.1 nm R1881 (Fig. 6A). The size of the most abundant mRNA is consistent with that of the CSGEF cDNA (3.1 kb). An approximately 4.4-kb transcript of lesser abundance was also detected in R1881-treated LNCaP cells. We believe that this transcript could result from the use of an alternate polyadenylation site. In agreement with the results of the ribonuclease protection experiments, we did not detect longer (⬎5 kb) transcripts that could correspond to SGEF in LNCaP total RNA. To determine the expression profile of SGEF and CSGEF mRNAs in human tissues, we performed Northern blot analyses of multiple tissue blots (CLONTECH Laboratories, Inc.) containing samples of polyadenylated RNA from 16 different human tissues (Fig. 6B). The B45 probe detected an approximately 6-kb transcript that we believe to be SGEF in tissues of the digestive system (liver, pancreas, small intestine, and colon), cardiovascular system (lung and heart), as well as brain, kidney, and tissues of the reproductive system (testis, placenta, and possibly uterus), including prostate. SGEF mRNA was not detected in skeletal muscle or tissues

of the immune or hemopoietic systems (spleen, thymus, and peripheral blood leukocytes). In contrast to the widely expressed SGEF, CSGEF mRNA was only detected in prostate, where it constitutes the major SGEF-derived transcript, and in liver. Interestingly, liver contained an abundance of SGEFderived (and/or SGEF-related) transcripts that ranged from approximately 1.4 – 6.0 kb in size. These transcripts were so abundant that CSGEF mRNA was clearly visible only after shorter exposures than the one presented in Fig. 6B. The fact that CSGEF mRNA is expressed in androgensensitive human prostate and LNCaP cells prompted us to examine CSGEF expression in a series of human prostate and breast cancer cell lines that display different biological responses to androgens. Northern blot analysis using the B45 cDNA probe revealed that CSGEF is expressed at very high levels in androgen-responsive LNCaP cells compared with the androgen-insensitive DU 145 and PC-3 cells in which CSGEF mRNA was not detected (Fig. 6C). Appreciable amounts of CSGEF mRNA were not detected in any of the human breast cancer cell lines by Northern blot analysis. However, a faint band that could correspond to SGEF mRNA

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FIG. 6. Expression profile of CSGEF and SGEF mRNA in human tissues and cell lines. Northern blot analyses (A–C) were performed using the radiolabeled B45 cDNA fragment as probe. The positions of major transcripts and molecular mass markers are indicated. A, Northern blot analysis of LNCaP cultures (10 ␮g total RNA) grown in the presence or absence of 0.1 nM R1881 for 24 h. B, CLONTECH Laboratories, Inc., human multiple tissue Northern blots (2 ␮g polyadenylated RNA/lane). C, Northern blot analysis of human breast and prostate cancer cells (10 ␮g total RNA/lane). Ribonuclease protection assays of the breast and prostate cancer cell lines shown in C were performed using the AK cRNA probe specific to SGEF (D) or the AL cRNA probe common to SGEF and CSGEF (E). Glyceraldehyde-3-phosphate dehydrogenase or ␤-actin mRNA levels served as loading controls.

was observed in several cell lines, particularly in breast BT474 and prostate DU 145 cells. To verify the presence of SGEF in these cells we performed ribonuclease protection assays

using the AK cRNA probe specific for SGEF. These experiments confirmed that SGEF is expressed, albeit at low levels, in all of the cell lines examined (Fig. 6D). To determine


whether any of these cell lines (besides LNCaP) expresses CSGEF, we performed ribonuclease protection assays using the AL cRNA probe that recognizes sequences common to SGEF and CSGEF mRNAs (Fig. 6E). We expected that cell lines that express CSGEF would display a stronger hybridization signal with the AL probe than with the AK probe. Except for LNCaP cells, which produced the expected pattern, none of the other cell lines, with the possible exception of BT-20 cells, was found to express CSGEF mRNA. Discussion

Androgens play an essential role in the development, growth, and maintenance of the prostate as well as in prostate cancer cell proliferation. The effects of androgens are mediated by the androgen receptor, a nuclear transcription factor that becomes activated upon ligand binding and subsequently regulates the expression of androgen target genes (12). The products of these target genes are the ultimate effectors of androgen action, and it is therefore important to identify the full complement of androgen-responsive genes in both normal and cancerous prostate cells to better understand the mechanism of androgen action in the prostate. In this report we describe the isolation and characterization of a novel androgen-regulated transcript, CSGEF, and the discovery of the SGEF gene from which it is derived. The human SGEF gene is localized on chromosome 3q25.2, a portion of chromosome 3 (3q25-q26.2) that has been identified by comparative genomic hybridization as an amplification unit in prostate tumors (13). Based on our analysis of SGEF/CSGEF mRNA levels in a limited number of cell lines, we could not confirm that SGEF mRNA is overexpressed in prostate cancer. In fact, SGEF mRNA levels were present at low levels in both breast and prostate cancer cell lines. In contrast, CSGEF mRNA levels were much higher in LNCaP cells compared with the other cell lines, in which CSGEF mRNA was not detectable by Northern blot analysis. We presume that the high level expression of CSGEF mRNA in LNCaP cells is not due to gene amplification, because a recent study that employed comparative genomic hybridization did not detect amplification of chromosome 3 in LNCaP cells (14). The structure of the SGEF gene proposed herein is consistent with the sequences of the most informative cDNAs that have been isolated to date, namely SGEF, CSGEF, AK022884/FLJ12822, and AL117429. Two other cDNAs that have not been discussed in detail, AK022655 and BC016628, are also consistent with the gene structure, because they are included in SGEF, except that AK022655 lacks a portion of exon 15 (see Fig. 3). This particularity of AK022655 cannot be explained at present. However, the existence of other alternatively spliced transcripts of SGEF cannot be excluded. SGEF was so named because it contains a DH domain in tandem with a PH domain, which are characteristic of GEFs for the Rho subfamily of guanine nucleotide-binding proteins. The prefix S was added because SGEF contains a Cterminal SH3 binding domain. SGEF is a full-length cDNA that encompasses a previously cloned partial cDNA designated FLJ12822 (GenBank accession no. AK022884). FLJ12822 contains the last 408 codons of the SGEF open reading frame,


which correspond to part of the DH domain and the complete PH and SH3 domains. We submitted the sequence of the SGEF cDNA to GenBank in November 2001, and it has since been included in a recently published comprehensive review of Rho GEFs (3). Based on this review, SGEF has been assigned to a subgroup of Rho GEFs that includes ephexin (15), TIM/Arhgef5 (16), KIAA0915/Arhgef15 (17), and NBR/ Arhgef16 (GenBank accession no. D89016/BC002681/ NM_014448), a Rho GEF thought to be a potential candidate gene for human neuroblastoma. An interesting feature of SGEF is the presence of an N-terminal proline-rich domain that could be involved in protein-protein interactions, especially of the intramolecular type, that are believed to inhibit Rho GEF activity (5). In fact, it is possible that SGEF could be maintained in an inactive form as a result of interactions between its N-terminal proline-rich region and its C-terminal SH3 domain (18). SGEF also contains two putative nuclear localization signals, which suggests that it could be translocated to the nucleus in response to certain stimuli. This possibility is supported by the fact that Vav1, another Rho GEF, is translocated to the nucleus of T cells in response to PRL (19). The biological properties of SGEF remain to be tested. For instance, we have yet to analyze the ability of SGEF to catalyze guanine nucleotide exchange on Rho GTPases. However, it is highly likely that SGEF possesses such catalytic activity, because it is structurally related to ephexin, which is known to catalyze guanine nucleotide exchange on Rho, Rac, and Cdc42 (15). To our knowledge, the catalytic activities of NBR/Arhgef16, KIAA0915/Arhgef15, and TIM/ Arhgef5 have not been demonstrated. It will also be of interest to determine whether SGEF, like most other Rho GEFs, possesses transforming activity. Another interesting function that will require testing is the possible role of SGEF in steroid hormone, particularly androgen, receptor action. For example, the Brx protein, another DH and PH domain-containing protein, was found to interact with the estrogen receptor and potentiate its transcriptional activity (20). Moreover, the recent discovery that the androgen receptor coactivator FHL2 is translocated to the nucleus after activation of the Rho signaling pathway may represent another possible mechanism by which androgen regulation of Rho GEF activity could be implicated in transcriptional regulation by androgen receptor (21). SGEF mRNA was detected in several human tissues of many different physiological systems (nervous, reproductive, digestive, etc.), which suggests that the SGEF protein could be involved in the activation of Rho GTPases in response to various stimuli. On the other hand, CSGEF mRNA expression is restricted to the prostate and liver, which suggests that CSGEF may play a much more specialized role in the control of Rho GTPase activity. Because CSGEF lacks the functional domains that are responsible for the biological activity of Rho GEFs, we hypothesize that CSGEF could act as a modulator of the activity of other Rho GEFs or even of SGEF itself. In one of several possible models, CSGEF protein produced in response to androgen would be targeted to the appropriate subcellular location by its partial PH domain, thereby allowing its SH3 domain to interact with compatible

1752 Endocrinology, May 2003, 144(5):1742–1752

functional domains of other proteins, including the prolinerich domain of SGEF. The consequences of such interactions remain speculative, but in theory an interaction between CSGEF and SGEF could result in the activation of SGEF. CSGEF could also act as a dominant negative regulator of the activity of SGEF or of other Rho GEFs. Dominant negative proteins are naturally occurring, physiologically important regulators that have been identified in several different signaling pathways. A recently identified dominant negative protein that is conceptually analogous to CSGEF is ⌬NSTAT92E (22). Like CSGEF, ⌬NSTAT92E lacks the Nterminal portion of the corresponding full-length protein (STAT92E), and it arises from a different promoter of the stat92e locus. The up-regulation of CSGEF mRNA levels by androgen in LNCaP cells is relatively rapid and occurs at the transcriptional level, as it is abrogated by an RNA synthesis inhibitor, but not by an inhibitor of protein synthesis. One would therefore expect the androgen receptor to interact, directly or via associated transcription factors, with the regulatory elements of the SGEF gene that control CSGEF expression. Intron 10 contains a putative TATA box (TATAAA) approximately 40 bp upstream of the 5⬘ end of the CSGEF cDNA as well as several half-sites of sequences TGTTCT, AGTGCT, GGAACA, GGAACA, AGTGCT, AGAACA, and GGTACA that partially match the consensus sequence of the androgen response element, 5⬘-GGA/TACANNNTGTTCT-3⬘ (23). A more detailed analysis of the SGEF gene will be required to define the regulatory elements that are involved in androgen-induced up-regulation of CSGEF. In summary, we have identified a new DH-PH domaincontaining protein termed SGEF that is likely to regulate Rho GTPase activity in a wide variety of human cell types. An interesting characteristic of SGEF is that it encodes an Nterminal truncated form of SGEF, CSGEF, whose expression is induced by androgens in LNCaP human prostate cancer cells. CSGEF may constitute a link between the androgen receptor and Rho GTPase signaling pathways in prostate cells. Future experiments will determine the specific role of SGEF/CSGEF in androgen-induced physiological changes in the prostate and prostate cancer cell proliferation. Acknowledgments We thank D. Turgeon for kindly providing the LNCaP cell library, and the CHUL Research Center art department for the figures. Received September 19, 2002. Accepted January 9, 2003. Address all correspondence and requests for reprints to: Dr. Claude Labrie, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 Laurier Boulevard, Que´ bec, Canada G1V 4G2. E-mail: [email protected]. This work was supported by a fellowship from Le Fonds de Recherche en Sante´ du Que´ bec (to C.L.), and by Endorecherche.


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