Oncogene (2001) 20, 1816 ± 1824 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc ORIGINAL PAPERS The small-GTPase RalA activates transcription of the urokinase plasminogen activator receptor (uPAR) gene via an AP1-dependent mechanism Emel Okan1, Victoria Drewett1, Peter E Shaw1 and Peter Jones*,1 1School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK The urokinase plasminogen activator receptor (uPAR) and beta-family integrins (Xue et al., 1997; Kindzelskii focuses extracellular protease activity to the cell surface, et al., 1996; Wei et al., 1996). It also has a role in signal modulates cell adhesion and activates intracellular signal transduction, possibly through interactions with trans- transduction pathways. In a range of cancers uPAR membrane molecules such as gp130 or integrins that expression often has a negative correlation with function as conduits of uPAR-mediated signal trans- prognosis. Here we show that uPAR transcription is duction (Koshelnick et al., 1997, 1999). That uPAR stimulated by V12 H-Ras, the eector loop mutant V12 plays an important role in determining malignancy of H-Ras G37 and constitutively-active RalA 72L. RalA- most human tumours is based on a large number of dependent transcription required the presence of the experimental studies of both human cancers and model ATF2-like AP1-site at 770 bp and the c-Jun binding systems and appears to be a negative prognostic motif at 7184 bp in the uPAR promoter. Consistent marker in a number of cancers (Andreasen et al., with this, both Gal4-c-Jun- and Gal4-ATF2-fusion 1997, 2000). proteins were activated by RalA signalling through As the level of uPAR expression critically aects the phosphorylation of their activation domains at Ser63 invasive capacity of a cell, its expression is subject to and Ser73 of c-Jun or Thr69 and Thr71 of ATF2. A complex regulation at the levels of translation and transdominant inhibitory mutant of c-Jun N-terminal transcription (Shetty et al., 1997; Aguirre-Ghiso et al., kinase (JNK) failed to inhibit uPAR transcription 1999a). Many of the growth factor- and cytokine- demonstrating that JNK activation is not a prerequisite activated signalling pathways that control uPAR for RalA-dependent uPAR transcription. A dominant transcription converge on the small GTP-binding negative inhibitor of c-Src eectively inhibited RalA- protein Ras (Aguirre-Ghiso et al., 1999a). The ras dependent uPAR transcription identifying it as a down- genes (H-ras,N-ras and K-ras) frequently acquire stream eector in the RalA signalling pathway. These activating mutations in human cancers (Bos, 1998; data provide evidence for the existence of a novel Hunter, 1997; Yamamoto et al., 1999) and at least H- signalling pathway that links RalA to the activation of Ras and K-Ras have been demonstrated to activate uPAR transcription via a c-Src intermediate and uPAR gene transcription (Aguirre-Ghiso et al., 1999a; activation of AP1. Oncogene (2001) 20, 1816 ± 1824. Allgayer et al., 1999; Muller et al., 2000). This suggests a mechanism whereby the activity of oncogenic Ras Keywords: RalA; c-Src; c-Jun; ATF2; uPAR; Ras can be linked to the increased invasiveness of malignant cells through the uPA/uPAR proteolytic system. Introduction Recent studies have demonstrated that Ras stimu- lates a number of dierent downstream eectors whose The urokinase plasminogen activator receptor (uPAR) activities are essential for its transforming properties is a glycosylphosphatidylinositol (GPI) membrane- (Shields et al., 2000; Yamamoto et al., 1999). The use anchored receptor that binds the serine protease of multiple eector pathways by Ras has been urokinase plasminogen activator (uPA). The receptor demonstrated through the use of constitutively-acti- anchors uPA to the leading edge of migrating cells vated mutants of Ras (V12 H-Ras) that also bear where the complex stimulates migration by concentrat- mutations in the eector loop, amino acids 32 ± 40 ing the proteolytic activity of uPA to the surface of the (White et al., 1995). These mutants interact with at invading cell. Cell adhesion is also aected by uPAR least three dierent eector molecules; c-Raf, Phos- through its interactions with the extracellular matrix phoinositide 3-kinase (PI3K) and RalGDS. The serine/ protein vitronectin (Wei et al., 1994; Waltz et al., 1997) threonine kinases c-Raf, activates the ERK (Extra- cellular Signal-Regulated Kinase) mitogen-activated protein kinase (MAPK) activity that regulates a diverse range of biological events especially gene expression. *Correspondence: P Jones Received 21 November 2000; revised 22 December 2000; accepted Phosphoinositide 3-kinase (PI3K) has an emerging role 15 January 2001 in promoting cell survival (Rodriguez-Viciana et al., RalA activation of uPAR transcription E Okan et al 1817 1997; Kaumann-Zeh et al., 1997). The third and most mutants containing 5'-deletions of increasing size. We recently established eector, is the Ral guanine found that proximal region of the uPAR promoter nucleotide exchange factor, RalGDS. RalGDS is a between 7139 bp and the major transcription start site guanine nucleotide exchange factor (GEF) for the (+1) was the minimum sequence required to retain the small GTPase Ral. Currently, ®ve Ral GEFs have been stimulation of transcription by the transforming hu- identi®ed; RalGDS, Rgl, Rlf, Rgr and RalGEF2 that man H-Ras (V12 H-Ras) (data not shown). Potential are direct targets for Ras (Bos, 1998; de Bruyn et al., binding sites for transcription factors in this region are 2000). They provide a mechanism for Ral activation by an AP1/GRE site, a binding site for NF-kB and a extracellular signals via a variety of receptors, includ- number of binding sites for Sp1 (Figure 1a). To ing G-protein-coupled receptors and receptor tyrosine determine if Ras utilized a preferred eector to activate kinases (Hofer et al., 1998; Wolthuis et al., 1998a,b). uPAR transcription from this region of the promoter, RalA is one of two highly similar (85% identity) we used luciferase reporter assays to compare the GTPases that constitute a distinct family of Ras-related eects of constitutively activated V12 H-Ras and the proteins (Bos, 1997). The demonstration that domi- eector-loop mutants RasV12/C40, RasV12/G37 and nant-negative mutants of Ras inhibit insulin- and RasV12/S35 on transcription in the cell line NIH3T3 epidermal growth factor-induced activation of Ral (Figure 1b). Transcription from the minimal uPAR has established that Ral de®nes a downstream promoter of 139 bp was stimulated approximately signalling pathway from Ras (Wolthuis et al., 1998a). ®vefold by V12 H-Ras and over twofold by Ras 35S However, Ral can also be activated by Ras-indepen- and Ras 40C, that utilize Raf-1 and PI3K as their dent mechanisms (Hofer et al., 1998; Wolthuis et al., downstream eectors respectively (White et al., 1995). 1998b). The most eective stimulation of transcription by a The role played by Ral-family proteins in the cell is unclear but they appear to be linked predominantly with cell proliferation and dierentiation (Urano et al., 1996; Wolthuis et al., 1997). These properties are proposed to result from of the ability of Ral to couple signals from Ras to the induction of gene transcription or to the inhibition of transcription factors like AFX (Murai et al., 1997; Okazaki et al., 1997; Kops et al., 1999; Medema et al., 2000). The signalling pathway that links Ral activity to transcription is currently unknown. Our previous work to identify the signalling pathway utilized by oncogenic Ras in activating uPAR gene transcription suggested that RalGDS could have some role in this process (Muller et al., 2000). In this study, we demonstrate that a constitutively activated RalA mutant, RalA 72L, stimulates transcription from the uPAR promoter and that this requires the presence of at least one of the AP1 sites located 7184 or 770 bp upstream of the major start point of transcription. Activation of transcription is inhibited by a dominant- negative mutant of c-Src, indicating that c-Src is a downstream eector of RalA. We also show that RalA activates the Jun-family members c-Jun and ATF2 in a manner dependent on the phosphorylation sites Ser63 and Ser73 of c-Jun and Thr69 and Thr71 of ATF2. The uPAR is the ®rst example of a gene that can be up-regulated by RalA through an AP1 dependent mechanism. Figure 1 (a) Diagram showing the location of potential transcription factor binding sites in the 1.55 kb upstream sequence of the uPAR promoter. The region between the SmaI site at 7139 bp and the major start point of transcription is Results expanded. (b) Eects of ras eector-loop mutants on the activation of transcription from the proximal region of the uPAR Differential stimulation of uPAR transcription by Ras promoter. NIH3T3 cells were co-transfected with the luciferase effector loop mutants through DNA response elements reporter construct and either V12 H-Ras or V12 H-Ras contain- ing a mutation in the eector loop. The downstream eectors of located within 139 bp of the transcription start site Ras 37G, Ras 35S and Ras 40C are RalGDS, c-Raf and PI3K To locate the Ras-response elements within the respectively. The RLA represents the relative luciferase activity after normalization for dierences in transfection eciencies. The promoter, we used luciferase reporter assays to data points represent the mean of three independent experiments measure expression from a series of uPAR promoter with standard deviations indicated by error bars Oncogene RalA activation of uPAR transcription E Okan et al 1818 Ras-eector loop mutant was obtained with Ras G37 potential binding sites for three classes of transcription that utilizes RalGDS as an eector. Stimulation of factor: NF-kB, SP1 and AP1 at an AP1-like site transcription by this mutant was similar to that designated AP1/GRE (Figure 1a).