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A new Rac target POSH is an SH3‐containing scaffold protein involved in the JNK and NF‐κB signalling pathways

A new Rac target POSH is an SH3‐containing scaffold protein involved in the JNK and NF‐κB... The EMBO Journal Vol.17 No.5 pp.1395–1404, 1998 A new Rac target POSH is an SH3-containing scaffold protein involved in the JNK and NF-κB signalling pathways Reif et al., 1996; Rodriguez-Viciana et al., 1997). Once Nicolas Tapon, Koh-ichi Nagata, 1,2 activated, Rac induces polymerization of monomeric actin Nathalie Lamarche and Alan Hall at the cell periphery to produce a dense meshwork of actin MRC Laboratory for Molecular Cell Biology, CRC Oncogene and filaments forming extending lamellipodia and membrane Signal Transduction Group and Department of Biochemistry, ruffles (Ridley et al., 1992). There is growing evidence University College London, Gower Street, London WC1E 6BT, UK that this pathway plays a major role in directed cell Corresponding author migration and axonal guidance (Van Aelst and D’Souza- e-mail: [email protected] Schorey, 1997). In addition to inducing actin poly- merization, Rac stimulates the formation of associated The Rho, Rac and Cdc42 GTPases coordinately regu- integrin-based adhesion complexes (Nobes and Hall, late the organization of the actin cytoskeleton and the 1995). These structures contain many of the same con- JNK MAP kinase pathway. Mutational analysis of Rac stituents as classical focal adhesions, though morphologic- has previously shown that these two activities are ally they are distinct; they do not seem to be required for mediated by distinct cellular targets, though their actin polymerization but instead may play a role in cell PAK identity is not known. Two Rac targets, p65 and movement or signalling (Machesky and Hall, 1997). MLK, are ser/thr kinases that have been reported to Members of the Rho GTPase family also regulate gene be capable of activating the JNK pathway. We present transcription. All three GTPases have been reported to evidence that neither is the Rac target mediating JNK stimulate the JNK/SAPK and p38/HOG1 MAP kinase activation in Cos-1 cells. We have used yeast two- cascades, the transcription factor NF-κB and the transcrip- hybrid selection and identified a new target of Rac, tion factor SRF (Bagrodia et al., 1995; Coso et al., 1995; POSH. This protein consists of four SH3 domains and Hill et al., 1995; Minden et al., 1995; Olson et al., 1995; ectopic expression leads to the activation of the JNK Sulciner et al., 1996; Teramoto et al., 1996b; Perona et al., pathway and to nuclear translocation of NF-κB. When 1997). To date, most of these observations have been overexpressed in fibroblasts, POSH is a strong inducer obtained by overexpressing GTPase constructs in trans- of apoptosis. We propose that POSH acts as a scaffold fected cells, but the genetic analysis of budding yeast and protein and contributes to Rac-induced signal trans- more recently Drosophila has confirmed that Rho GTPases duction pathways leading to diverse gene transcrip- coordinately regulate the organization of the actin cyto- tional changes. skeleton and the activity of MAP kinase pathways probably Keywords: apoptosis/JNK/NF-κB/POSH/Rac in all eukaryotic cells (Glise and Noselli, 1997; Leberer et al., 1997). In addition, Rho, Rac and Cdc42 can trigger G progression when introduced into quiescent fibroblasts Introduction and they are each required for serum-induced cell cycle progression and Ras-induced cell transformation (Olson Rho, Rac and Cdc42, three members of the Rho family et al., 1995; Qiu et al., 1995a,b, 1997). Moreover, many of small GTPases, act as molecular switches cycling of the Dbl family of Rho GEFs are potent oncogenes and between an active GTP-bound and an inactive GDP-bound will transform NIH-3T3 cells to a malignant phenotype state. Activation, in response to extracellular agonists (Cerione and Zheng, 1996). How they do this is not acting on membrane receptors, is mediated by the Dbl known, though stimulation of G progression and cell family of guanine nucleotide exchange factors (GEFs), transformation correlate well with the ability of the while down-regulation involves a poorly characterized GTPases to induce cytoskeletal changes and it is possible family of GTPase-activating proteins (GAPs) and that signals are induced in response to actin polymerization RhoGDIs (Lamarche and Hall, 1994; Cerione and Zheng, or integrin complex assembly (Joneson et al., 1996; 1996). In their active state, Rho, Rac and Cdc42 interact Lamarche et al., 1996; Westwick et al., 1997). with a variety of target (effector) proteins to elicit cellular To understand the biochemical mechanisms underlying responses (Van Aelst and D’Souza-Schorey, 1997). the various activities of the Rho GTPases, there has been Activation of Rho, Rac and Cdc42 in quiescent Swiss- intense activity to identify target proteins (Van Aelst and 3T3 fibroblasts induces rearrangement of filamentous actin D’Souza-Schorey, 1997). Mutational analysis of Rac has leading to the formation of actin stress fibres, lamellipodia provided evidence for bifurcating pathways controlling and filopodia, respectively (Ridley and Hall, 1992; Ridley cytoskeletal changes and MAP kinase activation; amino et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995). acid substitutions at codon 37, for example, block the Growth factors such as platelet-derived growth factor induction of lamellipodia without affecting JNK activation, (PDGF) and insulin, or constitutively activated (oncogenic) Ras protein stimulate Rac and in both cases this is while changes at codon 40 have the opposite effect mediated by PI 3-kinases (Kotani et al., 1994; Wennstrom (Joneson et al., 1996; Lamarche et al., 1996; Westwick et al., 1994; Hawkins et al., 1995; Nobes et al., 1995; et al., 1997). It appears, therefore, that Rac interacts © Oxford University Press 1395 N.Tapon et al. with at least two distinct target proteins to trigger these pathways. It is not clear whether activation of NF-κBor other transcription factors such as SRF are a consequence of, or are independent of, Rac’s effect on actin or the JNK pathway. To date, around 10 targets for Rac have been identified using yeast two-hybrid and affinity chromatography tech- niques (Van Aelst and D’Souza-Schorey, 1997). The first Rac target to be identified was a serine/threonine kinase PAK p65 . It is closely related to a yeast kinase, Ste20p, which is known to regulate MAP kinase pathways in this PAK organism and it has, therefore, been suggested that p65 probably mediates JNK activation by Rac in mammalian cells (Manser et al., 1994). In agreement with this, the PAK interaction of p65 is blocked by changes at codon 40, but not codon 37, of Rac (Joneson et al., 1996; Lamarche et al., 1996; Westwick et al., 1997). Furthermore, there have been a number of reports that a constitutively PAK activated version of p65 leads to JNK activation and one report that a dominant-negative version inhibits Rac- mediated JNK activation (Bagrodia et al., 1995; Knaus et al., 1995; Brown et al., 1996; Frost et al., 1996). PAK However, others have failed to find a role for p65 in JNK activation and its physiological role remains unclear (Teramoto et al., 1996a,b; Westwick et al., 1997). In PAK addition to p65 another target of Rac, MLK, is also a potential regulator of JNK pathways, since it belongs to the family of MAP kinase kinase kinases (Burbelo PAK Fig. 1. p65 and MLK2/3 do not mediate Rac-induced JNK et al., 1995). PAK activation in Cos-1 cells. (A) Activated p65 does not cause JNK1 We report here that in Cos-1 cells, the interaction of activation when overexpressed in Cos-1 cells. pCMV5FLAG-JNK1 PAK PAK Rac with p65 or MLK is not the trigger for JNK was co-transfected with myc-tagged activated (L107F) p65 , wild- PAK activation. Using a yeast two-hybrid screen, we have type p65 , V12 Rac or empty pRK5Myc vector (–), as described in PAK Materials and methods. Kinase activities of p65 and JNK1 were identified a new target for Rac, POSH, which contains measured on immune complexes (top panel) using Myelin Basic four SH3 domains. When transfected into Cos-1 cells, Protein (5 μg per reaction) or c-jun (2 μg per reaction). Expression POSH stimulates JNK activation. Expression of POSH PAK levels were visualized with anti-myc (for p65 ) and anti-JNK1 in fibroblasts leads to nuclear translocation of NF-κB, antibodies (bottom panel). (B) MLKs do not interact with F37A or Y40C effector mutants of Rac. Yeast strains containing Rac mutants in independently of either actin reorganization or JNK activ- the integrated bait vector pYTH9 were transformed with the pACTII ation. We conclude that POSH acts as a scaffold protein and prey vector containing MST1/MLK2 C-Terminus (aa 338–953), MLK3 participates in Rac-mediated signal transduction pathways C-Terminus (aa 348–847), RhoGAP as a positive control, or empty leading to gene transcriptional changes. vector as a negative control. Colonies of equal size were replated in the presence of 25 mM 3-aminotriazole and allowed to grow for 3 days at 30°C. Results PAK p65 and MLK do not mediate Rac-induced JNK (Lamarche et al., 1996); MST1/MLK2 and MLK3 (Figure activation PAK 1B) are no longer able to interact with Y40CRac in yeast To examine whether p65 could be a mediator of Rac- two-hybrid or dot-blot assays. However, we had shown induced JNK activation, Cos-1 cells were co-transfected PAK previously that Rac containing an F37A substitution was with a constitutively activated version of p65 (L107F, still able to activate JNK in Cos-1 cells (Lamarche et al., PAK1/PAKα isoform) and a JNK1 expression plasmid. 1996). As shown in Figure 1B, F37ARac does not interact As seen in Figure 1A, co-transfection with a constitutively with MST1/MLK2 or MLK3. We conclude, therefore, that activated Rac (V12) leads to a 21-fold stimulation of JNK PAK the interaction of Rac with MLKs cannot be the trigger activity, whereas activated p65 induced no significant PAK for JNK activation in this assay. activation. As a control, L107F-p65 was immunopre- cipitated from the transfected cells and shown to be active PAK using MBP as a substrate. It appears, therefore, that p65 Isolation and characterization of a novel does not activate the JNK kinase cascade in these cells. Rac-interacting protein It has been reported that another Rac target, MLK, is a In order to isolate potential downstream targets of Rac potent activator of the JNK pathway and we have con- that might account for its ability to activate JNK, we have firmed this in Cos-1 cells (Nagata et al., 1998). It is screened a yeast two-hybrid mouse cDNA library using possible, therefore, that the interaction of Rac with endo- L61 Rac as bait. Approximately 10 clones were screened genous MLK proteins is the trigger for JNK activation. as described in Materials and methods and of the 30 As shown previously, Rac containing a Y40C substitution fastest-growing clones picked from the selection plates, PAK can no longer activate JNK and as expected p65 one (clone 4) tested positive upon rescreening and was 1396 POSH, a new target for the Rac GTPase Fig. 2. Interaction of clone 4 with Rac. Clone 4 in the pGAD-10 prey vector was introduced into yeast strains containing integrated L61 Rac, L63 Rho, L61 Cdc42, F37A L61 Rac and Y40C L61 Rac. Colonies of equal size were replated in the presence of 25 mM 3-aminotriazole and allowed to grow for 3 days. negative in the absence of the Rac bait. Figure 2 shows that clone 4 does not interact with Rho or Cdc42 in yeast, and in addition it still interacts with F37ARac, but not with Y40CRac. These observations are consistent with clone 4 encoding a mediator of Rac-induced JNK activation. Sequence analysis of clone 4 (2.4 kb insert) revealed an open reading frame (ORF) for a protein of 67 kDa, but since no stop codon was present 5 of the first methionine, it was possible that clone 4 represented an incomplete ORF. Using a primer derived from a region close to the 5 end of clone 4 we looked for potential additional upstream sequences using a commercial 5 Fig. 3. Sequence and expression pattern of POSH. (A) Protein RACE PCR library. One major RACE product was sequence of full-length POSH. SH3 domains are underlined. The obtained (1.4 kbp in length) and sequence analysis of minimal Rac-binding site is indicated with a dotted line. DDBJ/ EMBL/GenBank accession number of POSH cDNA: AF030131. this confirmed that the original clone 4 represented an (B) Tissue expression of POSH. A mouse multiple tissue Northern blot incomplete ORF. The full ORF encodes a protein of 892 was probed as described in the methods. Lane 1, testis; lane 2, kidney; amino acids (predicted mol. wt 93 kDa) (see Figure 3A); lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6, spleen; the first methionine is surrounded by a consensus Kozak lane 7, brain; lane 8, heart. sequence and is preceded by an in-frame stop codon. Analysis of the protein sequence reveals a potential zinc finger structure (aa 18–82), but more interestingly analysis (Figure 3A) revealed that POSH does not contain four SH3 domains (underlined in Figure 3A). Accordingly, a CRIB site. To identify the region of the protein that we have called the protein POSH (Plenty Of SH3s). Using contains the Rac interaction site, a series of truncations a commercial mouse tissue Northern blot, a single mRNA were expressed as GST fusion proteins and tested for species for POSH (at ~5 kb) can be seen in all tissues binding to L61 Rac in dot-blot assays. As shown in Figure (Figure 3B), though skeletal muscle and spleen appear to 4C, Rac interacts with POSH in a region encompassing have relatively low levels. We conclude that POSH is 70 residues (aa 292–362, dotted underline in Figure 3A). ubiquitously expressed. Extensive searching of EST and non-redundant databases, which includes all other known GTPase targets showed Interaction of POSH with Rac no significant matches to sequences within this region. The A bona fide target protein would be expected to interact Rac interaction site in POSH is, therefore, so far unique. preferentially with the GTP-bound state of Rac. To test whether the interaction of POSH is GTP-dependent, POSH triggers programmed cell death we have used a modified dot-blot assay using To test whether POSH can induce changes to the actin [α- P]GTP-loaded Rac (see Materials and methods) and cytoskeleton, full-length POSH cDNA was first subcloned [α- P]GDP-loaded Rac (obtained by pre-incubating into the mammalian expression vector, pRK5, so as to [α- P]GTP-loaded Rac with a small amount of Rho introduce a myc epitope tag at its N-terminus. Plasmid GAP). As seen in Figure 4A, POSH interacts with the DNA was microinjected into the nuclei of serum-starved, GTP form of Rac but not the GDP form. Using a dot-blot confluent Swiss-3T3 fibroblasts and any effects on the assay, we also confirmed that POSH interacts with the actin cytoskeleton observed 2–8 h later using fluorescently F37A mutant of Rac, but not with the Y40C mutant labelled phalloidin. Under conditions where Rac induced (Figure 4B). strong actin polymerization and lamellipodia, POSH Many, though not all, previously identified Rac targets induced no detectable assembly of actin filaments (data contain a distinctive binding site, the CRIB site. Sequence not shown). It was noticed, however that POSH did induce 1397 N.Tapon et al. sion of POSH induces apoptotic cell death in primary and immortalized fibroblasts (Jacobson et al., 1997). POSH stimulates the JNK pathway To examine the effects of POSH on JNK activation, Cos-1 cells were co-transfected with POSH and JNK. As shown by Western blot analysis the toxic effects of full-length POSH, particularly when co-transfected with JNK (Figure 6A), were also apparent in this cell line [compare expres- sion levels of full-length POSH (lane 2) with truncated POSH (lane 3)]. The toxic effects could be largely over- come by including the caspase inhibitor BocD-fmk (Deshmukh et al., 1996; Weil et al., 1997) in the transfec- tion assays (Figure 6A, compare lane 1,  inhibitor with lane 2, – inhibitor). Under these conditions, it can be seen in Figure 6B that full-length POSH induces a 5.6-fold stimulation of JNK activity, compared with 11.0-fold by L61Rac. The truncated version of POSH was unable to stimulate JNK (Figure 6B), nor did it interfere with Rac- induced JNK activation (data not shown). In order to test whether POSH-induced cell death is dependent on JNK activation, we co-injected POSH with a 5-fold molar excess of dominant-negative SEK1 (SAPK/ ERK kinase 1) (S220A  T224L version or K129R version) into NIH-3T3 fibroblasts. Although both domin- ant-negative SEK1 constructs were expressed at relatively high levels, neither was able to prevent POSH-induced cell death (data not shown). POSH stimulates nuclear translocation of NF-κB To examine whether POSH might contribute to Rac- induced NF-κB activation, expression constructs were microinjected into quiescent Swiss-3T3 cells. Transloca- tion of NF-κB to the nucleus was visualized by immuno- Fig. 4. Interaction of POSH with Rac in vitro.(A) POSH binds to Rac fluorescence 5 h later. Full-length POSH, truncated POSH in a GTP-dependent manner. GST-POSH (10 μg, aa 292–892), containing the two C-terminal SH3 domains (aa 362– PAK GST-p65 (8 μg) and GST (10 μg) were spotted on strips of 32 32 892), activated L61 Rac and L61 F37A Rac were potent nitrocellulose which were probed with [α- P]GTP- or [α- P]GDP- inducers of NF-κB translocation (Figure 7A). However, bound wild-type Rac. (B) POSH binds to F37A but not Y40C Rac PAK in vitro. GST-POSH (10 μg, aa 292–892), GST-p65 (8 μg), GST- L61Y40CRac—which does not interact with POSH—was RhoGAP (10 μg, aa 198–439), and GST (10 μg) were spotted on severely impaired in its ability to cause translocation. nitrocellulose and probed with [γ- P]GTP-bound Rac mutants. Interestingly, translocation of NF-κB in injected cells was (C) Identification of a 70 amino acid fragment of POSH that is accompanied by NF-κB translocation in neighbouring non- sufficient for binding to L61 Rac. Ten μg of GST-fusion proteins of POSH truncations were spotted on nitrocellulose and tested for injected cells (Figure 7B, see arrowhead). We conclude that interaction with L61 Rac as in (B). From top to bottom: original two- Rac and POSH can induce nuclear translocation of NF-κB hybrid fragment (aa 292–892); Truncation 1 (aa 352–892); Truncation and that this may involve the induction of autocrine/ 2 (aa 292–398); Truncation 3 (aa 292–362). paracrine factors. Discussion significant cell death at the later time points in these conditions. Further analysis in NIH-3T3 cells revealed Rho, Rac and Cdc42 control the assembly and organization that 14 h after injection, full-length POSH induced cell of the actin cytoskeleton in eukaryotic cells. In response death in ~90% of injected cells, even in the presence of to extracellular signals, the active conformation of the serum (Figure 5, top) and that the few remaining cells three GTPases leads to the assembly of actin–myosin had shrunken and condensed (pyknotic) nuclei typical of contractile filaments, lamellipodia and filopodia, respect- apoptosis (Figure 5, bottom). In contrast, a POSH Trunca- ively, and it is likely that these proteins play important tion (aa 352–892) lacking the N-terminal two SH3 domains regulatory roles in cell movement (Murphy and Montell, and the Rac-binding domain, or activated L61Rac, did not 1996; Van Aelst and D’Souza-Schorey, 1997; Zipkin et al., induce significant cell death under these conditions (Figure 1997). In addition to their effects on actin, members 5). Time-lapse video microscopy revealed that cell contrac- of the Rho GTPase family regulate changes in gene tion and intense surface blebbing could be seen in the transcription. Rho, Rac and Cdc42 have each been reported majority of cells 5 h after injection (data not shown). to activate the JNK and p38 MAP kinase pathways, to Similar effects were observed in primary rat embryo activate the transcription factors NF-κB and SRF and to fibroblasts (data not shown). We conclude that overexpres- stimulate G progression in quiescent Swiss cells (Bagrodia 1398 POSH, a new target for the Rac GTPase Fig. 5. Full-length POSH induces apoptosis in NIH-3T3 cells. NIH-3T3 cells were replated on glass coverslips and left for 24 h in DMEM containing 10% DCS and the nuclei of 50 cells were injected with an expression vector containing full-length POSH, POSH Truncation 1 (aa 352– 892) or L61 Rac at a concentration of 0.04 mg/ml. The cells were fixed and stained as described after a 12 h incubation in the presence of serum. Bottom line shows typical nuclear morphology of injected cells (arrows) using Hoechst dye staining. In the middle panel, non-injected cell nuclei are in a different plane of focus from the rounded up POSH injected cells. Results were averaged over three independent experiments; error bars represent standard deviation. Scale bar  20 μm. et al., 1995; Coso et al., 1995; Hill et al., 1995; Minden that can no longer interact with MLK (MST1/MLK2 or et al., 1995; Olson et al., 1995; Sulciner et al., 1996; MLK3 isoforms) is still able to activate JNK. We conclude, Teramoto et al., 1996b; Perona et al., 1997). To what therefore, that neither target is likely to mediate JNK extent these pathways are interdependent is not clear, activation by Rac in Cos-1 cells. Although it remains a although G progression can be triggered by Rac mutants possibility that one of the other two PAK isoforms (PAK2/ that can no longer activate the JNK pathway (Joneson PAKγ or PAK3/PAKβ) or another member of the MLK et al., 1996; Lamarche et al., 1996; Westwick et al., 1997). family might trigger JNK activation in our Cos-1 cells, To characterize the biochemical pathways mediating this seems unlikely given their high degree of sequence the various cellular responses induced by GTPases, many homology. Two new MAP kinase kinase kinases, MEKK4 groups have used yeast two-hybrid and affinity chromato- and MEKK1, have recently been reported to interact graphy techniques to identify target proteins and approxim- directly with Rac in a GTP-dependent manner and to ately ten candidate targets for Rac have been isolated so activate the JNK cascade (Fanger et al., 1997). It is far (Van Aelst and D’Souza-Schorey, 1997). Little progress possible, therefore, that either of these could be the cellular has yet been made in identifying which is responsible for target of Rac in Cos-1 cells responsible for activation triggering actin polymerization after interacting with Rac of JNK. GTP, but several targets have been implicated in JNK We have searched for new Rac targets that might activation. A number of groups have reported that the contribute to JNK activation by screening a yeast two- PAK interaction of Rac with the ser/thr kinase, p65 , might hybrid library with constitutively active L61Rac. We have lead to activation of the JNK and p38 pathways and in identified a ubiquitously expressed protein, POSH, that PAK some cell types overexpression of p65 does lead to interacts with Rac (but not Rho or Cdc42) in a GTP- JNK activation (Bagrodia et al., 1995; Knaus et al., 1995; dependent manner. This 93 kDa protein contains four SH3 Brown et al., 1996; Frost et al., 1996). The MLK kinases domains (and a putative zinc finger) and it interacts with are also known to be potent activators of the JNK pathway F37ARac, but not Y40CRac. It is possible, therefore, that and these proteins have binding sites for Rac (Rana et al., this protein might play a role in Rac-mediated JNK 1996; Teramoto et al., 1996b; Nagata et al., 1998). We activation. Consistent with this, expression of POSH in PAK show here that constitutively activated p65 (PAK1/ Cos-1 cells induces activation of JNK. Activation of PAKα isoform) does not stimulate the JNK pathway in JNK by inflammatory cytokines or by stress is often Cos-1 cells. Furthermore, a Rac mutant (L61F37ARac) accompanied by nuclear translocation of the transcription 1399 N.Tapon et al. POSH, which can activate NF-κB but not JNK, do not induce cell death. Co-injection of POSH with dominant- negative SEK failed to block POSH-induced cell death, though this construct has been reported to block JNK activation induced by MEKK1 and to inhibit cell death induced by cis-platinum, UV irradiation or heat (Sanchez et al., 1994; Zanke et al., 1996). It is possible, therefore, that POSH induces cell death independently of JNK activation. Furthermore, even though POSH is a Rac target, overexpression of Rac does not lead to apoptosis. One possible explanation for this apparent discrepancy is that Rac, through its multiple downstream targets, might also activate survival signals. If this is the case, then the survival signal cannot be NF-κB, since this is also activated by POSH. SH3 domain-containing proteins have previously been shown to play important roles in mediating Rho GTPase signals. In phagocytic cells, p67 , a component of the phox NADPH oxidase enzyme complex is a target of Rac (Diekmann et al., 1994). p67 has no catalytic activity, phox but consists of two SH3 domains—the second of which shows close similarity to the first and fourth SH3 domains of POSH (51.9% with POSH 1, 42.6% with POSH 4). The membrane-bound oxidase is responsible for the generation of superoxide, which forms a part of the Fig. 6. POSH activates JNK1 in Cos-1 cells. (A) Cos-1 cells were co- transfected with pCMV5FLAG-JNK1 (5 μg) and pRK5myc-full-length pathogen-killing mechanism of professional phagocytes, POSH (lanes 1 and 2) or pRK5myc-POSH Truncation 1 (lane 3) (all and both Rac and p67 are essential for its activity phox 3 μg) in the presence () or absence (–) of BocD-fmk at 20 μM. (Abo et al., 1991; Segal and Abo, 1993; Diekmann et al., Cells were harvested in 300 μlof3 protein sample buffer and 30 μl 1994). Rac has also been reported to induce the formation of each lysate was analysed by Western blotting with anti-myc and anti-JNK1 antibodies. (B) pCMV5FLAG-JNK1 was co-transfected of reactive oxygen species (ROS) in non-phagocytic cells with empty pRK5myc vector (lane 1), pRK5myc-L61 Rac (lane 2), and in HeLa cells, this appears to account for Rac- pRK5myc-POSH Truncation 1 (aa 352–892) (lane 3), and pRK5myc- mediated NF-κB activation (Sulciner et al., 1996). Interes- full-length POSH (lane 4) all in the presence of 20 μM BocD-fmk tingly, during the POSH and Rac injection experiments, (Enzymes Systems Products). Aliquots of each transfection were we see translocation of NF-κB in neighbouring, non- electrophoresed on an SDS–PAGE gel and expression levels of transfected constructs were visualized on Western blots using anti-myc injected cells and although we cannot rule out mechanisms (for POSH, Rac), and anti-FLAG (for JNK1) antibodies followed by involving cell–cell contact, it raises the possibility that a I-labelled protein A (top panel). JNK1 activity was assayed on paracrine factor (perhaps ROS) is produced. We are immune complexes using GST-c-jun as a substrate and quantified on a currently looking at whether Rac can induce ROS in Bio-Rad Molecular Imager (middle panel). Levels of JNK1 in the immunoprecipitates were visualized using an anti JNK1 antibody Swiss-3T3 cells and if so, whether this is mediated by (bottom panel). p67 or perhaps POSH. phox In Saccharomyces cerevisiae, the SH3-containing pro- factor NF-κB (Verma et al., 1995). Furthermore, there tein Bem1p is a key component of Cdc42-mediated signals have been reports that Rac can activate NF-κB when during cell division and in the pheromone mating response transfected into cells (Sulciner et al., 1996; Perona et al., (Leberer et al., 1997). In the mating response, Bem1p 1997). Using an immunofluorescence assay, we have acts as a scaffold protein and interacts with multiple shown that expression of POSH in Swiss-3T3 cells leads proteins including Cdc24p (an exchange factor for to nuclear translocation of NF-κB. Interestingly, the two Cdc42p), with Ste20p (a target of Cdc42p and a relative PAK C-terminal SH3 domains are sufficient to induce NF-κB of mammalian p65 ), with Ste5p (a scaffold protein translocation, but not JNK activation. We conclude that required for MAP kinase activation), with actin and with POSH may play a role in both JNK and NF-κB activation Far1p (an inhibitor of the cell cycle) (Leberer et al., 1997). mediated by Rac. In agreement with this, Y40CRac— It is thought that a major role of Cdc42p in the pheromone which is inactive in both assays—no longer interacts with pathway is to localize this multi-molecular signalling POSH, while F37ARac—which interacts with POSH—is complex to the mating projection. Interestingly, in fission active in both assays. yeast the homologue of Bem1p, scd2, is also required for Finally, activation of JNK and NF-κB pathways is often the mating response. In this case, scd2 interacts directly seen in cells stimulated to undergo apoptosis though the with scd1 (the exchange factor for cdc42) and with cdc42 relative contributions of the two pathways to cell death (Chang et al., 1994). No Bem1p/scd2 homologues have are highly cell type-dependent (Liu et al., 1996; Baichwal been identified in higher eukaryotes and it is tempting to and Baeuerle, 1997; Chuang et al., 1997; Herdegen et al., speculate that POSH fulfils an analogous role and acts as 1997). We have found that overexpression of POSH in a scaffold protein in Rac-mediated signalling pathways in Swiss or NIH-3T3 cell lines, in primary rat embryo mammalian cells. If this is the case, POSH might be fibroblasts or in Cos cells is highly toxic and leads to cell predicted to interact with other components of the Rac death via apoptosis. The two C-terminal SH3 domains of signal transduction pathway (e.g. an exchange factor or 1400 POSH, a new target for the Rac GTPase Fig. 7. POSH induces nuclear translocation of NF-κB in Swiss-3T3 cells. (A) Myc-tagged pRK5 plasmids (0.04 mg/ml) encoding POSH (full-length), POSH Truncation1 (aa 352–892), L61Y40CRac, L61F37ARac, L61Rac or empty vector (co-injected with FITC-dextran) were microinjected into serum-starved, confluent Swiss-3T3 cells and the cells fixed 5 h later. POSH/Rac expression was visualized using anti-myc antibody, while NF-κB localization was visualized using an anti-NF-κB antibody (Santa Cruz). The percentage of myc-positive cells in which clear nuclear fluorescence of NF-κB was seen were scored as positive. Results were averaged over four independent experiments; between 29 and 85 myc-expressing cells were analysed per experiment and the error bar represents standard deviation. (B) Right panel: a typical phenotype of a myc-positive, nuclear NF-κB-positive cell after injection with truncated POSH. The arrowhead points to a neighbouring, non-injected cell that is nuclear NF-κB positive. Left panel: a typical phenotype of a myc-positive, nuclear NF-κB-negative cell after injection with L61Y40CRac. Scale bar  20 μm. other targets). We have been unable to detect an interaction NF-κB and apoptosis or acts by titrating out inhibitors of PAK between p65 and POSH after Cos cell transfections these processes. (data not shown) and we are currently using yeast two- In conclusion, we have shown that the two Rac targets, PAK hybrid screens to identify binding partners of POSH. p65 and MLK are unlikely to account for Rac-induced JNK activation in Cos-1 cells. We have identified a new The biochemical mechanisms through which over- SH3-containing Rac target, POSH, which activates JNK expression of POSH leads to activation of JNK in when transfected into Cos-1 cells and induces nuclear fibroblasts is unclear. Expression of other adaptor-like translocation of NF-κB. We propose that POSH acts as molecules with SH3 domains can also have profound scaffold protein required for the assembly of signalling cellular effects; the SH2/SH3-containing protein v-Crk, complexes that control gene transcriptional events down- for example, can induce malignant transformation (Mayer stream of Rac. et al., 1989). Furthermore, overexpression of Bem1p in yeast has been reported to activate the mating pheromone Materials and methods pathway independently of mating pheromone when expressed in a STE11-4 mutant background (Lyons et al., Yeast two-hybrid screen 1996). We propose that POSH either triggers the formation A Ras-transformed NIH-3T3 cDNA library fused to the GAL-4 activation of a signalling complex leading to activation of JNK, domain in the pGAD-10 vector (kind gift of Dr C.C.Kumar, USA) was 1401 N.Tapon et al. screened using L61 Rac in an integrated pYTH6 vector as a bait, as Purification and expression of recombinant proteins previously described (Aspenstrom et al., 1996; Lamarche et al., 1996). Rac GST fusion proteins were purified on glutathione–Sepharose beads Approximately 10 yeast colonies were screened for their ability to grow (Sigma) and cleaved using human thrombin as previously described PAK on selective medium containing 25 mM 3-aminotriazole. The 30 fastest- (Self and Hall, 1995). POSH, p65 and RhoGAP were produced using growing clones were replated and plasmids were rescued using the a modification of this protocol. A culture of bacteria bearing pGEX-4T3 Wizard clean-up kit (Promega) and retransformed into the original yeast containing the relevant insert was grown in 500 ml of L-Broth containing strain. One of these, clone 4 was strongly positive in the plate lift assay 100 μg/ml ampicillin overnight at 37°C with vigorous agitation. The for expression of the LacZ reporter gene after the second round of cells were diluted with 500 ml of fresh L-Broth containing ampicillin transformation. The 2.4 kb insert of clone 4 was sequenced and found and left to grow for 2 h. Fusion protein was induced for2hby addition to be a novel cDNA potentially encoding a 67 kDa protein. of IPTG to 1 mM. The cells were harvested and lysed as previously described (Self and Hall, 1995). Following purification on glutathione– Sepharose beads, the proteins were eluted with three washes of 500 μl Cloning of full-length clone 4 resuspension buffer (15 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM The full-length clone 4 sequence was isolated using the Clonetech MgCl , 0.1 mM DTT) containing 1.5 mM glutathione. Eluted proteins Marathon RACE PCR kit (Mouse whole embryo) with Advantage 2 were concentrated on centricon-10 columns and stored in liquid nitrogen. Klentaq. A primer was generated corresponding to the minus strand of Protein concentration was assayed using the Bradford method and the bp 156–178 of the original yeast clone (5-GTCACCGGGCTGCTA- quality was checked on SDS–PAGE gels using Coomassie blue. GGGGGTGGGG-3). This primer was used with the Clonetech adaptor primer in the standard touchdown PCR protocol described in the GTP dependence Clonetech manual. Analysis of the RACE reaction by agarose gel To determine the relative binding of RacGTP and RacGDP to target electrophoresis revealed a major RACE product at 1.4 kb. The product proteins a modified dot-blot protocol was used. GST-fusion proteins was ligated into pYTH6 after NcoI–NotI digestion and sequenced. Two were spotted onto nitrocellulose strips. 200 ng of wild-type Rac were independent clones of the 1.4 kb fragment were analysed to eliminate loaded with [α- P]GTP and the exchange reaction stopped by addition any errors due to PCR amplification. of MgCl on ice as previously described (Diekmann et al., 1994). The sample was split in two and 10 ng of RhoGAP were added to one of Mammalian cell transfections the tubes. This tube was incubated for 10 min at 30°C (to produce Cos-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium predominantly [α- P]GDP), while the other tube was left on ice (DMEM) (Gibco) containing 10% FCS (Sigma). These were transfected (predominantly [α- P]GTP). The GTPase aliquots were used in a dot- using the DEAE–dextran method as previously described (Olson et al., blot assay as described above. Bound radioactivity was quantified by 1995). Plasmid amounts per 10 cm dish were as follows: pRK5myc- autoradiography and densitometry. POSH, 5 μg; pRK5myc-POSH Truncation T1, 2 μg; pRK5myc- PAK L107Fp65 (PAK1/PAKα isoform), 5 μg; pRK5myc-V12/L61Rac1, DNA constructs 1.5 μg; pCMV-FLAG-JNK1, 5 μg. Empty vector was added where Standard DNA protocols were used (Sambrook et al., 1989). pGEX- appropriate to ensure all transfections contained equal amounts of DNA. 4T3-clone 4 was generated by digestion of pGAD10-clone 4 with BamHI Cells were serum-starved 24 h after transfection and harvested 16 h and HindIII followed by ligation of clone 4 cDNA into pGEX-4T3. later. After transfection, the cells were kept in serum-containing or Truncations of clone 4 in pGEX were made by PCR with Pfu polymerase serum-free media supplemented with the caspase inhibitor BocD-fmk (Stratagene). Clone 4 and the truncations were transferred from pGEX- (Boc-Aspartic acid-fluoromethylketone; Enzyme Systems Products) at 4T3, using BamHI and HindIII, into the eukaryotic expression vector, 20 μM (from a 10 mM stock in DMSO). PRK5-myc (Lamarche et al., 1996). Full-length POSH in PRK5-myc was generated by amplifying the RACE product with Pfu polymerase Kinase assays using the following oligonucleotides: 5-GCCGGATCCGATGAGT- Transfected Cos-1 cells were harvested in lysis buffer (20 mM Tris CTGCCTTGTTGGAC-3, and 5-GACTTGTTGGCCATGG TGAGGG- pH 8.0, 40 mM Na pyrophosphate, 50 mM NaF, 5 mM MgCl , 100 μM AGGTGAAGG-3. This fragment was then combined with the remainder Na vanadate, 10 mM EGTA, 1% Triton X-100, 3 mM PMSF, 20 μg/ml of the POSH coding sequence and inserted into PRK5-myc using BamHI PAK leupeptin/aprotinin) using a cell scraper (Falcon). JNK1 or p65 were and NcoI. The amplified fragment was checked by sequencing. A immunoprecipitated using anti-FLAG antibody (Scientific Imaging) for pRK5FLAG vector was constructed by digesting pRK5myc with ClaI PAK JNK1 or anti-myc antibody for p65 (Olson et al., 1995). Kinase and HindIII and introducing the FLAG epitope using the following activity of immunocomplexes was measured using GST-c-jun for JNK1 oligonucleotides: 5-CGATAGCCACCATGGACTACAAGGACGATG- PAK or Myelin Basic Protein for p65 as substrates, as described (Olson ACGATAAGGGATCCCGGGTCTAGAATTCGGGA-3 and 5-AGCT- et al., 1995; Lamarche et al., 1996). The relative levels of substrate TCCCGAATTCTAGACCCTGGATCCCTTATCGTCATCGTCCTTGT- phosphorylation were determined on a Bio-Rad PhosphorImager, follow- AGTCCATGGTGGCTAT-3. Full-length POSH was inserted into ing SDS–PAGE and transfer to nitrocellulose. Amounts of immunopre- pRK5FLAG from pRK5myc using BamHI and HindIII. cipitated kinases were checked using anti-JNK1 antibody (Santa Cruz) The following constructs were kind gifts from colleagues: human PAK or anti-myc antibody for p65 . MST-1/MLK2 from Dr M.Terada, National Cancer Center Research Institute, Tokyo; PCMV-FLAG-JNK1 and pGEX-c-Jun from Dr M.Karin, PAK Co-precipitation of p65 and POSH UC San Diego and Dr J.Ham, Eisai London Research Laboratories; 65PAK Cos-1 cells were transfected with pRK5myc-L107Fp or pRK5mycSEK-1 (K129R) from Dr M.Olson, Chester Beatty Labora- pRK5FLAG-full-length POSH (3 μg each). Cell were harvested as above tories, London; HA-tagged SEK-1 AL (S220A, T224L) from Dr L.Zon, and the lysates were combined. Half of the lysate was used for Dana Farber Cancer Institute, Boston; and PAK (L107F) from Dr immunoprecipitation using anti-myc antibody and the other half using J.Chant, Harvard University. anti-FLAG antibody (Scientific Imaging). Immunoprecipitations and washing were carried out in 40 mM Tris, pH 7.5, 50 mM NaCl, 0.2% Microinjection NP-40, 50 mM NaF, 20 μg/ml leupeptin/aprotinin as for JNK assays Swiss-3T3 cells were maintained in DMEM containing 10% FCS and (Olson et al., 1995). antibiotics. NIH-3T3 cells were maintained in DMEM containing 10% donor calf serum (DCS) and antibiotics. Swiss-3T3 cells were plated on Dot-blot assay acid-washed, round 13 mm coverslips at 610 cells/coverslip. At 7– Interaction of GTPases with their targets was determined by dot-blot 10 days after plating, the confluent quiescent cells were serum-starved assay (Diekmann et al., 1994). 10 μg of GST-fusion proteins were for 16 h in DMEM containing 2 g/l NaHCO . NIH-3T3 cells were plated spotted on strips of nitrocellulose. The strips were air-dried and incubated on acid-washed coverslips at 510 cells/coverslip and left for 24 h in for 1 h in 1 M glycine, 5% milk powder, 1% ovalbumin, and 5% fetal DMEM with 10% DCS before injection. Cells were microinjected as calf serum. The strips were then washed in buffer A [50 mM Tris, described (Nobes and Hall, 1995). pH 7.5, 100 mM NaCl, 5 mM MgCl , 0.1 mM dithiothreitol (DTT)] and incubated for 5 min at 4°C with the indicated GTPase radiolabelled Immunofluorescence microscopy with [γ- P]GTP in a total volume of 2.5 ml of buffer A. The strips After microinjection and incubation at 37°C for the indicated times, the were washed three times with 5 ml of cold buffer A containing 0.1% coverslips were rinsed in PBS and fixed for 10 min with 4% (w/v) Tween. Remaining radioactivity was visualized by autoradiography and paraformaldehyde in PBS. Coverslips were rinsed in PBS between each quantified by densitometry. step of the staining procedure. Following fixation, the cells were 1402 POSH, a new target for the Rac GTPase permeabilized for 5 min in 0.2% Triton X-100 in PBS (10 min for and Decapentaplegic signaling pathways in Drosophila morphogenesis. NF-κB staining). Free aldehyde groups were reduced by treatment with Genes Dev., 11, 1738–1747. 0.5 mg/ml sodium borohydride for 10 min. Labelling of the cells was Hawkins,P.T. et al. (1995) PDGF stimulates an increase in GTP-Rac via as previously described (Nobes and Hall, 1995). Anti-myc (9E10) activation of phosphoinositide 3-kinase. Curr. Biol., 5, 393–403. antibody (kind gift of D.Drechsel) was diluted 1/200 in PBS; anti-NF-kB Herdegen,T., Skene,P. and Bahr,M. (1997) The c-Jun transcription factor– antibody (Santa Cruz) was diluted 1/200 in PBS. Primary antibodies bipotential mediator of neuronal death, survival and regeneration. were left on the coverslips for 1 h. After washing, the coverslips were Trends Neurosci., 20, 227–231. incubated for 30 min with secondary antibodies: goat anti-mouse FITC Hill,C.S., Wynne,J. and Treisman,R. (1995) The Rho family GTPases (Pierce), donkey anti-rabbit TRITC (Jackson), diluted 1/100 in PBS. RhoA, Rac1, and CDC42Hs regulate transcriptional activation by Coverslips were mounted on moviol mountant containing p-phenylenedi- SRF. Cell, 81, 1159–1170. amine as an anti-bleaching agent. After1hat 37°C, the coverslips were Jacobson,M.D., Weil,M. and Raff,M.C. (1997) Programmed cell death examined and the cells counted on a Zeiss axiophot microscope using in animal development. Cell, 88, 347–354. Zeiss 401.3 and 631.4 oil-immersion objectives. Pictures were taken Joneson,T., White,M.A., Wigler,M.H. and Bar-Sagi,D. (1996) Stimulation with a Hamamatsu C5985-10 video camera, then transferred to Kodak of membrane ruffling and MAP kinase activation by distinct effectors T-MAX 400 ASA film. of RAS. Science, 271, 810–812. Knaus,U.G., Morris,S., Dong,H.J., Chernoff,J. and Bokoch,G.M. (1995) Regulation of human leukocyte p21-activated kinases through G Acknowledgements protein-coupled receptors. Science, 269, 221–223. Kotani,K. et al. (1994) Involvement of phosphoinositide 3-kinase in We are grateful to Dr S.Courtneidge for computer alignments of POSH SH3 domains, Dr J.Chant for the activated PAK, Dr M.Terada for human insulin- or IGF-1-induced membrane ruffling. EMBO J., 13, 2313– MST1/MLK2 and Dr C.Kumar for the Ras-transformed NIH cDNA 2321. library. We thank Miss T.Bridges for recombinant proteins, Dr C.Nobes Kozma,R., Ahmed,S., Best,A. and Lim,L. (1995) The Ras-related protein for Swiss-3T3 cells and helpful discussions, Dr M.Jacobson for reagents Cdc42Hs and bradykinin promote formation of peripheral actin and helpful discussions, Dr P.Burbelo for two-hybrid library DNA and microspikes and filopodia in Swiss-3T3 fibroblasts. Mol. Cell. Biol., Dr D.Diekmann for helpful discussions. 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A new Rac target POSH is an SH3‐containing scaffold protein involved in the JNK and NF‐κB signalling pathways

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Abstract

The EMBO Journal Vol.17 No.5 pp.1395–1404, 1998 A new Rac target POSH is an SH3-containing scaffold protein involved in the JNK and NF-κB signalling pathways Reif et al., 1996; Rodriguez-Viciana et al., 1997). Once Nicolas Tapon, Koh-ichi Nagata, 1,2 activated, Rac induces polymerization of monomeric actin Nathalie Lamarche and Alan Hall at the cell periphery to produce a dense meshwork of actin MRC Laboratory for Molecular Cell Biology, CRC Oncogene and filaments forming extending lamellipodia and membrane Signal Transduction Group and Department of Biochemistry, ruffles (Ridley et al., 1992). There is growing evidence University College London, Gower Street, London WC1E 6BT, UK that this pathway plays a major role in directed cell Corresponding author migration and axonal guidance (Van Aelst and D’Souza- e-mail: [email protected] Schorey, 1997). In addition to inducing actin poly- merization, Rac stimulates the formation of associated The Rho, Rac and Cdc42 GTPases coordinately regu- integrin-based adhesion complexes (Nobes and Hall, late the organization of the actin cytoskeleton and the 1995). These structures contain many of the same con- JNK MAP kinase pathway. Mutational analysis of Rac stituents as classical focal adhesions, though morphologic- has previously shown that these two activities are ally they are distinct; they do not seem to be required for mediated by distinct cellular targets, though their actin polymerization but instead may play a role in cell PAK identity is not known. Two Rac targets, p65 and movement or signalling (Machesky and Hall, 1997). MLK, are ser/thr kinases that have been reported to Members of the Rho GTPase family also regulate gene be capable of activating the JNK pathway. We present transcription. All three GTPases have been reported to evidence that neither is the Rac target mediating JNK stimulate the JNK/SAPK and p38/HOG1 MAP kinase activation in Cos-1 cells. We have used yeast two- cascades, the transcription factor NF-κB and the transcrip- hybrid selection and identified a new target of Rac, tion factor SRF (Bagrodia et al., 1995; Coso et al., 1995; POSH. This protein consists of four SH3 domains and Hill et al., 1995; Minden et al., 1995; Olson et al., 1995; ectopic expression leads to the activation of the JNK Sulciner et al., 1996; Teramoto et al., 1996b; Perona et al., pathway and to nuclear translocation of NF-κB. When 1997). To date, most of these observations have been overexpressed in fibroblasts, POSH is a strong inducer obtained by overexpressing GTPase constructs in trans- of apoptosis. We propose that POSH acts as a scaffold fected cells, but the genetic analysis of budding yeast and protein and contributes to Rac-induced signal trans- more recently Drosophila has confirmed that Rho GTPases duction pathways leading to diverse gene transcrip- coordinately regulate the organization of the actin cyto- tional changes. skeleton and the activity of MAP kinase pathways probably Keywords: apoptosis/JNK/NF-κB/POSH/Rac in all eukaryotic cells (Glise and Noselli, 1997; Leberer et al., 1997). In addition, Rho, Rac and Cdc42 can trigger G progression when introduced into quiescent fibroblasts Introduction and they are each required for serum-induced cell cycle progression and Ras-induced cell transformation (Olson Rho, Rac and Cdc42, three members of the Rho family et al., 1995; Qiu et al., 1995a,b, 1997). Moreover, many of small GTPases, act as molecular switches cycling of the Dbl family of Rho GEFs are potent oncogenes and between an active GTP-bound and an inactive GDP-bound will transform NIH-3T3 cells to a malignant phenotype state. Activation, in response to extracellular agonists (Cerione and Zheng, 1996). How they do this is not acting on membrane receptors, is mediated by the Dbl known, though stimulation of G progression and cell family of guanine nucleotide exchange factors (GEFs), transformation correlate well with the ability of the while down-regulation involves a poorly characterized GTPases to induce cytoskeletal changes and it is possible family of GTPase-activating proteins (GAPs) and that signals are induced in response to actin polymerization RhoGDIs (Lamarche and Hall, 1994; Cerione and Zheng, or integrin complex assembly (Joneson et al., 1996; 1996). In their active state, Rho, Rac and Cdc42 interact Lamarche et al., 1996; Westwick et al., 1997). with a variety of target (effector) proteins to elicit cellular To understand the biochemical mechanisms underlying responses (Van Aelst and D’Souza-Schorey, 1997). the various activities of the Rho GTPases, there has been Activation of Rho, Rac and Cdc42 in quiescent Swiss- intense activity to identify target proteins (Van Aelst and 3T3 fibroblasts induces rearrangement of filamentous actin D’Souza-Schorey, 1997). Mutational analysis of Rac has leading to the formation of actin stress fibres, lamellipodia provided evidence for bifurcating pathways controlling and filopodia, respectively (Ridley and Hall, 1992; Ridley cytoskeletal changes and MAP kinase activation; amino et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995). acid substitutions at codon 37, for example, block the Growth factors such as platelet-derived growth factor induction of lamellipodia without affecting JNK activation, (PDGF) and insulin, or constitutively activated (oncogenic) Ras protein stimulate Rac and in both cases this is while changes at codon 40 have the opposite effect mediated by PI 3-kinases (Kotani et al., 1994; Wennstrom (Joneson et al., 1996; Lamarche et al., 1996; Westwick et al., 1994; Hawkins et al., 1995; Nobes et al., 1995; et al., 1997). It appears, therefore, that Rac interacts © Oxford University Press 1395 N.Tapon et al. with at least two distinct target proteins to trigger these pathways. It is not clear whether activation of NF-κBor other transcription factors such as SRF are a consequence of, or are independent of, Rac’s effect on actin or the JNK pathway. To date, around 10 targets for Rac have been identified using yeast two-hybrid and affinity chromatography tech- niques (Van Aelst and D’Souza-Schorey, 1997). The first Rac target to be identified was a serine/threonine kinase PAK p65 . It is closely related to a yeast kinase, Ste20p, which is known to regulate MAP kinase pathways in this PAK organism and it has, therefore, been suggested that p65 probably mediates JNK activation by Rac in mammalian cells (Manser et al., 1994). In agreement with this, the PAK interaction of p65 is blocked by changes at codon 40, but not codon 37, of Rac (Joneson et al., 1996; Lamarche et al., 1996; Westwick et al., 1997). Furthermore, there have been a number of reports that a constitutively PAK activated version of p65 leads to JNK activation and one report that a dominant-negative version inhibits Rac- mediated JNK activation (Bagrodia et al., 1995; Knaus et al., 1995; Brown et al., 1996; Frost et al., 1996). PAK However, others have failed to find a role for p65 in JNK activation and its physiological role remains unclear (Teramoto et al., 1996a,b; Westwick et al., 1997). In PAK addition to p65 another target of Rac, MLK, is also a potential regulator of JNK pathways, since it belongs to the family of MAP kinase kinase kinases (Burbelo PAK Fig. 1. p65 and MLK2/3 do not mediate Rac-induced JNK et al., 1995). PAK activation in Cos-1 cells. (A) Activated p65 does not cause JNK1 We report here that in Cos-1 cells, the interaction of activation when overexpressed in Cos-1 cells. pCMV5FLAG-JNK1 PAK PAK Rac with p65 or MLK is not the trigger for JNK was co-transfected with myc-tagged activated (L107F) p65 , wild- PAK activation. Using a yeast two-hybrid screen, we have type p65 , V12 Rac or empty pRK5Myc vector (–), as described in PAK Materials and methods. Kinase activities of p65 and JNK1 were identified a new target for Rac, POSH, which contains measured on immune complexes (top panel) using Myelin Basic four SH3 domains. When transfected into Cos-1 cells, Protein (5 μg per reaction) or c-jun (2 μg per reaction). Expression POSH stimulates JNK activation. Expression of POSH PAK levels were visualized with anti-myc (for p65 ) and anti-JNK1 in fibroblasts leads to nuclear translocation of NF-κB, antibodies (bottom panel). (B) MLKs do not interact with F37A or Y40C effector mutants of Rac. Yeast strains containing Rac mutants in independently of either actin reorganization or JNK activ- the integrated bait vector pYTH9 were transformed with the pACTII ation. We conclude that POSH acts as a scaffold protein and prey vector containing MST1/MLK2 C-Terminus (aa 338–953), MLK3 participates in Rac-mediated signal transduction pathways C-Terminus (aa 348–847), RhoGAP as a positive control, or empty leading to gene transcriptional changes. vector as a negative control. Colonies of equal size were replated in the presence of 25 mM 3-aminotriazole and allowed to grow for 3 days at 30°C. Results PAK p65 and MLK do not mediate Rac-induced JNK (Lamarche et al., 1996); MST1/MLK2 and MLK3 (Figure activation PAK 1B) are no longer able to interact with Y40CRac in yeast To examine whether p65 could be a mediator of Rac- two-hybrid or dot-blot assays. However, we had shown induced JNK activation, Cos-1 cells were co-transfected PAK previously that Rac containing an F37A substitution was with a constitutively activated version of p65 (L107F, still able to activate JNK in Cos-1 cells (Lamarche et al., PAK1/PAKα isoform) and a JNK1 expression plasmid. 1996). As shown in Figure 1B, F37ARac does not interact As seen in Figure 1A, co-transfection with a constitutively with MST1/MLK2 or MLK3. We conclude, therefore, that activated Rac (V12) leads to a 21-fold stimulation of JNK PAK the interaction of Rac with MLKs cannot be the trigger activity, whereas activated p65 induced no significant PAK for JNK activation in this assay. activation. As a control, L107F-p65 was immunopre- cipitated from the transfected cells and shown to be active PAK using MBP as a substrate. It appears, therefore, that p65 Isolation and characterization of a novel does not activate the JNK kinase cascade in these cells. Rac-interacting protein It has been reported that another Rac target, MLK, is a In order to isolate potential downstream targets of Rac potent activator of the JNK pathway and we have con- that might account for its ability to activate JNK, we have firmed this in Cos-1 cells (Nagata et al., 1998). It is screened a yeast two-hybrid mouse cDNA library using possible, therefore, that the interaction of Rac with endo- L61 Rac as bait. Approximately 10 clones were screened genous MLK proteins is the trigger for JNK activation. as described in Materials and methods and of the 30 As shown previously, Rac containing a Y40C substitution fastest-growing clones picked from the selection plates, PAK can no longer activate JNK and as expected p65 one (clone 4) tested positive upon rescreening and was 1396 POSH, a new target for the Rac GTPase Fig. 2. Interaction of clone 4 with Rac. Clone 4 in the pGAD-10 prey vector was introduced into yeast strains containing integrated L61 Rac, L63 Rho, L61 Cdc42, F37A L61 Rac and Y40C L61 Rac. Colonies of equal size were replated in the presence of 25 mM 3-aminotriazole and allowed to grow for 3 days. negative in the absence of the Rac bait. Figure 2 shows that clone 4 does not interact with Rho or Cdc42 in yeast, and in addition it still interacts with F37ARac, but not with Y40CRac. These observations are consistent with clone 4 encoding a mediator of Rac-induced JNK activation. Sequence analysis of clone 4 (2.4 kb insert) revealed an open reading frame (ORF) for a protein of 67 kDa, but since no stop codon was present 5 of the first methionine, it was possible that clone 4 represented an incomplete ORF. Using a primer derived from a region close to the 5 end of clone 4 we looked for potential additional upstream sequences using a commercial 5 Fig. 3. Sequence and expression pattern of POSH. (A) Protein RACE PCR library. One major RACE product was sequence of full-length POSH. SH3 domains are underlined. The obtained (1.4 kbp in length) and sequence analysis of minimal Rac-binding site is indicated with a dotted line. DDBJ/ EMBL/GenBank accession number of POSH cDNA: AF030131. this confirmed that the original clone 4 represented an (B) Tissue expression of POSH. A mouse multiple tissue Northern blot incomplete ORF. The full ORF encodes a protein of 892 was probed as described in the methods. Lane 1, testis; lane 2, kidney; amino acids (predicted mol. wt 93 kDa) (see Figure 3A); lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6, spleen; the first methionine is surrounded by a consensus Kozak lane 7, brain; lane 8, heart. sequence and is preceded by an in-frame stop codon. Analysis of the protein sequence reveals a potential zinc finger structure (aa 18–82), but more interestingly analysis (Figure 3A) revealed that POSH does not contain four SH3 domains (underlined in Figure 3A). Accordingly, a CRIB site. To identify the region of the protein that we have called the protein POSH (Plenty Of SH3s). Using contains the Rac interaction site, a series of truncations a commercial mouse tissue Northern blot, a single mRNA were expressed as GST fusion proteins and tested for species for POSH (at ~5 kb) can be seen in all tissues binding to L61 Rac in dot-blot assays. As shown in Figure (Figure 3B), though skeletal muscle and spleen appear to 4C, Rac interacts with POSH in a region encompassing have relatively low levels. We conclude that POSH is 70 residues (aa 292–362, dotted underline in Figure 3A). ubiquitously expressed. Extensive searching of EST and non-redundant databases, which includes all other known GTPase targets showed Interaction of POSH with Rac no significant matches to sequences within this region. The A bona fide target protein would be expected to interact Rac interaction site in POSH is, therefore, so far unique. preferentially with the GTP-bound state of Rac. To test whether the interaction of POSH is GTP-dependent, POSH triggers programmed cell death we have used a modified dot-blot assay using To test whether POSH can induce changes to the actin [α- P]GTP-loaded Rac (see Materials and methods) and cytoskeleton, full-length POSH cDNA was first subcloned [α- P]GDP-loaded Rac (obtained by pre-incubating into the mammalian expression vector, pRK5, so as to [α- P]GTP-loaded Rac with a small amount of Rho introduce a myc epitope tag at its N-terminus. Plasmid GAP). As seen in Figure 4A, POSH interacts with the DNA was microinjected into the nuclei of serum-starved, GTP form of Rac but not the GDP form. Using a dot-blot confluent Swiss-3T3 fibroblasts and any effects on the assay, we also confirmed that POSH interacts with the actin cytoskeleton observed 2–8 h later using fluorescently F37A mutant of Rac, but not with the Y40C mutant labelled phalloidin. Under conditions where Rac induced (Figure 4B). strong actin polymerization and lamellipodia, POSH Many, though not all, previously identified Rac targets induced no detectable assembly of actin filaments (data contain a distinctive binding site, the CRIB site. Sequence not shown). It was noticed, however that POSH did induce 1397 N.Tapon et al. sion of POSH induces apoptotic cell death in primary and immortalized fibroblasts (Jacobson et al., 1997). POSH stimulates the JNK pathway To examine the effects of POSH on JNK activation, Cos-1 cells were co-transfected with POSH and JNK. As shown by Western blot analysis the toxic effects of full-length POSH, particularly when co-transfected with JNK (Figure 6A), were also apparent in this cell line [compare expres- sion levels of full-length POSH (lane 2) with truncated POSH (lane 3)]. The toxic effects could be largely over- come by including the caspase inhibitor BocD-fmk (Deshmukh et al., 1996; Weil et al., 1997) in the transfec- tion assays (Figure 6A, compare lane 1,  inhibitor with lane 2, – inhibitor). Under these conditions, it can be seen in Figure 6B that full-length POSH induces a 5.6-fold stimulation of JNK activity, compared with 11.0-fold by L61Rac. The truncated version of POSH was unable to stimulate JNK (Figure 6B), nor did it interfere with Rac- induced JNK activation (data not shown). In order to test whether POSH-induced cell death is dependent on JNK activation, we co-injected POSH with a 5-fold molar excess of dominant-negative SEK1 (SAPK/ ERK kinase 1) (S220A  T224L version or K129R version) into NIH-3T3 fibroblasts. Although both domin- ant-negative SEK1 constructs were expressed at relatively high levels, neither was able to prevent POSH-induced cell death (data not shown). POSH stimulates nuclear translocation of NF-κB To examine whether POSH might contribute to Rac- induced NF-κB activation, expression constructs were microinjected into quiescent Swiss-3T3 cells. Transloca- tion of NF-κB to the nucleus was visualized by immuno- Fig. 4. Interaction of POSH with Rac in vitro.(A) POSH binds to Rac fluorescence 5 h later. Full-length POSH, truncated POSH in a GTP-dependent manner. GST-POSH (10 μg, aa 292–892), containing the two C-terminal SH3 domains (aa 362– PAK GST-p65 (8 μg) and GST (10 μg) were spotted on strips of 32 32 892), activated L61 Rac and L61 F37A Rac were potent nitrocellulose which were probed with [α- P]GTP- or [α- P]GDP- inducers of NF-κB translocation (Figure 7A). However, bound wild-type Rac. (B) POSH binds to F37A but not Y40C Rac PAK in vitro. GST-POSH (10 μg, aa 292–892), GST-p65 (8 μg), GST- L61Y40CRac—which does not interact with POSH—was RhoGAP (10 μg, aa 198–439), and GST (10 μg) were spotted on severely impaired in its ability to cause translocation. nitrocellulose and probed with [γ- P]GTP-bound Rac mutants. Interestingly, translocation of NF-κB in injected cells was (C) Identification of a 70 amino acid fragment of POSH that is accompanied by NF-κB translocation in neighbouring non- sufficient for binding to L61 Rac. Ten μg of GST-fusion proteins of POSH truncations were spotted on nitrocellulose and tested for injected cells (Figure 7B, see arrowhead). We conclude that interaction with L61 Rac as in (B). From top to bottom: original two- Rac and POSH can induce nuclear translocation of NF-κB hybrid fragment (aa 292–892); Truncation 1 (aa 352–892); Truncation and that this may involve the induction of autocrine/ 2 (aa 292–398); Truncation 3 (aa 292–362). paracrine factors. Discussion significant cell death at the later time points in these conditions. Further analysis in NIH-3T3 cells revealed Rho, Rac and Cdc42 control the assembly and organization that 14 h after injection, full-length POSH induced cell of the actin cytoskeleton in eukaryotic cells. In response death in ~90% of injected cells, even in the presence of to extracellular signals, the active conformation of the serum (Figure 5, top) and that the few remaining cells three GTPases leads to the assembly of actin–myosin had shrunken and condensed (pyknotic) nuclei typical of contractile filaments, lamellipodia and filopodia, respect- apoptosis (Figure 5, bottom). In contrast, a POSH Trunca- ively, and it is likely that these proteins play important tion (aa 352–892) lacking the N-terminal two SH3 domains regulatory roles in cell movement (Murphy and Montell, and the Rac-binding domain, or activated L61Rac, did not 1996; Van Aelst and D’Souza-Schorey, 1997; Zipkin et al., induce significant cell death under these conditions (Figure 1997). In addition to their effects on actin, members 5). Time-lapse video microscopy revealed that cell contrac- of the Rho GTPase family regulate changes in gene tion and intense surface blebbing could be seen in the transcription. Rho, Rac and Cdc42 have each been reported majority of cells 5 h after injection (data not shown). to activate the JNK and p38 MAP kinase pathways, to Similar effects were observed in primary rat embryo activate the transcription factors NF-κB and SRF and to fibroblasts (data not shown). We conclude that overexpres- stimulate G progression in quiescent Swiss cells (Bagrodia 1398 POSH, a new target for the Rac GTPase Fig. 5. Full-length POSH induces apoptosis in NIH-3T3 cells. NIH-3T3 cells were replated on glass coverslips and left for 24 h in DMEM containing 10% DCS and the nuclei of 50 cells were injected with an expression vector containing full-length POSH, POSH Truncation 1 (aa 352– 892) or L61 Rac at a concentration of 0.04 mg/ml. The cells were fixed and stained as described after a 12 h incubation in the presence of serum. Bottom line shows typical nuclear morphology of injected cells (arrows) using Hoechst dye staining. In the middle panel, non-injected cell nuclei are in a different plane of focus from the rounded up POSH injected cells. Results were averaged over three independent experiments; error bars represent standard deviation. Scale bar  20 μm. et al., 1995; Coso et al., 1995; Hill et al., 1995; Minden that can no longer interact with MLK (MST1/MLK2 or et al., 1995; Olson et al., 1995; Sulciner et al., 1996; MLK3 isoforms) is still able to activate JNK. We conclude, Teramoto et al., 1996b; Perona et al., 1997). To what therefore, that neither target is likely to mediate JNK extent these pathways are interdependent is not clear, activation by Rac in Cos-1 cells. Although it remains a although G progression can be triggered by Rac mutants possibility that one of the other two PAK isoforms (PAK2/ that can no longer activate the JNK pathway (Joneson PAKγ or PAK3/PAKβ) or another member of the MLK et al., 1996; Lamarche et al., 1996; Westwick et al., 1997). family might trigger JNK activation in our Cos-1 cells, To characterize the biochemical pathways mediating this seems unlikely given their high degree of sequence the various cellular responses induced by GTPases, many homology. Two new MAP kinase kinase kinases, MEKK4 groups have used yeast two-hybrid and affinity chromato- and MEKK1, have recently been reported to interact graphy techniques to identify target proteins and approxim- directly with Rac in a GTP-dependent manner and to ately ten candidate targets for Rac have been isolated so activate the JNK cascade (Fanger et al., 1997). It is far (Van Aelst and D’Souza-Schorey, 1997). Little progress possible, therefore, that either of these could be the cellular has yet been made in identifying which is responsible for target of Rac in Cos-1 cells responsible for activation triggering actin polymerization after interacting with Rac of JNK. GTP, but several targets have been implicated in JNK We have searched for new Rac targets that might activation. A number of groups have reported that the contribute to JNK activation by screening a yeast two- PAK interaction of Rac with the ser/thr kinase, p65 , might hybrid library with constitutively active L61Rac. We have lead to activation of the JNK and p38 pathways and in identified a ubiquitously expressed protein, POSH, that PAK some cell types overexpression of p65 does lead to interacts with Rac (but not Rho or Cdc42) in a GTP- JNK activation (Bagrodia et al., 1995; Knaus et al., 1995; dependent manner. This 93 kDa protein contains four SH3 Brown et al., 1996; Frost et al., 1996). The MLK kinases domains (and a putative zinc finger) and it interacts with are also known to be potent activators of the JNK pathway F37ARac, but not Y40CRac. It is possible, therefore, that and these proteins have binding sites for Rac (Rana et al., this protein might play a role in Rac-mediated JNK 1996; Teramoto et al., 1996b; Nagata et al., 1998). We activation. Consistent with this, expression of POSH in PAK show here that constitutively activated p65 (PAK1/ Cos-1 cells induces activation of JNK. Activation of PAKα isoform) does not stimulate the JNK pathway in JNK by inflammatory cytokines or by stress is often Cos-1 cells. Furthermore, a Rac mutant (L61F37ARac) accompanied by nuclear translocation of the transcription 1399 N.Tapon et al. POSH, which can activate NF-κB but not JNK, do not induce cell death. Co-injection of POSH with dominant- negative SEK failed to block POSH-induced cell death, though this construct has been reported to block JNK activation induced by MEKK1 and to inhibit cell death induced by cis-platinum, UV irradiation or heat (Sanchez et al., 1994; Zanke et al., 1996). It is possible, therefore, that POSH induces cell death independently of JNK activation. Furthermore, even though POSH is a Rac target, overexpression of Rac does not lead to apoptosis. One possible explanation for this apparent discrepancy is that Rac, through its multiple downstream targets, might also activate survival signals. If this is the case, then the survival signal cannot be NF-κB, since this is also activated by POSH. SH3 domain-containing proteins have previously been shown to play important roles in mediating Rho GTPase signals. In phagocytic cells, p67 , a component of the phox NADPH oxidase enzyme complex is a target of Rac (Diekmann et al., 1994). p67 has no catalytic activity, phox but consists of two SH3 domains—the second of which shows close similarity to the first and fourth SH3 domains of POSH (51.9% with POSH 1, 42.6% with POSH 4). The membrane-bound oxidase is responsible for the generation of superoxide, which forms a part of the Fig. 6. POSH activates JNK1 in Cos-1 cells. (A) Cos-1 cells were co- transfected with pCMV5FLAG-JNK1 (5 μg) and pRK5myc-full-length pathogen-killing mechanism of professional phagocytes, POSH (lanes 1 and 2) or pRK5myc-POSH Truncation 1 (lane 3) (all and both Rac and p67 are essential for its activity phox 3 μg) in the presence () or absence (–) of BocD-fmk at 20 μM. (Abo et al., 1991; Segal and Abo, 1993; Diekmann et al., Cells were harvested in 300 μlof3 protein sample buffer and 30 μl 1994). Rac has also been reported to induce the formation of each lysate was analysed by Western blotting with anti-myc and anti-JNK1 antibodies. (B) pCMV5FLAG-JNK1 was co-transfected of reactive oxygen species (ROS) in non-phagocytic cells with empty pRK5myc vector (lane 1), pRK5myc-L61 Rac (lane 2), and in HeLa cells, this appears to account for Rac- pRK5myc-POSH Truncation 1 (aa 352–892) (lane 3), and pRK5myc- mediated NF-κB activation (Sulciner et al., 1996). Interes- full-length POSH (lane 4) all in the presence of 20 μM BocD-fmk tingly, during the POSH and Rac injection experiments, (Enzymes Systems Products). Aliquots of each transfection were we see translocation of NF-κB in neighbouring, non- electrophoresed on an SDS–PAGE gel and expression levels of transfected constructs were visualized on Western blots using anti-myc injected cells and although we cannot rule out mechanisms (for POSH, Rac), and anti-FLAG (for JNK1) antibodies followed by involving cell–cell contact, it raises the possibility that a I-labelled protein A (top panel). JNK1 activity was assayed on paracrine factor (perhaps ROS) is produced. We are immune complexes using GST-c-jun as a substrate and quantified on a currently looking at whether Rac can induce ROS in Bio-Rad Molecular Imager (middle panel). Levels of JNK1 in the immunoprecipitates were visualized using an anti JNK1 antibody Swiss-3T3 cells and if so, whether this is mediated by (bottom panel). p67 or perhaps POSH. phox In Saccharomyces cerevisiae, the SH3-containing pro- factor NF-κB (Verma et al., 1995). Furthermore, there tein Bem1p is a key component of Cdc42-mediated signals have been reports that Rac can activate NF-κB when during cell division and in the pheromone mating response transfected into cells (Sulciner et al., 1996; Perona et al., (Leberer et al., 1997). In the mating response, Bem1p 1997). Using an immunofluorescence assay, we have acts as a scaffold protein and interacts with multiple shown that expression of POSH in Swiss-3T3 cells leads proteins including Cdc24p (an exchange factor for to nuclear translocation of NF-κB. Interestingly, the two Cdc42p), with Ste20p (a target of Cdc42p and a relative PAK C-terminal SH3 domains are sufficient to induce NF-κB of mammalian p65 ), with Ste5p (a scaffold protein translocation, but not JNK activation. We conclude that required for MAP kinase activation), with actin and with POSH may play a role in both JNK and NF-κB activation Far1p (an inhibitor of the cell cycle) (Leberer et al., 1997). mediated by Rac. In agreement with this, Y40CRac— It is thought that a major role of Cdc42p in the pheromone which is inactive in both assays—no longer interacts with pathway is to localize this multi-molecular signalling POSH, while F37ARac—which interacts with POSH—is complex to the mating projection. Interestingly, in fission active in both assays. yeast the homologue of Bem1p, scd2, is also required for Finally, activation of JNK and NF-κB pathways is often the mating response. In this case, scd2 interacts directly seen in cells stimulated to undergo apoptosis though the with scd1 (the exchange factor for cdc42) and with cdc42 relative contributions of the two pathways to cell death (Chang et al., 1994). No Bem1p/scd2 homologues have are highly cell type-dependent (Liu et al., 1996; Baichwal been identified in higher eukaryotes and it is tempting to and Baeuerle, 1997; Chuang et al., 1997; Herdegen et al., speculate that POSH fulfils an analogous role and acts as 1997). We have found that overexpression of POSH in a scaffold protein in Rac-mediated signalling pathways in Swiss or NIH-3T3 cell lines, in primary rat embryo mammalian cells. If this is the case, POSH might be fibroblasts or in Cos cells is highly toxic and leads to cell predicted to interact with other components of the Rac death via apoptosis. The two C-terminal SH3 domains of signal transduction pathway (e.g. an exchange factor or 1400 POSH, a new target for the Rac GTPase Fig. 7. POSH induces nuclear translocation of NF-κB in Swiss-3T3 cells. (A) Myc-tagged pRK5 plasmids (0.04 mg/ml) encoding POSH (full-length), POSH Truncation1 (aa 352–892), L61Y40CRac, L61F37ARac, L61Rac or empty vector (co-injected with FITC-dextran) were microinjected into serum-starved, confluent Swiss-3T3 cells and the cells fixed 5 h later. POSH/Rac expression was visualized using anti-myc antibody, while NF-κB localization was visualized using an anti-NF-κB antibody (Santa Cruz). The percentage of myc-positive cells in which clear nuclear fluorescence of NF-κB was seen were scored as positive. Results were averaged over four independent experiments; between 29 and 85 myc-expressing cells were analysed per experiment and the error bar represents standard deviation. (B) Right panel: a typical phenotype of a myc-positive, nuclear NF-κB-positive cell after injection with truncated POSH. The arrowhead points to a neighbouring, non-injected cell that is nuclear NF-κB positive. Left panel: a typical phenotype of a myc-positive, nuclear NF-κB-negative cell after injection with L61Y40CRac. Scale bar  20 μm. other targets). We have been unable to detect an interaction NF-κB and apoptosis or acts by titrating out inhibitors of PAK between p65 and POSH after Cos cell transfections these processes. (data not shown) and we are currently using yeast two- In conclusion, we have shown that the two Rac targets, PAK hybrid screens to identify binding partners of POSH. p65 and MLK are unlikely to account for Rac-induced JNK activation in Cos-1 cells. We have identified a new The biochemical mechanisms through which over- SH3-containing Rac target, POSH, which activates JNK expression of POSH leads to activation of JNK in when transfected into Cos-1 cells and induces nuclear fibroblasts is unclear. Expression of other adaptor-like translocation of NF-κB. We propose that POSH acts as molecules with SH3 domains can also have profound scaffold protein required for the assembly of signalling cellular effects; the SH2/SH3-containing protein v-Crk, complexes that control gene transcriptional events down- for example, can induce malignant transformation (Mayer stream of Rac. et al., 1989). Furthermore, overexpression of Bem1p in yeast has been reported to activate the mating pheromone Materials and methods pathway independently of mating pheromone when expressed in a STE11-4 mutant background (Lyons et al., Yeast two-hybrid screen 1996). We propose that POSH either triggers the formation A Ras-transformed NIH-3T3 cDNA library fused to the GAL-4 activation of a signalling complex leading to activation of JNK, domain in the pGAD-10 vector (kind gift of Dr C.C.Kumar, USA) was 1401 N.Tapon et al. screened using L61 Rac in an integrated pYTH6 vector as a bait, as Purification and expression of recombinant proteins previously described (Aspenstrom et al., 1996; Lamarche et al., 1996). Rac GST fusion proteins were purified on glutathione–Sepharose beads Approximately 10 yeast colonies were screened for their ability to grow (Sigma) and cleaved using human thrombin as previously described PAK on selective medium containing 25 mM 3-aminotriazole. The 30 fastest- (Self and Hall, 1995). POSH, p65 and RhoGAP were produced using growing clones were replated and plasmids were rescued using the a modification of this protocol. A culture of bacteria bearing pGEX-4T3 Wizard clean-up kit (Promega) and retransformed into the original yeast containing the relevant insert was grown in 500 ml of L-Broth containing strain. One of these, clone 4 was strongly positive in the plate lift assay 100 μg/ml ampicillin overnight at 37°C with vigorous agitation. The for expression of the LacZ reporter gene after the second round of cells were diluted with 500 ml of fresh L-Broth containing ampicillin transformation. The 2.4 kb insert of clone 4 was sequenced and found and left to grow for 2 h. Fusion protein was induced for2hby addition to be a novel cDNA potentially encoding a 67 kDa protein. of IPTG to 1 mM. The cells were harvested and lysed as previously described (Self and Hall, 1995). Following purification on glutathione– Sepharose beads, the proteins were eluted with three washes of 500 μl Cloning of full-length clone 4 resuspension buffer (15 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM The full-length clone 4 sequence was isolated using the Clonetech MgCl , 0.1 mM DTT) containing 1.5 mM glutathione. Eluted proteins Marathon RACE PCR kit (Mouse whole embryo) with Advantage 2 were concentrated on centricon-10 columns and stored in liquid nitrogen. Klentaq. A primer was generated corresponding to the minus strand of Protein concentration was assayed using the Bradford method and the bp 156–178 of the original yeast clone (5-GTCACCGGGCTGCTA- quality was checked on SDS–PAGE gels using Coomassie blue. GGGGGTGGGG-3). This primer was used with the Clonetech adaptor primer in the standard touchdown PCR protocol described in the GTP dependence Clonetech manual. Analysis of the RACE reaction by agarose gel To determine the relative binding of RacGTP and RacGDP to target electrophoresis revealed a major RACE product at 1.4 kb. The product proteins a modified dot-blot protocol was used. GST-fusion proteins was ligated into pYTH6 after NcoI–NotI digestion and sequenced. Two were spotted onto nitrocellulose strips. 200 ng of wild-type Rac were independent clones of the 1.4 kb fragment were analysed to eliminate loaded with [α- P]GTP and the exchange reaction stopped by addition any errors due to PCR amplification. of MgCl on ice as previously described (Diekmann et al., 1994). The sample was split in two and 10 ng of RhoGAP were added to one of Mammalian cell transfections the tubes. This tube was incubated for 10 min at 30°C (to produce Cos-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium predominantly [α- P]GDP), while the other tube was left on ice (DMEM) (Gibco) containing 10% FCS (Sigma). These were transfected (predominantly [α- P]GTP). The GTPase aliquots were used in a dot- using the DEAE–dextran method as previously described (Olson et al., blot assay as described above. Bound radioactivity was quantified by 1995). Plasmid amounts per 10 cm dish were as follows: pRK5myc- autoradiography and densitometry. POSH, 5 μg; pRK5myc-POSH Truncation T1, 2 μg; pRK5myc- PAK L107Fp65 (PAK1/PAKα isoform), 5 μg; pRK5myc-V12/L61Rac1, DNA constructs 1.5 μg; pCMV-FLAG-JNK1, 5 μg. Empty vector was added where Standard DNA protocols were used (Sambrook et al., 1989). pGEX- appropriate to ensure all transfections contained equal amounts of DNA. 4T3-clone 4 was generated by digestion of pGAD10-clone 4 with BamHI Cells were serum-starved 24 h after transfection and harvested 16 h and HindIII followed by ligation of clone 4 cDNA into pGEX-4T3. later. After transfection, the cells were kept in serum-containing or Truncations of clone 4 in pGEX were made by PCR with Pfu polymerase serum-free media supplemented with the caspase inhibitor BocD-fmk (Stratagene). Clone 4 and the truncations were transferred from pGEX- (Boc-Aspartic acid-fluoromethylketone; Enzyme Systems Products) at 4T3, using BamHI and HindIII, into the eukaryotic expression vector, 20 μM (from a 10 mM stock in DMSO). PRK5-myc (Lamarche et al., 1996). Full-length POSH in PRK5-myc was generated by amplifying the RACE product with Pfu polymerase Kinase assays using the following oligonucleotides: 5-GCCGGATCCGATGAGT- Transfected Cos-1 cells were harvested in lysis buffer (20 mM Tris CTGCCTTGTTGGAC-3, and 5-GACTTGTTGGCCATGG TGAGGG- pH 8.0, 40 mM Na pyrophosphate, 50 mM NaF, 5 mM MgCl , 100 μM AGGTGAAGG-3. This fragment was then combined with the remainder Na vanadate, 10 mM EGTA, 1% Triton X-100, 3 mM PMSF, 20 μg/ml of the POSH coding sequence and inserted into PRK5-myc using BamHI PAK leupeptin/aprotinin) using a cell scraper (Falcon). JNK1 or p65 were and NcoI. The amplified fragment was checked by sequencing. A immunoprecipitated using anti-FLAG antibody (Scientific Imaging) for pRK5FLAG vector was constructed by digesting pRK5myc with ClaI PAK JNK1 or anti-myc antibody for p65 (Olson et al., 1995). Kinase and HindIII and introducing the FLAG epitope using the following activity of immunocomplexes was measured using GST-c-jun for JNK1 oligonucleotides: 5-CGATAGCCACCATGGACTACAAGGACGATG- PAK or Myelin Basic Protein for p65 as substrates, as described (Olson ACGATAAGGGATCCCGGGTCTAGAATTCGGGA-3 and 5-AGCT- et al., 1995; Lamarche et al., 1996). The relative levels of substrate TCCCGAATTCTAGACCCTGGATCCCTTATCGTCATCGTCCTTGT- phosphorylation were determined on a Bio-Rad PhosphorImager, follow- AGTCCATGGTGGCTAT-3. Full-length POSH was inserted into ing SDS–PAGE and transfer to nitrocellulose. Amounts of immunopre- pRK5FLAG from pRK5myc using BamHI and HindIII. cipitated kinases were checked using anti-JNK1 antibody (Santa Cruz) The following constructs were kind gifts from colleagues: human PAK or anti-myc antibody for p65 . MST-1/MLK2 from Dr M.Terada, National Cancer Center Research Institute, Tokyo; PCMV-FLAG-JNK1 and pGEX-c-Jun from Dr M.Karin, PAK Co-precipitation of p65 and POSH UC San Diego and Dr J.Ham, Eisai London Research Laboratories; 65PAK Cos-1 cells were transfected with pRK5myc-L107Fp or pRK5mycSEK-1 (K129R) from Dr M.Olson, Chester Beatty Labora- pRK5FLAG-full-length POSH (3 μg each). Cell were harvested as above tories, London; HA-tagged SEK-1 AL (S220A, T224L) from Dr L.Zon, and the lysates were combined. Half of the lysate was used for Dana Farber Cancer Institute, Boston; and PAK (L107F) from Dr immunoprecipitation using anti-myc antibody and the other half using J.Chant, Harvard University. anti-FLAG antibody (Scientific Imaging). Immunoprecipitations and washing were carried out in 40 mM Tris, pH 7.5, 50 mM NaCl, 0.2% Microinjection NP-40, 50 mM NaF, 20 μg/ml leupeptin/aprotinin as for JNK assays Swiss-3T3 cells were maintained in DMEM containing 10% FCS and (Olson et al., 1995). antibiotics. NIH-3T3 cells were maintained in DMEM containing 10% donor calf serum (DCS) and antibiotics. Swiss-3T3 cells were plated on Dot-blot assay acid-washed, round 13 mm coverslips at 610 cells/coverslip. At 7– Interaction of GTPases with their targets was determined by dot-blot 10 days after plating, the confluent quiescent cells were serum-starved assay (Diekmann et al., 1994). 10 μg of GST-fusion proteins were for 16 h in DMEM containing 2 g/l NaHCO . NIH-3T3 cells were plated spotted on strips of nitrocellulose. The strips were air-dried and incubated on acid-washed coverslips at 510 cells/coverslip and left for 24 h in for 1 h in 1 M glycine, 5% milk powder, 1% ovalbumin, and 5% fetal DMEM with 10% DCS before injection. Cells were microinjected as calf serum. The strips were then washed in buffer A [50 mM Tris, described (Nobes and Hall, 1995). pH 7.5, 100 mM NaCl, 5 mM MgCl , 0.1 mM dithiothreitol (DTT)] and incubated for 5 min at 4°C with the indicated GTPase radiolabelled Immunofluorescence microscopy with [γ- P]GTP in a total volume of 2.5 ml of buffer A. The strips After microinjection and incubation at 37°C for the indicated times, the were washed three times with 5 ml of cold buffer A containing 0.1% coverslips were rinsed in PBS and fixed for 10 min with 4% (w/v) Tween. Remaining radioactivity was visualized by autoradiography and paraformaldehyde in PBS. Coverslips were rinsed in PBS between each quantified by densitometry. step of the staining procedure. Following fixation, the cells were 1402 POSH, a new target for the Rac GTPase permeabilized for 5 min in 0.2% Triton X-100 in PBS (10 min for and Decapentaplegic signaling pathways in Drosophila morphogenesis. NF-κB staining). Free aldehyde groups were reduced by treatment with Genes Dev., 11, 1738–1747. 0.5 mg/ml sodium borohydride for 10 min. Labelling of the cells was Hawkins,P.T. et al. (1995) PDGF stimulates an increase in GTP-Rac via as previously described (Nobes and Hall, 1995). Anti-myc (9E10) activation of phosphoinositide 3-kinase. Curr. Biol., 5, 393–403. antibody (kind gift of D.Drechsel) was diluted 1/200 in PBS; anti-NF-kB Herdegen,T., Skene,P. and Bahr,M. (1997) The c-Jun transcription factor– antibody (Santa Cruz) was diluted 1/200 in PBS. Primary antibodies bipotential mediator of neuronal death, survival and regeneration. were left on the coverslips for 1 h. After washing, the coverslips were Trends Neurosci., 20, 227–231. incubated for 30 min with secondary antibodies: goat anti-mouse FITC Hill,C.S., Wynne,J. and Treisman,R. (1995) The Rho family GTPases (Pierce), donkey anti-rabbit TRITC (Jackson), diluted 1/100 in PBS. RhoA, Rac1, and CDC42Hs regulate transcriptional activation by Coverslips were mounted on moviol mountant containing p-phenylenedi- SRF. Cell, 81, 1159–1170. amine as an anti-bleaching agent. After1hat 37°C, the coverslips were Jacobson,M.D., Weil,M. and Raff,M.C. (1997) Programmed cell death examined and the cells counted on a Zeiss axiophot microscope using in animal development. Cell, 88, 347–354. Zeiss 401.3 and 631.4 oil-immersion objectives. Pictures were taken Joneson,T., White,M.A., Wigler,M.H. and Bar-Sagi,D. (1996) Stimulation with a Hamamatsu C5985-10 video camera, then transferred to Kodak of membrane ruffling and MAP kinase activation by distinct effectors T-MAX 400 ASA film. of RAS. Science, 271, 810–812. 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Journal

The EMBO Journal – Springer Journals

Published: Mar 2, 1998

Keywords: apoptosis; JNK; NF‐κB; POSH; Rac

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