Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1–dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.

The production of immunostimulatory cytokines by epithelial cells is an essential part of the biological response to the infection with human pathogenic microorganisms (1). Any inflammatory reaction requires the de novo synthesis of defined proteins. These include chemokines that attract macrophages and inflammatory cytokines that serve to amplify and spread the primary pathogenic signal. The mechanism by which these proteins are newly synthesized involves an inducible transcriptional initiation of their respective genes. This is governed by several transcription factors playing a role in regulating immune response genes including the early transcription factor activator protein 1 (AP-1; reference 2).1

Human pathogenic Neisseria gonorrhoeae (Ngo) induce a number of cytokine genes in epithelial cells involving the activity of the transcription factor AP-1 (3). The natural host benefit of cytokine release in response to pathogen infection is exploited by Ngo which cause a massive localized inflammation characterized by the infiltration of polymorphonuclear leukocytes and mononuclear phagocytes into the infected tissue of the urethra or the endocervix (4). As a consequence, the infected epithelial cell layer is destroyed and the bacteria may gain better access to deeper tissues (5). The colonization of the epithelia is a prerequisite of infection by Ngo and is largely dependent on the expression of a set of variable surface proteins. The hair-like type 4 pili mediate the first contact between bacteria and host cells and are indispensable for infection in vivo (6, 7). Variant outer membrane proteins of the opacity-associated outer membrane protein family (Opa proteins) tightly interact with the host cell surface and trigger the engulfment of Ngo (8). There are at least three independent routes by which opa proteins induce the uptake of Ngo into host cells each mediated by interaction with a certain receptor. Heparan-sulfate proteoglycan (HSP) receptors (9) or the concerted action of HSPR and integrin receptors (10, 11) account for the major pathways for uptake into epithelial cells. Uptake of Ngo by the HSPR pathway involves the action of phosphatidylcholine-dependent phospholipase C (PLC) and the acidic sphingomyelinase (ASM; reference 12), the HSPR– integrin pathway possibly PKC (11). In phagocytic cells, Opa proteins occupy receptors of the CD66 family that results in a rapid, opsonin-independent phagocytosis of Ngo (1315) involving Src kinases and Rac1 (16).

In contrast to the well-studied Neisseria adherence and invasion process, very little is known about the nature of the pathogen-induced proinflammatory signals. Our previous results showed that the infection of epithelial cells with gonococci induced the activation of the transcription factor AP-1 (3). In line with the known immunostimulatory function of AP-1 and nuclear factor κB (NF-κB; references 17, 18) epithelial cells infected with Ngo produced increased amounts of numerous proinflammatory cytokines (3). The secretion of cytokines in the cultured epithelial cell model was not initiated by LPS suggesting that the endotoxin-mediated mechanisms known to trigger inflammatory reactions of lymphoid cells are not relevant for epithelial cells (3). However, neither the signals nor the signaling pathways activating AP-1, which lead to production of cytokines in Ngo-infected epithelial cells, are defined.

Environmental stress or engagement of cytokine receptors leads to the activation of AP-1 and a family of mitogen-activated protein kinases (MAPK), which consecutively activate their members by phosphorylation (18, 19). In such a cascade, the AP-1 subunit c-Jun is activated by the c-Jun NH2-terminal kinase (JNK); JNK itself is activated by MAPK kinase 4 and 7 (MKK4, MKK7); and MKK4 is activated by MAP/ERK (extracellular signal–regulated kinase) kinase 1 (MEKK1) at the level of MAPK, MKK, and MKK kinase (MKKK), respectively. Numerous MKKK beside MEKK1 have recently been identified, all able to activate the JNK pathway (2025). The reason why mammalian cells express so many MKKK involved in signaling to stress-activated transcription factors is still unknown. It is still unclear in particular how the MKKK themselves are activated in mammalian cells in response to stress signals or cytokine receptor activation.

The small GTPases Rac1 and Cdc42 have been identified as far upstream regulators of the JNK-kinase cascade (20, 26, 27). The p21-activated kinases (PAKs) were the first kinases identified as direct effectors for the active GTPases Rac1 and Cdc42 (28). GTP-loaded Rac1 or Cdc42 bind to a Cdc42/Rac interactive binding (CRIB) domain (29) and thereby increase the activity of PAK significantly (28). Recently, MEKK1 and MEKK4 have also been shown to interact with Rac and Cdc42 (30). Blocking either PAK or MEKK1 activity prevents the activation of JNK either in response to activated GTPases (30, 31) or to apoptotic stimuli independent of GTPases (32, 33). However, blocking MEKK kinase does not prevent JNK activation by active PAK suggesting that both classes of kinases signal independently.

Here we show that Ngo induces the activation of the AP-1 transcription factor by a distinct kinase cascade involving GTPases, PAK, MKK4, and JNK1, but not MEK kinase 1. The identification of pathogen-specific signaling pathways to inflammatory cytokine production might be a basis for the therapeutic intervention of infections.

Human Cell Culture and Infection.

Epithelial cells (HeLa and Chang) were grown in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 4 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS in a humidified 5% CO2 atmosphere. Epithelial cells were seeded in tissue culture plates for 48 h before infection. 16 h before infection, the medium was replaced by fresh RPMI 1640 medium supplemented with 10% FCS. The epithelial cells were infected with Ngo given in multiplicities of infection (MOI) as described in the figure legends for different periods of time. For infection experiments, bacteria were centrifuged onto the epithelial cell monolayer for 5 min at 500 g. In the experiments using 10–50 ng/ml Toxin B (supplied by F. Hofmann and K. Aktories, University of Freiburg, Freiburg, Germany), the cells were preincubated for 15 min before the bacteria were added. Stimulation of the cells with 10 ng/ ml TNF-α (Promega, Heidelberg, Germany) or 50 nM PMA (Sigma Chemical Co., St. Louis, MO) was performed for the indicated time points.

Bacteria.

Three different Ngo strains were used for infection of human epithelial cell lines: the nonpiliated, but invasive Opa+ strain N242, (VP1, P.IA, P, Opa27, Opa27.5, Opa28, Opa29, Opa30, lipopolysaccharide type L1) has been described (8). The adherent but noninvasive P+ strain N138 (P+ PilEF3) and the P Opa control strain N300 (PilEB1, opaC30::cat, pTH7) are derivatives of strain MS11 (34, 35). Gonococci were routinely grown on GC phosphate agar as described previously (36). Strain N242 was also grown in RPMI 1640 containing 25 mM Hepes, pH 7.2, for 2 h at 37°C before infection.

Transfections and Luciferase Reporter Assays.

To measure the transactivating activity of AP-1, HeLa cells at 50–70% confluence were cotransfected by cationic liposomes (Promega) with 1 μg of a luciferase expression plasmid containing three repeats of the AP-1 binding site as an enhancer element and expression constructs: dominant negative MEKK1 (K432M), dominant negative (DN) MKK4 (K116R, DNJNKK), DNPAK1 (K299R), PAK2 (K278R) or PAK1 (H83L, H86L, and K299R), or wild-type PAK2 or PAK1. 24 h after transfection, cells were left untreated, infected with Ngo strains, treated with 50 nM PMA, or preincubated with Toxin B. Luciferase assays were performed at the indicated time points as recommended by the manufacturer's instructions (Promega). The results were recorded on a Wallac 409 β-counter (Berthold-Wallac, Bad Wildbad, Germany), and given as fold induction against untreated cells. The data presented are representative of more than three separate experiments.

Immunoprecipitation, Immunoblotting, and Protein Kinase Assays.

To assess the kinase activity of JNK and p38, cells were lysed in RIPA buffer as described previously (37). For PAK, cells were lysed in 50 mM Tris, pH 7.5, 1 mM EGTA, 5 mM MgCl2, 1% NP-40, 2.5% glycerol, 150 mM NaCl, 1 mM Na3VO4, 10 mM Na4P2O4, 50 mM NaF, 100 μM PMSF, 10 μM Pepstatin A, and 4 μM Aprotinin. The cells were disrupted by passing the lysate several times through a 21-gauge syringe. The cell debris was collected by centrifugation and the supernatant was subsequently incubated with appropriate antisera (Santa Cruz Biotechnology, Santa Cruz, CA) coupled to protein A– or G–Sepharose (Amersham Biotechnology Inc., Piscataway, NJ). The antisera were coupled to the protein A or G beads in PBS for 1 h at 4°C. In a first preadsorption step the supernatants were incubated with protein A or G beads alone for 1 h at 4°C. After this preadsorption the supernatants were incubated with the appropriate antisera for 2–3 h at 4°C. Immunocomplexes were recovered and washed several times with RIPA buffer and twice with kinase buffer. Immunoprecipitates of JNK were resuspended in 30 μl of kinase buffer containing 100 mK KCl, 0.1 mM CaCl2, 6 mM MgCl2, 30 mM Tris, pH 7.5, 0.1 mM Na3VO4, 10 mM K2HPO4, 10 mM NaF, 1 mM DTT, and 1 μg of the substrate (GST–c-Jun for JNK, GST-ATF2 for p38; Santa Cruz Biotechnology). Immunoprecipitates of PAK were resuspended in 30 μl of kinase buffer containing 50 mM Hepes, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 0.2 mM dithiothreitol, 0.1 mM Na3VO4, 14 μM ATP, and 1 μg of myelin basic protein (MBP; Sigma Chemical Co.). The kinase reactions were initiated by addition of 10 μCi γ-[32P]ATP (5,000 Ci/mmol; Amersham Pharmacia Biotechnology Inc.). After 30 min incubation at 30°C, reactions were terminated by adding SDS loading buffer. Samples were separated by SDS-PAGE, blotted, and developed as described (38) to show similar protein amounts in all lanes. After immunostaining the blots were analyzed further by autoradiography or PhosphorImager analysis.

Activation of JNK in Response to Ngo–Epithelial Cell Contact.

In our previous report, we showed that the early response transcription factor AP-1 and inflammatory cytokine gene expression are coordinately induced in Ngo infected epithelial cells (3). Activation of the dimeric sequence specific enhancer factor AP-1 is mediated through phosphorylation of c-Jun by members of the MAPK family. Therefore, we examined whether the JNKs and/or p38, also known as stress activated kinases (3941) are involved in the signaling leading to Ngo-induced AP-1 activation and inflammatory cytokine gene expression. Cell lysates from Ngo-infected epithelial cells were used at different time points after infection for immunoprecipitation of the endogenous kinases and solid-phase kinase assays using appropriate substrates were performed.

The Ngo strains tested differed with regard to the infection of epithelial cells. Strain N138 (P+ Opa) expresses pili that confer adherence to epithelial cells, but does not express any Opa proteins and therefore is not invasive (42). Strain N242 (Opa+) enters epithelial cells by binding to heparan sulfate containing surface proteoglycan receptor proteins (8, 9). Strain N300 (P Opa) is neither piliated, nor does it express Opa proteins (35) and, hence, is a very weak adherent. Infection with Ngo strains was routinely monitored by light microscopy.

The activity of JNK1 was induced several fold in subconfluent monolayers of HeLa cells by adherence of Ngo strain N138 (P+ Opa; Fig. 1, top) within 15 min after infection. The immediate JNK1 induction was followed by a sustained JNK1 kinase activation for at least 90 min. Activation of JNK1 in Chang epithelial cells was at least 10 times weaker than in HeLa cells, probably due to lower adherence levels of Ngo to Chang cells. To test whether cellular JNK1 activation is at variance in epithelial cells infected with adherent versus invasive Ngo strains, we compared JNK1 kinase activity in HeLa cells and Chang cells infected either with the P+ or Opa+ strain. At a MOI of 50, the invasive Opa+ strain induced a slightly stronger activation of JNK1 activity than the P+ strain, whereas weak JNK1 activation was observed in cells treated with the nonpathogenic strain N300 (P Opa) or LPS. The activation of JNK1 in response to Ngo was delayed compared to the JNK1 induction in TNF-α–treated cells, but showed a similar potential to induce JNK1 activity. The lower panel shows a Western blot probed with an anti-JNK1 antibody, to show similar protein amounts in all lanes. Weak activation of JNK2 was observed in HeLa cells in response to Ngo infection (data not shown). In contrast to JNK1, we did not detect any p38 induction paralleled with JNK1 activation (data not shown).

DNMKK4 Inhibits Ngo-mediated Induction of AP-1.

Besides the various stimuli (growth factors, UV light irradiation, cytokines, protein synthesis inhibitors, etc.) that could induce the JNK cascade (19), pathogens represent new potent inducers in this regard. Thus it is a challenge to understand whether pathogens inducing inflammation use similar or different signaling pathways compared to known stimuli. For JNK, MKK4/SEK1 (stress-activated protein kinase/ extracellular-regulated kinase 1 [SAPK/ERK]) has been identified as a direct activator, phosphorylating JNK at threonine and tyrosine residue in the motif TPY (4345). In addition, recent studies using sek1−/− cells indicated that there are MKK4/SEK1-dependent and MKK4/SEK1- independent signaling pathways for JNK activation (46, 47).

To address the possible role of MKK4 in Ngo-induced transcriptional activity of AP-1, we examined the effect of inhibitory DNMKK4 on the transcription of an AP-1– dependent reporter construct (Fig. 2). In transient transfection assays the overexpressed DNMKK4 significantly inhibited P+ Opa and P Opa+ induced AP-1–dependent reporter gene expression (Fig. 2, A and B), but not of an NF-κB–dependent reporter gene (data not shown). This is in agreement with the earlier observation that the JNK activation is not linked to NF-κB activation (48, 49). Similarly, PMA induced AP-1 activation was blocked by DNMKK4 (Fig. 2,D). Ngo strain P Opa did not induce AP-1 transactivation activity (Fig. 2 C). The strong inhibitory effect of DNMKK4 on pathogenic Ngo and PMA induced AP-1 activation indicates a major role of MKK4 for the JNK-MAPK signal transduction pathway. DNMKK4 might inhibit Ngo-induced signal transduction pathways, in which MKK4 is intimately involved by strongly interacting with its upstream activators and/or its downstream elements.

Toxin B Inhibits AP-1 and JNK Activation in Ngo-stimulated Cells.

Attachment of epithelial cells by Ngo suggests the involvement of cytoskeletal rearrangements (36) as shown for Helicobacter pylori (50) and other pathogenic microbes (51). Cytoskeletal rearrangements are known to be regulated by small GTPases of the Rho family (52). Therefore, we studied whether GTPases were involved in Ngo- induced signaling pathways leading to JNK and AP-1 activation. The GTPases Rac1 and Cdc42 have been identified as important intermediates to the JNK signaling cascades (20, 27, 53). The activity of Rho GTPases but not of Ras is specifically inhibited by enterotoxin Toxin B from Clostridium difficile, which glycosylates and inactivates Cdc42, Rac1, and Rho (54, 55). Pretreatment of HeLa cells for 15 min with 10 ng/ml Toxin B is sufficient to reduce AP-1 transactivation activity in the presence of adherent Ngo strain (P+ Opa) to the baseline levels (Fig. 3, compare right and left panel) indicating the involvement of Rho-GTPases in Ngo-induced AP-1 activation. As a control we studied the PMA-induced AP-1–dependent reporter activity, which appears not to be affected by Toxin B (Fig. 3). Similarly to AP-1, JNK activation (assayed in an in vitro kinase assay) in response to the adherent Ngo strain (P+ Opa) was inhibited in the presence of Toxin B (Fig. 4, lanes 1 and 3 versus 4 and 6). Treatment of HeLa cells with Toxin B already induces JNK activity (Fig. 4, lanes 1 and 4). In contrast to Ngo, PMA-stimulated cells could induce JNK activity in Toxin B–treated cells (Fig. 4, lanes 1, 2, 4, and 5).

AP-1 Activation in Ngo-stimulated Epithelial Cells Involves PAK Signaling.

There are potentially many different kinases that could contribute to small GTPase-mediated JNK and AP-1 activation. The most prominent kinases acting downstream of Rho-GTPases are the PAKs (p21-activated kinases) and the MEKK (56). The PAKs show homology to STE20p (28), which mediate specific responses involving Cdc42p in S. cerevisiae (57). In analogy to S. cerevisiae, it was suggested that PAKs are intermediate in Cdc42/ Rac1-mediated activation of MEKK (26, 27). Recent results show that MEKK1 directly interacts with Rac1 and Cdc42 (30), indicating that PAK and MEKK1 independently associate with Rho-GTPases. To examine whether PAK or MEKK1 are involved in transcriptional responses characteristic of Ngo-infected epithelial cells, we compared their capacity to contribute to Ngo-induced AP-1 activation.

In transient transfection assays we used PAK2 and PAK1 dominant inhibitory mutant kinases to investigate whether PAK might be involved upstream in the Ngo-induced signaling cascade leading to MKK4, JNK, and AP-1 activation. The PAK2 mutant contains a K278R and PAK1 a K299R mutation that inactivate the catalytic activity of the kinase domain. The mutations in the amino acids arginine 278 and 299 have been demonstrated to be important for the kinase activity of PAKs (58). HeLa cells transfected with DNPAK2 and treated either with adherent (P+ Opa) or invasive (P Opa+) Ngo strains for 30 min strongly reduced the AP-1 transactivation activity, compared to mock transfected cells (Fig. 5,A). Similar to PAK2 we observed reduced transactivation activity of AP-1 in transfected DNPAK1 and Ngo-stimulated cells (data not shown). The experiments were performed after careful titration of the cDNAs to allow specific inhibition of the AP-1 transactivation activity. Since mammalian PAKs (PAK1, 2 and 3) exert >90% homology in their COOH-terminal kinase domain, they will putatively inhibit downstream signaling of all three PAK isoenzymes. To exclude potential effects on the putative upstream components due to titration of Rho-GTPases, we performed experiments with a PAK1 construct mutated in histidine 83 and 86, which prevent the binding of GTPases. Using such construct, similar results as described above were obtained (data not shown). Therefore, these results indicate that PAK could lie upstream of MKK4 and JNK leading to AP-1 activation. As already shown in Fig. 2,C, nonpathogenic Ngo strain P Opa slightly induces the transactivation activity of AP-1, which is reduced in DNPAK2-transfected cells. In PMA-treated cells, AP-1 transactivation activity was substantially increased but unaffected in DNPAK2-transfected cells (Fig. 5 A) suggesting that PMA induces JNK/AP-1 activation via a PAK-independent pathway, as previously shown by Rudel et al. (33).

To address the possible role of wild-type PAK in transcriptional activity of AP-1 in response to Ngo (P+ Opa), we examined the effect of expression of PAK on the transcription of an AP-1–dependent reporter construct. Overexpression of PAK2 (wtPAK2) slightly enhanced transcription of an AP-1–dependent reporter gene in the absence of Ngo (Fig. 5,B). Similar data were obtained using a wild-type PAK1 construct (data not shown). This may be in agreement with the recent observation that the PAK activation depends on active GTPases (28) and confirms previous data showing small JNK protein kinase activity of wild-type PAK3 overexpression (20). In the presence of Ngo P+ Opa we observed an increased and additive effect on the AP-1 transactivation activity compared to mock transfected cells (Fig. 5 B).

To study the JNK/AP-1 activation by the kinase activity of PAK2, we immunoprecipitated PAK2 from Ngo (P+ Opa)-stimulated HeLa cells after the indicated periods of time. The activity of PAK2 (p-PAK2) was induced several fold in HeLa cells by the Ngo strain N138 (P+ Opa; Fig. 5,C) within 15 min after infection measuring phosphorylation activity of PAK2 with MBP as a substrate. The immediate PAK2 induction further increased within 45 min and declined to a reduced activity within 90 min. The lower panel of Fig. 5 shows the Western blot probed with an anti-PAK2 antibody, to show similar protein amounts in all lanes.

In an attempt to explore whether MEKK1 is involved when Ngo bacteria induce MKK4/JNK/AP-1 activity, we tested if inhibitory DNMEKK1 blocks this pathway. In contrast to kinase-inactive inhibitory mutants of PAKs, DNMEKK1 does not block Ngo-induced AP-1 transcriptional activity (Fig. 6, A and B). In contrast to Ngo, PMA-induced AP-1 transactivation activity clearly appears inhibited by DNMEKK1 (Fig. 6 C). A role for PMA-activated PKC in the activation of MEKK1 was recently described (59).

Therefore, these results indicate that PAKs do not lie upstream of MEKK1 in a pathway leading to AP-1 activation. Rather, the data suggest that PAKs independently mediate JNK/AP-1 activation in a Ngo-induced signaling pathway.

In most pathogen infections, epithelial cells represent the first barrier for the microorganisms in the host tissue and could function as an early warning system that activates the immune response using signals that lead to the attraction of phagocytic cells (1). The activatory signaling leading to posttranslational modification and activation of the transcription factor AP-1 plays a critical role in the regulation of proinflammatory cytokine genes and other gene promoters whose gene products have immunomodulatory functions (60). The ability to respond appropriately to such signaling events is essential for the survival of the eukaryotic cell. Perturbations in the normal regulation of these specific responses can result in pathogenic events that lead to acute or chronic disease. AP-1 transcriptional activity and subsequent upregulation of proinflammatory cytokines is induced upon the initial contact of Neisseria gonorrhoeae with epithelial cells, which influence the immune surveillance. Adherent P+ Ngo or invasive Opa+ Ngo induce a rapid and specific activation of AP-1 and subsequently a panel of cytokine genes. The physical invasion of epithelial cells by Ngo is not a prerequisite of AP-1 activation and cytokine upregulation (3). Thus, it appears that the Ngo- eukaryotic cell contact is already sufficient to induce an efficient downstream signaling. To a better understanding of the crucial outcome of the Ngo infection, we studied the upstream components involved in AP-1 activation.

In this study we analyzed the capability of adherent and invasive Ngo strains to induce stress response kinase pathways leading to AP-1 activation in epithelial cells. Components of kinase cascades modulate AP-1 activation at different control points including regulation at the transcriptional and posttranslational level. Exposure of mammalian cells to a variety of physiological stress stimuli, including Ngo cell contact (3), activate the transcription factor AP-1 and many target genes (19). Moreover, overexpression of AP-1 in response to a variety of stress factors has been implicated in a number of immunoinflammatory and proliferative diseases (60). c-Jun is the central component of all AP-1 dimeric protein complexes and phosphorylation of c-Jun in the activation domain is exerted only by the JNKs (19). We found that Ngo stimulates JNK in epithelial cells (Fig. 1), which phosphorylates c-Jun at serines 63/73 (61), and AP-1– dependent transcriptional activity (Fig. 2). The JNK kinase activity in response to adherent (P+ Opa) or invasive (P Opa+) Ngo strains, which induce a variety of proinflammatory cytokines, was observed rapidly within 15 min after infection at a MOI of 50 and further increased within 90 min, whereas the nonpathogenic strain (P Opa) or LPS induced weak and no JNK activity, respectively. Our data indicate that contact between Ngo and epithelial cells is sufficient to induce an efficient downstream signaling leading to the activation of cytokine genes. The TNF-α–treated cells showed JNK activation to a similar extent, but with a maximum within 15 min. Activation of JNK was observed in both cell lines studied, but was at least 10 times less effective and delayed in Chang cells compared to HeLa cells. This points to the assumption that epithelial cells are capable of responding differently to external stimuli. For example, the opa-dependent invasion of Ngo in Chang conjunctiva cells occurs irrespective of the presence of serum but involves PLC and ASM (12). In many other epithelial cells, this signaling pathway appears to be less prominent, whereas the serum factor vitronectin was shown to allow HSPR-dependent invasion (10). The observation that JNK, but not p38 kinase was activated by Ngo suggests that the activatory mechanisms of these kinases by this stimulus are at least partially different. Our finding that Ngo induced stress response kinase JNK contributing in the activation of cytokine genes in epithelial cells is consistent with another report on Salmonella typhimurium (62), which supports the notion that epithelial cells are an integral component of the host immune system. LPS does not effectively induce cytokines AP-1 and JNK in epithelial cells, which could be explained in part by lack of CD14 on epithelial cells. In contrast to Ngo epithelial cell contact, infection of macrophages by Yersinia enterocolitica suppresses the LPS-mediated activation of JNK as well as p38 kinase activation (63). Thus, different pathogen stimulants trigger multiple signal transduction pathways in the target cell.

Stimulation of cells with epidermal growth factor (EGF; references 4345) or IL-1 (64) leads to JNK activation by a pathway that involves the activation of MKK4. Like other physiologic stress inducers (e.g. UV light and osmotic shock; reference 2), the human pathogenic adherent (P+ Opa) or invasive (P Opa+) Ngo strains also induce MKK4, and overexpression of the kinase negative MKK4 inhibits activation of AP-1 (Fig. 2, A and B). The dominant inhibitory effect of MKK4 on Ngo-induced AP-1 activation indicates that JNK is required to mediate full transcriptional activation of AP-1 in response to Ngo and suggests either the formation of a stable JNK/MKK4 complex or sequestration of crucial elements immediately upstream of MKK4. A number of kinases have been postulated to regulate the JNK/AP-1 activation. These include the MEKKs 1, 2, and 4 (6567) and tumor progression locus 2 (Tpl-2; reference 24), which phosphorylate and activate MKK4. Germinal center kinase (21), the mixed lineage kinases (MLK), MLK2 and 3/SPRK, DLK/MUK (23, 6870), and TGF-β–activated kinase (22) show selectivity for the activation of JNK and phosphorylate MKK4 in vitro. In contrast to other stimuli like EGF, we could exclude that MEKK1 is involved in Ngo-induced JNK/ AP-1 activation. DNMEKK1 does not affect the pathogen-induced (P+ Opa or P Opa+) activation of AP-1, whereas PMA-induced AP-1 activity, which presumably involves activated PKC (59), is inhibited (Fig. 6, A–C). Similar to a number of other kinases, PAKs are shown to be activators of JNK signaling cascades (20), although PAKs do not directly phosphorylate MKK4 (53). In Ngo-stimulated cells, PAK mediates its effect on the JNK/AP-1 pathway independent of MEKK1. The ability of DNPAKs (DNPAK2 and DNPAK1) to block AP-1 activation by Ngo (P+ Opa or P Opa+), and the activation of AP-1 by PAK (wtPAK2 and wtPAK1) overexpression and activation of endogenous PAK2 in response to Ngo (P+ Opa) indicate that PAK may function as a component of the same signaling transduction pathway that leads to MKK4/ JNK and AP-1 activation (Fig. 5). In the future it will be necessary to address which components downstream of PAKs lead to JNK/AP-1 activation. A suggested candidate kinase that might play such a role could be SPRK, a mixed lineage kinase (23). p21-activated kinase is the most upstream component of the Rac1/Cdc42 pathway linking these GTPases to JNK (56). By studying the involvement of GTPases in Ngo-induced signaling, we used Toxin B from Clostridium difficile, which specifically inhibits the activity of the members of the Rho family (54). AP-1 transcriptional activity and JNK activation is strongly reduced in the presence of Toxin B (Fig. 3). These data indicate that GTPases and PAKs may function as components of the same signal transduction pathway that leads to JNK protein kinase and the activation of the transcription factor AP-1.

From these findings, we suggest a pathway, depicted in Fig. 7, through which human pathogenic Ngo stimulate the JNK/AP-1 activity. Activation of GTPases by upstream signals leads to increased activity of PAK kinase(s). PAK does not directly phosphorylate JNK. Activation of JNK by dual phosphorylation is mediated by MKK4, which becomes phosphorylated by MKKK. Thus PAKs presumably regulate the activity of MKKK. The signaling pathway through GTPases, PAK, MKKK, MKK4, JNK, and AP-1 are orchestrated to yield a coordinated response of cytoskeletal changes and transcriptional activity leading to the release of immune response mediators.

Neisseria strains used in our experiments produce either pili (P+) or Opa proteins (Opa+) capable of interacting with cells, or neither pili nor Opa proteins. In phagocytes the Opa-CD66 receptor interaction could be important for the invasion-dependent downstream signaling involving Src-kinases and Rac1 (16). Internalization of Opa-expressing Ngo in epithelial cells has been shown to be HSP- dependent (9) and occur by at least two different mechanisms. Invasion in Chang cells depends on the activation of ASM and the second messengers diacylglycerol (DAG) and ceramide (12). In other epithelial cells including HeLa, another pathway depending on the serum factor vitronectin allows HSP-dependent invasion. Vitronectin-dependent invasion of Opa+ Ngo (10) involves integrin receptors (11). Vitronectin also possesses a HSP-binding site and is generally known to interact with certain integrins (71). In this respect internalization of Ngo by epithelial cells could be mediated by integrin-dependent endocytosis of vitronectin, which results from interaction with heparan-sulfate proteoglycans (HSPGs). Since Ngo-induced cytokines, the activation of AP-1 (3) and stress response kinases are already induced by noninvasive Ngo (P+ Opa), we suggest that Ngo adhesion activates signals leading to the activation of immune response mediators. Thus it seems, that Ngo trigger a number of signaling pathways, which are either essential for the pathogen infection, or represent the host reaction in response to the pathogenic microbes.

Cytokines produced from epithelial cells represent specific modulators of the immune response and could be an advantage in the eradication of the parasite, but could also be a disadvantage by leading to chronic inflammatory diseases instead. The ability to respond appropriately to such signaling events is essential for the survival of the cell and ultimately the whole organism. A chronic inflammatory state, induction of adhesion molecules, chemotaxis and activation of leukocytes, the production of mediators of inflammation and continuous T cell activation leads to self-perpetuating destruction of the epithelial structure. All these events are controlled at the level of transcription of genes necessary for the production of new proteins such as growth factors, interleukins, proteinases and cytokines. Regulating this process, e.g., by disrupting the signal transduction pathways of transcription factors, has the potential to attenuate the production of immune response mediators, thereby halting or reversing the course of the disease.

We are grateful to F. Hofmann and K. Aktories for the generous gift of Toxin B protein. We thank G. Bokoch and J. Chernoff for providing PAK cDNA constructs and M. Karin for providing the DNMEKK1 and DNJNKK (DNMKK4) cDNA constructs.

This work was supported in part by the Fonds der Chemischen Industrie.

     
  • AP-1

    activator protein 1

  •  
  • ASM

    acidic sphingomyelinase

  •  
  • CRIB

    Cdc42/Rac interactive binding

  •  
  • DAG

    diacylglycerol

  •  
  • DN

    dominant negative

  •  
  • EGF

    epidermal growth factor

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • HSP

    heparan-sulfate proteoglycan

  •  
  • JNK

    c-Jun NH2-terminal kinase

  •  
  • MAP

    mitogen-activated protein

  •  
  • MAPK

    MAP kinase

  •  
  • MEK

    MAP/ERK

  •  
  • MEKK

    MEK kinase

  •  
  • MKK

    MAPK kinase

  •  
  • MKKK

    MKK kinase

  •  
  • MOI

    multiplicities of infection

  •  
  • NF-κB

    nuclear factor κB

  •  
  • Ngo

    Neisseria gonorrhoeae

  •  
  • PAK

    p21-activated kinase

  •  
  • PLC

    phosphatidylcholine–dependent phospholipase C

  •  
  • SAPK

    stress-activated protein kinase

1
Hedges
SR
,
Agace
WW
,
Svanborg
C
Epithelial cytokine responses and mucosal cytokine networks
Trends Microbiol
1995
3
266
270
[PubMed]
2
Rosette
C
,
Karin
M
Ultraviolet light and osmotic stress—activation of the JNK cascade through multiple growth factor and cytokine receptors
Science
1996
274
1194
1197
[PubMed]
3
Naumann
M
,
Wessler
S
,
Bartsch
C
,
Wieland
B
,
Meyer
TF
Neisseria gonorrheaeepithelial cell interaction leads to the activation of the transcription factors nuclear factor κB and activator protein 1 and the induction of inflammatory cytokines
J Exp Med
1997
186
247
258
[PubMed]
4
Witt
K
,
Veale
DR
,
Smith
H
Resistance of Neisseria gonorrhoeaeto ingestion and digestion by phagocytes of human buffy coat
J Med Microbiol
1976
9
1
12
[PubMed]
5
McGee
ZA
,
Johnson
AP
,
Taylor-Robinson
D
Pathogenic mechanisms of Neisseria gonorrhoeae: observation on damage to human fallopian tubes in organ culture by gonococci of colony type 1 or type 4
J Infect Dis
1981
143
413
422
[PubMed]
6
Kellogg
DS
,
Peacock
WL
,
Deacon
WE
,
Brown
L
,
Pirkle
CI
Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation
J Bacteriol
1963
85
1274
1282
[PubMed]
7
Swanson
J
,
Robbins
K
,
Barrera
O
,
Corwin
D
,
Boslego
J
,
Ciak
J
,
Blake
MS
,
Koomey
JM
Gonococcal pilin variants in experimental gonorrhoea
J Exp Med
1987
165
1344
1357
[PubMed]
8
Makino
S
,
van Putten
JP
,
Meyer
TF
Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeaeinto human epithelial cells
EMBO (Eur Mol Biol Organ) J
1991
10
1307
1315
[PubMed]
9
van Putten
JP
,
Paul
SM
Binding of syndecan-like cell surface proteoglycan receptors is required for Neisseria gonorrhoeaeentry into human mucosal cells
EMBO (Eur Mol Biol Organ) J
1995
14
2144
2154
[PubMed]
10
Gómez-Duarte
OG
,
Dehio
M
,
Guzmán
CA
,
Chhatwal
GS
,
Dehio
C
,
Meyer
TF
Binding of vitronectin to Opa-expressing Neisseria gonorrhoeaemediates invasion of HeLa cells
Infect Immun
1997
65
3857
3866
[PubMed]
11
Dehio
M
,
Gomez-Duarte
OG
,
Dehio
C
,
Meyer
TF
Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeaeinvolves alpha(v) integrin receptors
FEBS Letters
1998
424
84
88
[PubMed]
12
Grassme
H
,
Gulbins
E
,
Brenner
B
,
Ferlinz
K
,
Sandhoff
K
,
Harzer
K
,
Lang
F
,
Meyer
TF
Acidic sphingomyelinase mediates entry of N. gonorrhoeaeinto nonphagocytic cells
Cell
1997
91
605
615
[PubMed]
13
Chen
T
,
Gotschlich
EC
CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins
Proc Natl Acad Sci USA
1996
93
14851
14860
[PubMed]
14
Gray-Owen
S
,
Dehio
C
,
Haude
A
,
Grunert
F
,
Meyer
TF
CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeaeand human polymorphonuclear phagocytes
EMBO (Eur Mol Biol Organ) J
1997
16
3435
3445
[PubMed]
15
Virji
M
,
Makepeace
K
,
Ferguson
DJP
,
Watt
SM
Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic Neisseriae.
Mol Microbiol
1996
22
941
950
[PubMed]
16
Hauck
CR
,
Meyer
TF
,
Lang
F
,
Gulbins
E
CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeaerequires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway
EMBO (Eur Mol Biol Organ) J
1998
17
443
454
[PubMed]
17
Baeuerle
PA
,
Baichwal
VR
NF-κB as a frequent target for immunosuppressive and anti-inflammatory molecules
Adv Immunol
1997
65
111
137
[PubMed]
18
Ip
YT
,
Davis
RJ
Signal transduction by the c-jun NH2-terminal kinase (JNK)—from inflammation to development
Curr Opin Cell Biol
1998
10
205
219
[PubMed]
19
Karin
M
,
Zg
L
,
Zandi
E
AP-1 function and regulation
Curr Opin Cell Biol
1997
9
240
246
[PubMed]
20
Bagrodia
S
,
Derijard
B
,
Davis
RJ
,
Cerione
RA
Cdc42 and PAK-mediated signaling leads to jun kinase and p38 mitogen-activated protein kinase activation
J Biol Chem
1995
270
27995
27998
[PubMed]
21
Pombo
CM
,
Kehrl
JH
,
Sanchez
I
,
Katz
P
,
Avruch
J
,
Zon
LI
,
Woodgett
JR
,
Force
T
,
Kyriakis
JM
Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase
Nature
1995
377
750
754
[PubMed]
22
Yamaguchi
K
,
Shirakabe
K
,
Shibuya
H
,
Irie
K
,
Oishi
I
,
Ueno
N
,
Taniguchi
T
,
Nishida
E
,
Matsumoto
K
Identification of a member of the MAPKKK family as a potential mediator of TGF-beta signal transduction
Science
1995
270
2008
2011
[PubMed]
23
Rana
A
,
Gallo
K
,
Godowski
P
,
Hirai
S
,
Ohno
S
,
Zon
L
,
Kyriakis
JM
,
Avruch
J
The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1
J Biol Chem
1996
271
19025
19028
[PubMed]
24
Salmeron
A
,
Ahmad
TB
,
Carlile
GW
,
Pappin
D
,
Narsimhan
RP
,
Ley
SC
Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase
EMBO (Eur Mol Biol Organ) J
1996
15
817
826
[PubMed]
25
Ichijo
H
,
Nishida
E
,
Irie
K
,
Dijke
P
,
Saitho
M
,
Moriguchi
T
,
Takagi
M
,
Matsumoto
K
,
Miyanzono
K
,
Gotoh
Y
Induction of apoptosis by ASK1, a mammalian MAPKKK that activates the SAPK/JNK and p38 signaling pathway
Science
1997
275
90
95
[PubMed]
26
Coso
OA
,
Chiariello
M
,
Yu
JC
,
Teramoto
H
,
Crespo
P
,
Xu
N
,
Miki
T
,
Gutkind
JS
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway
Cell
1995
81
1137
1146
[PubMed]
27
Minden
A
,
Lin
A
,
Claret
FX
,
Abo
A
,
Karin
M
Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs
Cell
1995
81
1147
1157
[PubMed]
28
Manser
E
,
Leung
T
,
Salihuddin
H
,
Zhao
ZS
,
Lim
L
A brain serine/threonine protein kinase activated by Cdc42 and Rac1
Nature
1994
367
40
46
[PubMed]
29
Burbelo
PD
,
Drechsel
D
,
Hall
A
A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases
J Biol Chem
1995
270
29071
29074
[PubMed]
30
Fanger
GR
,
Johnson
NL
,
Johnson
GL
MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42
EMBO (Eur Mol Biol Organ) J
1997
16
4961
4972
[PubMed]
31
Brown
JL
,
Stowers
L
,
Baer
M
,
Trejo
J
,
Coughlin
S
,
Chant
J
Human STE20 homologue HPAK1 links GTPases to the JNK/MAP kinase pathway
Curr Biol
1996
6
598
605
[PubMed]
32
Cardone
MH
,
Salvesen
GS
,
Widmann
C
,
Johnson
G
,
Frisch
SM
The regulation of anoikis: MEKK-1 activation requires cleavage by caspases
Cell
1997
90
315
325
[PubMed]
33
Rudel
T
,
Zenke
FT
,
Chuang
TH
,
Bokoch
GM
Cutting edge—p21-activated kinase (pak) is required for fas-induced jnk activation in jurkat cells
J Immunol
1998
160
7
11
[PubMed]
34
Haas
R
,
Schwarz
H
,
Meyer
TF
Release of soluble pilin antigen coupled with gene conversion in Neisseria gonorrhoeae.
Proc Natl Acad Sci USA
1987
84
9079
9083
[PubMed]
35
Kahrs
AF
,
Bihlmaier
A
,
Facius
D
,
Meyer
TF
Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants
Mol Microbiol
1994
12
819
832
[PubMed]
36
Grassmé
HUC
,
Ireland
RM
,
van Putten
JPM
Gonococcal opacity protein promotes bacterial entry-associated re-arrangements of the epithelial cell actin cytoskeleton
Infect Immun
1996
64
1621
1630
[PubMed]
37
Naumann
M
,
Scheidereit
C
Activation of NF-κB in vivo is regulated by multiple phosphorylations
EMBO (Eur Mol Biol Organ) J
1994
13
4597
4607
[PubMed]
38
Naumann
M
,
Wulczyn
FG
,
Scheidereit
C
The NF-κB precursor p105 and the proto-oncogene product Bcl-3 are I κB molecules and control nuclear translocation of NF-kappa B
EMBO (Eur Mol Biol Organ) J
1993
12
213
222
[PubMed]
39
Hibi
M
,
Lin
A
,
Smeal
T
,
Minden
A
,
Karin
M
Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain
Genes Dev
1993
7
2135
2148
[PubMed]
40
Derijard
B
,
Hibi
M
,
Wu
IH
,
Barrett
T
,
Su
B
,
Deng
T
,
Karin
M
,
Davis
RJ
JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain
Cell
1994
76
1020
1028
41
Kyriakis
JM
,
Banerjee
P
,
Nikolakaki
E
,
Dai
T
,
Rubie
EA
,
Ahmad
MF
,
Avruch
J
,
Woodgett
JR
The stress activated protein kinase subfamily of c-Jun kinases
Nature
1994
368
156
160
[PubMed]
42
Rudel
T
,
van Putten
JPM
,
Gibbs
CP
,
Haas
R
,
Meyer
TF
Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrheaeto human epithelial cells
Mol Microbiol
1992
6
3439
3450
[PubMed]
43
Sanchez
I
,
Hughes
RT
,
Mayer
BJ
,
Yee
K
,
Woodgett
JR
,
Avruch
J
,
Kyriakis
JM
,
Zon
LI
Role of sapk/erk kinase-1 in the stress-activated pathway regulating transcription factor c-jun
Nature
1994
372
794
798
[PubMed]
44
Derijard
B
,
Raingeaud
J
,
Barrett
T
,
Wu
IH
,
Han
JH
,
Ulevitch
RJ
,
Davis
RJ
Independent human map kinase signal transduction pathways defined by mek and mkk isoforms
Science
1995
267
682
685
[PubMed]
45
Lin
AN
,
Minden
A
,
Martinetto
H
,
Claret
FX
,
Langecarter
C
,
Mercurio
F
,
Johnson
GL
,
Karin
M
Identification of a dual specificity kinase that activates the jun kinases and p38-mpk2
Science
1995
268
286
290
[PubMed]
46
Nishina
H
,
Fischer
KD
,
Radvanyi
L
,
Shahinian
A
,
Hakem
R
,
Rubie
EA
,
Bernstein
A
,
Mak
TW
,
Woodgett
JR
,
Penninger
JM
Stress-signalling kinase sek1 protects thymocytes from apoptosis mediated by cd95 and cd3
Nature
1997
385
350
353
[PubMed]
47
Yang
D
,
Tournier
C
,
Wysk
M
,
Lu
HT
,
Xu
J
,
Davis
RJ
,
Flavell
RA
Targeted disruption of the mkk4 gene causes embryonic death, inhibition of c-jun nh2-terminal kinase activation, and defects in ap-1 transcriptional activity
Proc Natl Acad Sci USA
1997
94
3004
3009
[PubMed]
48
Liu
ZG
,
Hsu
HL
,
Goeddel
DV
,
Karin
M
Dissection of tnf receptor 1 effector functions—jnk activation is not linked to apoptosis while NF-κB activation prevents cell death
Cell
1996
87
565
576
[PubMed]
49
Reinhard
C
,
Shamoon
B
,
Shyamala
V
,
Williams
LT
Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by traf2
EMBO (Eur Mol Biol Organ) J
1997
6
1080
1092
[PubMed]
50
Segal
ED
,
Lange
C
,
Covacci
A
,
Tompkins
LS
,
Falkow
S
Induction of host signal transduction pathways by Helicobacter pylori.
Proc Natl Acad Sci USA
1997
94
7595
7599
[PubMed]
51
Bliska
JB
,
Galan
JE
,
Falkow
S
Signal transduction in the mammalian cell during bacterial attachment and entry
Cell
1993
73
903
920
[PubMed]
52
Hall
A
Small GTP-binding proteins and the regulation of the actin cytoskeleton
Annu Rev Cell Biol
1994
10
31
54
[PubMed]
53
Zhang
SJ
,
Han
JH
,
Sells
MA
,
Chernoff
J
,
Knaus
UG
,
Ulevitch
RJ
,
Bokoch
GM
Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator pak1
J Biol Chem
1995
270
23934
23936
[PubMed]
54
Just
I
,
Selzer
J
,
Wilm
M
,
von Eichel-Streiber
C
,
Mann
M
,
Aktories
K
Glucosylation of Rho proteins by Clostridium difficiletoxin B
Nature
1995
375
500
504
[PubMed]
55
Aktories
K
Rho proteins: targets for bacterial toxins
Trends Microbiol
1997
5
282
285
[PubMed]
56
Kyriakis
JM
,
Avruch
J
Protein kinase cascades activated by stress and inflammatory cytokines
Bioessays
1996
18
567
577
[PubMed]
57
Leberer
E
,
Wu
C
,
Leeuw
T
,
Fourest-Lieuvin
A
,
Segall
JE
,
Thomas
DY
Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase
EMBO (Eur Mol Biol Organ) J
1997
16
83
97
[PubMed]
58
Sells
MA
,
Knaus
UG
,
Bagrodia
S
,
Ambrose
DM
,
Bokoch
GM
,
Chernoff
J
Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells
Curr Biol
1997
7
202
210
[PubMed]
59
Siow
YL
,
Kalmar
GB
,
Sanghera
JS
,
Tai
G
,
Oh
SS
,
Pelech
SL
Identification of two essential phosphorylated threonine residues in the catalytic domain of Mekk1. Indirect activation by Pak3 and protein kinase C
J Biol Chem
1997
272
7586
7594
[PubMed]
60
Rao
A
NF-ATp: a transcription factor required for the co-ordinate induction of several cytokine genes
Immunol Today
1994
15
274
278
[PubMed]
61
Pulverer
BJ
,
Kyriakis
JM
,
Avruch
J
,
Nikolakaki
E
,
Woodgett
JR
Phosphorylation of c-jun mediated by MAP kinases
Nature
1991
353
670
674
[PubMed]
62
Chen
LM
,
Hobbie
S
,
Galan
JE
Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses
Science
1996
274
2115
2119
[PubMed]
63
Ruckdeschel
K
,
Machold
J
,
Roggenkamp
A
,
Schubert
S
,
Pierre
J
,
Zumbihl
R
,
Liautard
JP
,
Heesemann
J
,
Rouot
B
Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-alpha production
J Biol Chem
1997
272
15920
15927
[PubMed]
64
Whitmarsh
AJ
,
Yang
SH
,
Su
MSS
,
Sharrocks
AD
,
Davis
RJ
Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors
Mol Cell Biol
1997
17
2360
2371
[PubMed]
65
Blank
JL
,
Gerwins
P
,
Elliott
EM
,
Sather
S
,
Johnson
GL
Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase
J Biol Chem
1996
271
5361
5368
[PubMed]
66
Xu
S
,
Robbins
DJ
,
Christerson
LB
,
English
JM
,
Vanderbilt
CA
,
Cobb
MH
Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain
Proc Natl Acad Sci USA
1996
93
5291
5296
[PubMed]
67
Gerwins
P
,
Blank
JL
,
Johnson
GL
Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway
J Biol Chem
1997
272
8288
8295
[PubMed]
68
Hirai
S
,
Izawa
M
,
Osada
S
,
Spyrou
G
,
Ohno
S
Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK
Oncogene
1996
12
641
650
[PubMed]
69
Teramoto
H
,
Coso
OA
,
Miyata
H
,
Igishi
T
,
Miki
T
,
Gutkind
JS
Signaling from the small GTP-binding proteins rac1 and cdc42 to the c-jun NH2-terminal kinase stress-activated protein kinase pathway—a role for mixed lineage kinase 3 protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family
J Biol Chem
1996
271
27225
27228
[PubMed]
70
Hirai
S
,
Katoh
M
,
Terada
M
,
Kyriakis
JM
,
Zon
LI
,
Rana
A
,
Avruch
J
,
Ohno
S
MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates sek1, an activator of c-jun NH2-terminal kinase/stress-activated protein kinase
J Biol Chem
1997
272
15167
15173
[PubMed]
71
Preissner
KT
Structure and biological role of vitronectin
Annu Rev Cell Biol
1991
7
275
310
[PubMed]

Author notes

Address correspondence to Dr. Michael Naumann, Max-Planck-Institut für Infektionsbiologie, Abt. Molekulare Biologie, Monbijoustr. 2, 10117 Berlin, Germany. Phone: 49-30-28026317; Fax: 49-30-28026611; E-mail: naumann@mpiib-berlin.mpg.de