Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1

The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-acti- vated kinase 1 (Pak1) is a potential mediator of Rac/ Cdc42 signaling, and we observe that Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK. Rac and Cdc42 are members of the Rho family of small guanosine 5 (cid:57) -triphosphate (GTP)-binding proteins. These GTPases regulate assembly of actin cytoskeletal structures associated with cell motility and metastasis, as well as the generation of bactericidal oxygen metabolites by the phagocyte NADPH oxidase (1, 2). Rac was also shown to be an important component of cellular transformation by Ras oncogenes, al-though the mechanisms by which Rac contributes to the transformation process are unknown (3). Regulation of nuclear signaling by Rho family GTPases has recently been described (4), EDTA, 3 m M EGTA, 250 m M NaCl, 1% Nonidet P-40, 1 m M dithiothreitol, 0.1 m M orthovanadate, 1 m M phenylmethylsulfonyl fluoride, 10 (cid:109) g/ml leupep- tin, 0.078 trypsin inhibitory units/ml aprotinin) for 30 min with shaking at 4 °C and then cleared by centrifugation at 100,000 (cid:51) g for 30 min at 4 °C prior to immunoprecipitation and kinase assay. Expression levels of cDNA constructs after transient transfection were verified using the respective epitope tag antibodies. Kinase Assays— Mouse monoclonal antibodies against the Flag epitope, M2 (Kodak Scientific Imaging Systems), the c- myc epitope, 9E10 (Santa Cruz Biotechnology), HA epitope, and 12CA5 (kindly pro- vided by I. Wilson, Scripps Research Institute) or rabbit polyclonal Pak1 antibody (15) were prebound to protein G-Sepharose or protein A-Sepharose beads, respectively. 20 (cid:109) l of a 1:1 suspension of beads was added to 300- (cid:109) l cell lysates and gently shaken for 3 h at 4 °C. The precipitates were washed 6 times with 1 ml of wash buffer containing 25

The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-activated kinase 1 (Pak1) is a potential mediator of Rac/ Cdc42 signaling, and we observe that Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1-and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK.
Rac and Cdc42 are members of the Rho family of small guanosine 5Ј-triphosphate (GTP)-binding proteins. These GTPases regulate assembly of actin cytoskeletal structures associated with cell motility and metastasis, as well as the generation of bactericidal oxygen metabolites by the phagocyte NADPH oxidase (1,2). Rac was also shown to be an important component of cellular transformation by Ras oncogenes, although the mechanisms by which Rac contributes to the transformation process are unknown (3). Regulation of nuclear signaling by Rho family GTPases has recently been described (4), possibly through their stimulatory effects on c-Jun amino-terminal kinase (JNK) 1 (5,6).
JNKs or stress-activated protein kinases represent a second class of the mammalian mitogen-activated protein (MAP) kinases, which includes the "classical" extracellular signal-regulated kinases (ERK) (7,8). An additional class, which presents substantial similarity to the Saccharomyces cerevisiae HOG1 kinase involved in responses to increased extracellular osmolarity (reviewed by Herskowitz (9)), is p38 MAP kinase. Like HOG1, p38 can be activated by changes in osmolarity but also appears to participate in the inflammatory response to lipopolysaccharides or to inflammatory mediators such as interleukin-1 (IL-1) or tumor necrosis factor (10 -12). The mechanisms by which p38 activation occurs in response to external stimuli remain to be determined. Induction of p38 activity by IL-1 or tumor necrosis factor ␣ has little effect on ERK activity, suggesting upstream signaling via Ras does not play an important role in p38 activation.
In their active GTP-bound forms, both Rac and Cdc42 bind to and stimulate the activity of a group of 65-68-kDa Ser/Thr kinases in mammalian cells (13)(14)(15). These p21-activated kinases (Paks) are homologous to the yeast Ste20 kinase involved in regulating yeast MAP kinase cascades controlling the mating pheromone response pathway, invasive growth of haploid yeast, and pseudohyphal differentiation in diploid yeast (9). As in the yeast mating factor pathway, we have recently established that Pak activity can be regulated by mammalian G protein-coupled receptors through a pertussis toxin-sensitive G protein (15). In the present communication, we show that Pak and its upstream regulators, Rac and Cdc42, couple to and regulate the activity of p38 MAP kinase and are an integral part of the signaling pathway linking cell surface proinflammatory receptors to p38 activation.
Transient Cell Expression-COS-7 and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% bovine serum. Cells on 35-mm plates were transiently transfected with 1 g of each plasmid DNA (see below) using Lipofectamine reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. Transfection efficiency was evaluated using a luciferase co-transfection assay (Promega). After 48 h, the cells were treated with or without UV radiation or IL-1 as described (12,20). Cells were solubilized with lysis buffer (25 mM Hepes, pH 7.6, 3 mM ␤-glycerophosphate, 3 mM EDTA, 3 mM EGTA, 250 mM NaCl, 1% Nonidet P-40, 1 mM dithiothreitol, 0.1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 0.078 trypsin inhibitory units/ml aprotinin) for 30 min with shaking at 4°C and then cleared by centrifugation at 100,000 ϫ g for 30 min at 4°C prior to immunoprecipitation and kinase assay. Expression levels of cDNA constructs after transient transfection were verified using the respective epitope tag antibodies.

RESULTS AND DISCUSSION
The proinflammatory cytokine IL-1 is a physiological regulator of p38 (12), causing a marked and rapid stimulation of p38 activity in HeLa cells (Fig. 1). We observed that IL-1 also stimulated Pak1, with Pak1 activation slightly preceding that of p38 (Fig. 1). Since Rac and Cdc42 are known regulators of Pak1 (13-15), we speculated that IL-1 might be linked to Pak1 activation through these GTPases and that this pathway might be involved in regulation of p38. In support of this hypothesis, we observed that expression of dominant negative forms of both Rac and Cdc42 effectively inhibited the ability of IL-1 to stimulate p38 activity (Fig. 2). Inhibition was directly dependent upon the amount of the dominant negative plasmid used.
While dominant negative forms of Rac and Cdc42 inhibited p38 activation by IL-1, we wanted to determine whether Rac and Cdc42 were sufficient to stimulate p38 activity. Co-expression of active Rac or Cdc42 with p38 in COS cells caused a large enhancement of p38 activity, comparable with that seen with stimulation by UV radiation, which maximally activates the enzyme and which serves an indicator of the total levels of p38 expressed and present in the immune precipitates (Fig. 3, A  and B). Expression of wild type Rac had only a slight effect on p38 activity (data not shown). This effect was specific for the GTPases Rac and Cdc42, as we failed to observe stimulation when activated forms of H-Ras, Raf, or RhoA were co-transfected with p38 (Fig. 3C).
The role of Pak in the p38 activation process was also assessed. Co-expression of wild type Pak1 itself with p38 caused a marked increase in p38 activity (Fig. 3, A and B). Pak1 appears to become activated when expressed in a COS cell environment, possibly due to the presence of low levels of active GTP-bound Cdc42. 2 However, when we co-expressed Pak1 with constitutively GTP-bound Rac or Cdc42, we observed a greater increase in p38 activity, indicating that the action of Pak could be enhanced by these known activators of the enzyme's catalytic function.
Taking into account the ability of IL-1 to stimulate Pak1 activity with a similar time course as that for p38 activation, the ability of a dominant negative Pak1 to block p38 activation by IL-1, and the ability of Pak1 itself to stimulate p38 activity, we conclude that the activity of Pak1, regulated by the upstream GTPases Rac and/or Cdc42, is an integral component of the signaling process linking cytokine receptors to p38 activation. The regulatory effects of Pak1 are not limited to the p38 pathway. The JNKs form an additional branch of the mammalian MAP kinase family, which are regulated by many of the same upstream stimuli as p38 (7)(8)(9). In addition to the recently reported ability of Rac and Cdc42 to stimulate JNK activity (5, 6), we observed that Pak1 could activate JNK activity as well (data not shown). In contrast, we could detect no stimulatory effect of activated Rac, Cdc42, or Pak1 on the ERK branch of the MAP kinase family; the latter are responsive to upstream regulators quite distinct from the "stress-activated" MAP kinases (7,8). Since we have shown that Pak(s) can be activated by mammalian G-protein-coupled receptors (15) and growth factor receptors, 5 it is likely that signaling through Pak contributes to the activation of stress-activated MAP kinases by such stimuli as well (25). Based on these data, we suggest a pathway, depicted in Fig. 4, through which a variety of up-stream signaling molecules can stimulate activity of the p38 and JNK kinases. Activation of Rac and/or Cdc42 by upstream signals leads to increased activity of Pak kinase(s). Pak does not directly phosphorylate p38 or JNK1, and both p38 and JNK are known to require phosphorylation of both Thr and Tyr residues for activation to occur (12). This dual phosphorylation is mediated by the action of upstream MAP kinase kinases, which are in turn controlled by MAP kinase kinase kinases in a typical MAP kinase regulatory cascade (21)(22)(23). We therefore suggest it is likely that, by analogy with the Ste20 kinase cascade in S. cerevisiae, Paks regulate the activity of MAP kinase kinase kinases, which act in turn on MAP kinase kinases to directly phosphorylate and regulate p38 and JNK. Potentially, Paks may serve to coordinate stress responses at the transcriptional level with morphological and cytoskeletal changes that occur concomitantly. Thus, regulation of Rac and Cdc42 function may be an important component of the mammalian response to shock and other inflammatory disorders.