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The Small GTP-Binding Proteins Racl, and Cdc42 Regulate the Activity of the JNK/SAPK Signaling Pathway

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The Small GTP-Binding Proteins Racl, and Cdc42 Regulate the Activity of the JNK/SAPK Signaling Pathway View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 81, 1137-1146, June 30, 1995, Copyright © 1995 by Cell Press The Small GTP-Binding Proteins Racl, and Cdc42 Regulate the Activity of the JNK/SAPK Signaling Pathway Omar A. Coso,* Mario Chiariello,* Jin-Chen Yu,t closely related to MAPKs have been identified. One class Hidemi Teramoto,* Piero Crespo,* Ningzhi Xu,* presents extended similarity to the Saccharomyces cere- Toru Miki,t and J. Silvio Gutkind* visiae HOG1 kinase (Han et al., 1994), which is involved *Molecular Signaling Unit in protecting S. cerevisiae from hyperosmotic solutions Laboratory of Cellular Development and Oncology (reviewed by Herskowitz, 1995). The role of this mamma- National Institute of Dental Research lian HOG1 homolog is largely unknown. Although it can tLaboratory of Cellular and Molecular Biology also be activated by changes in osmolarity, it appears to National Cancer Institute participate in the inflammatory response to lipopolysac- National Institutes of Health charides or to inflammatory mediators such as interleu- Bethesda, Maryland 20892 kin-1 (IL-1) (Han et al., 1994; Freshney et al., 1994). The other class of MAPKs represents a family of closely related enzymes activated by cellular stress, which have been Summary named stress-activated protein kinases (SAPKs) (Kyriakis et al., 1994). SAPKs were independently identified by vir- c-Jun amino-terminal kinases (JNKs) and mitogen- tue of their ability to phosphorylate the amino terminus of activated protein kinases (MAPKs) are closely related; the c-Jun transcription factor; hence, they have been also however, they are independently regulated by a variety termed c-Jun amino-terminal kinases (JNKs) (D~rijard et of environmental stimuli. Although molecules linking al., 1994). JNKs can be potently activated by inhibitors of growth factor receptors to MAPKs have been recently protein synthesis such as cycloheximide and anisomycin, identified, little is known about pathways controlling inflammatory cytokines such as tumor necrosis factor JNK activation. Here, we show that in COS-7 cells, acti- (TNFa) and IL-1, changes in osmolarity, heat shock, and vated Ras effectively stimulates MAPK but poorly in- ultraviolet irradiation (D6rijard et al., 1994; Kyriakis et al., duces JNK activity. In contrast, mutationally activated 1994). JNKs are thought to be responsible for phosphory- Racl and Cdc42 GTPases potently activate JNK without lating the transactivating domain of c-Jun protein in vivo affecting MAPK, and oncogenic guanine nucleotide (D~rijard et al., 1994; Kyriakis et al., 1994), and, in turn, exchange factors for these Rho-Iike proteins selec- phosphorylated c-Jun homodimers have potent AP-1 ac- tively stimulate JNK activity. Furthermore, expression tivity and can control the expression of a number of genes, of inhibitory molecules for Rho-related GTPases and including c-jun itself (Angel et al., 1988). dominant negative mutants of Racl and Cdc42 block The mechanism of activation of JNKs has just begun JNK activation by oncogenic exchange factors or after tO be explored. Although initial observations suggested induction by inflammatory cytokines and growth fac- that these enzymes were located downstream of Ras (D6- tors. Taken together, these findings strongly support rijard et al., 1994), this hypothesis is in conflict with recently a critical role for Racl and Cdc42 in controlling the available data (Sun et al., 1994), including'the lack of acti- JNK signaling pathway. vation of JNK in certain cellular systems by agonists acting on receptors that are known to couple to the Ras-MAPK Introduction pathway (Kyriakis et al., 1994; Coso et al., 1995). Further- more, agonists such as TNFa and IL-1 potently induce Mitogen-activated protein kinases (MAPKs), also known JNK, but they activate MAPK poorly (Kyriakis et al., 1994). as extracellular signal-regulated kinases, are protein- Taken together, these observations suggest the existence serine/threonine kinases that are rapidly activated upon of distinct pathways leading to the activation of either stimulation of a variety of cell surface receptors (Ray and MAPK or JNK. In this study, we have used the expression Sturgill, 1987). Their function is to convert extracellular of hemagglutinin (HA) epitope-tagged JNK1 (HA-JNK) in stimuli to intracellular signals controlling the expression COS-7 cells to explore the mechanism of activation of J N K. of genes essential for many cellular processes, including We present evidence that the small GTP-binding proteins cell growth and differentiation (Marshall, 1995). These ki- Racl and Cdc42 are an integral part of a novel signaling nases play a central role in mitogenic signaling, as imped- pathway linking cell surface receptors to JNK activation. ing their function prevents cell proliferation in response to a number of growth-stimulating agents (Pages et al., Results 1993). Furthermore, aberrant functioning of proteins known to be upstream of MAPK can induce cells to acquire Functional Expression of an Epitope-Tagged JNK the transformed phenotype, and constitutive activation of The JNK1 cDNA was obtained by the polymerase chain the MAPK pathway is itself sufficient for tumorigenesis reaction (PCR)technique, its coding sequence was altered (Cowley et al., 1994; Mansour et al., 1994). Thus, MAPKs by the insertion of an amino-t~rminal tag of 9 amino acids appear to be a critical component of growth-promoting representing influenza HA1 protein (Wilson et al., 1984), pathways. and the cDNA was cloned in the expression vector pcDNA3. Recently, two novel classes of mammalian enzymes As shown in Figure 1 (top), this epitope-tagged JNK1 cDNA Cell 1138 MEK. Transfection with the wild-type form of MEK and treatment with anisomycin served as controls. As shown in Figure 2, activated forms of Ras, Raf, and MEK potently wa ..... induced MAPK activity, while anisomycin induced this ac- tivity to a lesser extent and the wild-type MEK lacked a demonstrable effect. In contrast, anisomycin potently in- duced JNK activity, v-ras induced a modest increase, and L lysate HA-IPj ~sate HA-IPj Raf and both MEK constructs failed to stimulate JNK. antibody JNK1 HA Thus, in COS-7 cells, v.ras can weakly signal JNK activa- tion, however, through a biochemical route different from that of the Raf-MEK-MAPK pathway. DNA - + anisomycin The Small GTP-Binding Proteins Racl and Cdc42 Can Potently Activate JNK JNK ~ ~ ~ GST-cjun (79) The recent observation that two members of the Rho fam- .... ~ ~'~ GST-ATF2 (96) ily of small GTP-binding proteins, Racl and Cdc42, can bind and activate certain intracellular kinases (Manser et Figure 1. Expression of an Epitope-Tagged JNK1 in COS-7 Cells al., 1994), a situation analogous to that of Ras acting on Lysates containing 40 l~g of protein from COS-7 cells transfected with Raf (see McCormick, 1994), prompted us to explore whether pcDNA3 vector (control) or with an expression plasmid carrying an this family of GTPases can initiate a pathway leading to epitope-tagged human JNK1 cDNA (HA-JNK) (1 ~g per plate) were JNK activation. We began by expressing wild-type and subjected to Western blot (WB) analysis with antibodies against HA activated forms of members of the Rho family of GTP- or JNK1, as indicated. Additional lysates containing 800 pg of cellular protein were first i mmunoprecipitated with the anti-HA antibody (HA-IP) binding proteins in COS--/cells. This family consists of and then subjected to the same analysis. Bands were visualized by ,.4he enhanced chemiluminescence technique using the appropriate horseradish peroxidase-conjugated goat antiserum. Arrows indicate the position of the epitope-tagged J NK1 protein. Parallel immunopre- A ~ B ~ cipitates from serum-starved cells untreated (minus) or exposed to anisomycin (10 l~g/ml; 20 min) (plus) were assayed for JNK activity Kinase Kinase (JNK) ~;~described in Experimental Procedures, using GST-c-Jun(79) or GST-ATF2(96) as substrates. Bands corresponding to the radiola- MBP ~ ~ GST-cjun(79)~ beled substrate are indicated. (HA-JNK) was efficiently expressed when transfected into COS-7 cells, as judged by its immunodetection with the 61 anti-HA murine monoclonal antibody 12CA5 (Wilson et al., >.~ J= m 1984) or with a JNKl-specific antiserum. Furthermore, only HA-JNK-transfected cells exhibited in vitro phosphor- ~:~ 4 ylating activity in an immunocomplex kinase assay, using purified glutathione S-transferase (GST)-c-Jun(79) or ~ 2 GST-ATF2(96) as substrates (Figure 1, bottom). Exposure of serum-starved cells to anisomycin, a potent JNK/SAPK 0 activator (Kyriakis et al., 1994), induced a striking increase WB WB in JNK activity (Figure 1, bottom). Thus, this epitope-tagged HA-JNK ~ ~ ~ ~ O ~" JNK1 encoded by the HA-JNK cDNA construct functions as a JNK. Figure 2. Effect of Activated Ras, Raf, and MEK on MAPK and JNK Activity Activation of JNK Involves a Signaling Route COS-7 cells were transfected with pcDNA3-HA-JNK1 (1 I~g per plate) or pcDNA3-HA-MAPK (1 pg per plate) for the MAPK (A) or JNK (B) Different from That of the assay, respectively, together with pcDNA3 vector (control) or expres- Ras-MAPK Pathway sion vectors carrying cDNAs for v-ras, a constitutively active form of Although a number of studies have demonstrated that the Raf (BXB-raf) (previously described by Crespo et al., 1994), MEK, or activity of c-Jun and JNK is elevated in cells transformed the mutationally activated MEK (MEKEE), as indicated (2 I~g per plate in each case). Treatment of cells with 10 i~g/ml anisomycin for 20 min by oncogenic ras genes (Angel and Karin, 1991; D~rijard was used as a control. Kinase reactions or Western blot (WB) analysis et al., 1994; Westwick et al., 1994), many growth factors were performed in anti-HA immunoprecipitates from the correspond- known to activate the Ras-MAPK pathway fail to elevate ing cellular lysates as described in Experimental Procedures.
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