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• W2079712059 abstract "Article15 August 1997free access MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42 Gary R. Fanger Gary R. Fanger Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO, 80206 USA Search for more papers by this author Nancy Lassignal Johnson Nancy Lassignal Johnson Program in Molecular Signal Transduction, National Jewish Medical and Research Center, Denver, CO, 80206 USA Search for more papers by this author Gary L. Johnson Corresponding Author Gary L. Johnson Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO, 80206 USA Department of Pharmacology, University of Colorado Medical School, Denver, CO, 80262 USA Search for more papers by this author Gary R. Fanger Gary R. Fanger Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO, 80206 USA Search for more papers by this author Nancy Lassignal Johnson Nancy Lassignal Johnson Program in Molecular Signal Transduction, National Jewish Medical and Research Center, Denver, CO, 80206 USA Search for more papers by this author Gary L. Johnson Corresponding Author Gary L. Johnson Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO, 80206 USA Department of Pharmacology, University of Colorado Medical School, Denver, CO, 80262 USA Search for more papers by this author Author Information Gary R. Fanger2, Nancy Lassignal Johnson1 and Gary L. Johnson 2,3 1Program in Molecular Signal Transduction, National Jewish Medical and Research Center, Denver, CO, 80206 USA 2Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO, 80206 USA 3Department of Pharmacology, University of Colorado Medical School, Denver, CO, 80262 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4961-4972https://doi.org/10.1093/emboj/16.16.4961 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1–4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli. Introduction The mitogen-activated protein kinase (MAPK) family of serine/threonine kinases is composed of the extracellular regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs) and p38 kinases (for review, see Kyriakis and Avruch, 1996). The MAPKs are components of sequential kinase cascades which are phosphorylated and activated by an intermediate MAPK kinase in the pathway; MAPK kinases are themselves phosphorylated and activated by MAPK kinase kinases (for reviews, see Seger and Krebs, 1995; Denhardt, 1996). The MAPK kinase kinases include Raf which regulates the ERK pathway (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992) and a group of MEK (MAP/ERK kinase) kinases (MEKKs) which, although they have the ability to activate ERK (Lange-Carter et al., 1993; Blank et al., 1996), appear to preferentially regulate the JNK pathway (Kyriakis et al., 1994; Minden et al., 1994; Yan et al., 1994). In addition to Raf and MEKKs, other kinases have been proposed to function as MAPK kinase kinases including germinal center kinase (GCK), p21-activated kinase (PAK), TGF-β-activated kinase (TAK), tumor progression locus-2 (Tpl-2), mixed lineage kinase-3 (MLK-3), mitogen-activated protein kinase kinase kinase 5 (MAPKKK5) and apoptosis signal regulating kinase 1 (ASK1) (Bagrodia et al., 1995; Pombo et al., 1995; Yamaguchi et al., 1995; Brown et al., 1996; Rana et al., 1996; Salmeron et al., 1996; Teramoto et al., 1996a; Wang et al., 1996; Ichijo et al., 1997). Thus, there are potentially many different kinases where biochemical and limited genetic evidence suggests these kinases could function as MAPK kinase kinases. Why so many kinases might function as MAPK kinase kinases is unclear; an obvious explanation would be that these kinases allow input into the MAPK pathways from many diverse stimuli (for review, see Fanger et al., 1997). Possibly the best example of multiple cellular stimuli feeding into the MAPK pathways is illustrated by the JNK pathway. In many cell types, growth factors such as EGF and cytokines including IL-1 and TNFα activate the JNK pathway (Coso et al., 1995). Also, stress stimuli including heat shock, DNA damaging agents like cisplatin and irradiation can activate the JNK pathway (Xu et al., 1996; for review, see Kyriakis and Avruch, 1996). Certainly, agents like cisplatin and growth factors such as EGF have dramatically different mechanisms of action and different initial cellular signaling responses leading to JNK activation. We have cloned four MEKKs, referred to as MEKK1, 2, 3 and 4 based on the order in which they were cloned (Lange-Carter et al., 1993; Blank et al., 1996; Gerwins et al., 1997). Each functions as a MAPK kinase kinase capable of activating the JNK pathway. MEKK1, 2 and 3 are also capable of activating the ERK pathway to varying degrees in transfection experiments, whereas MEKK4 has little ability to activate the ERKs. MEKK1–4 do not appear to be involved in the regulation of the p38 pathway. The deduced primary sequence of MEKK1, 2, 3 and 4 predicts specific properties for each (for review, see Fanger et al., 1997). The kinase domain of each MEKK is encoded at the COOH-terminal end, with unique features in the NH2-terminal regulatory moiety. MEKK1 is a 196 kDa protein having a proline-rich motif near its NH2-terminus, one or two predicted pleckstrin homology domains and a modest cysteine-rich domain. The MEKK1 kinase domain region and a small sequence just upstream of the kinase domain have been shown to bind Ras in a GTP-dependent manner (Russell et al., 1995). MEKK2 and 3 are smaller kinases being ∼70 kDa with no demonstrable regulatory domains except possible bipartite nuclear localization signals in their NH2-terminus (Blank et al., 1996). MEKK4 is a 180 kDa protein which is similar to MEKK1 and has a proline-rich motif near its NH2-terminus, a predicted pleckstrin homology domain and a modified Cdc42/Rac interactive binding (CRIB) domain adjacent to its kinase domain. The kinase domains of MEKK2 and 3 are 96% conserved at the amino acid level indicating they are extremely homologous in their catalytic regions, whereas their NH2-terminal moieties are quite different. Relative to MEKK2 and 3 the kinase domains of MEKK1 and 4 are ∼55% conserved in amino acid sequence and similarly conserved towards each other. As with MEKK2 and 3, the NH2-terminal domains of MEKK1 and 4 are also dramatically different. Therefore the sequences of MEKK1, 2, 3 and 4 indicate their regulation may be quite different even though they all appear capable of activating the JNK pathway. In this report, we demonstrate that the MEKK family members have distinct intracellular distributions. In addition, we show activation of MEKK family members by epidermal growth factor and utilizing kinase-inactive mutants more clearly define the role of each MEKK, and the specificity inherent to each kinase, in mediating the effects of this growth factor on MAPK family members. Furthermore, evidence that the MEKK family members differentially interact with GTP-binding proteins, and are not required for JNK activation by PAK, provide a better mechanistic understanding of the sequence of events involved in regulation of not only the MEKKs, but also the MAPK family members. These findings define the MEKK proteins as localized sensors in the cell for regulation of MAPK pathways. Results Subcellular localization of MEKKs Figure 1 shows immunoblots for type-specific MEKK antibodies using lysates from control and MEKK1, 2, 3 and 4 transfected COS cells. Endogenous MEKK1 is 196 kDa and, due to different phosphorylation states, the recombinantly expressed MEKK1 migrates as a triplet at about this size. MEKK1 also has a 90 kDa proteolytic cleavage fragment (C.Widmann and G.Johnson, submitted for publication) which due to low abundance is difficult to observe, but is present in untransfected lysates and readily apparent when expressed by transfection. Recombinant MEKK4 migrates as a slightly larger protein than the endogenous 180 kDa MEKK4 protein because of its hyperphosphorylated state and its epitope-tag. Endogenous MEKK2 and 3 are difficult to observe in immunoblots because of their low abundance. Transfected MEKK2 and 3 migrate as bands larger than their calculated size due in part to their phosphorylation, which also generates multiple gel-shifted bands. The antibodies for MEKK1 and 2 were immunoprecipitating, whereas those for MEKK3 and 4 were not. The antibody for MEKK3 did not stain cells using indirect immunofluorescence procedures that were successful with the antibodies raised against epitopes for MEKK1, 2 and 4. Figure 1.Characterization of type-specific MEKK antibodies used for immunofluorescence. Peptides corresponding to unique sequence within each MEKK family member were generated and used to derive MEKK type-specific antibodies (see Materials and methods). Western blot analysis was performed on lysates from COS cells transfected with either empty mammalian expression vector (pCMV5) or pCMV5 vector expressing the full-length forms of (A) MEKK1, (B) MEKK4, (C) MEKK2 or (D) MEKK3. Relative sizes are indicated by molecular weight markers. Download figure Download PowerPoint Figure 2 shows the subcellular localization of MEKK1, 2 and 4 that was determined using confocal immunofluorescence microscopy. Analysis was performed by co-staining with either wheat germ agglutinin (WGA) to identify plasma membrane and Golgi or anti-adaptin (AP-1) antibody, a marker specific for the Golgi adaptor complex AP-1 (Ahle et al., 1988). For reference, J, K and L show the profiles of Golgi, mitochondria and the endoplasmic reticulum, respectively. Figure 2A and B demonstrate that MEKK1 is found in the nucleus and dispersed in punctate vesicular-like structures in the cytoplasm. Figure 2D and E show that MEKK2 localization is different from that of MEKK1 in that it is found predominantly in the Golgi, as well as a distinct cytoplasmic punctate pattern. Similar to MEKK2, but in contrast to MEKK1, MEKK4 appears strictly Golgi-associated (Figure 2G and H). Using AP-1 as a marker for clathrin-coated membranes of the trans-Golgi network and brefeldin A (BFA) to induce rapid redistribution of coat proteins associated with clathrin-coated vesicles from the trans-Golgi (Orci et al., 1991), Figure 2B/C, E/F and H/I demonstrate that MEKK2 and 4, but not MEKK1, are associated with structures that lead to their redistribution in response to BFA. These findings demonstrate that MEKK2 and 4 co-localize with Golgi-associated structures. However, a component of MEKK2 is also found localized in BFA resistant structures in the cytoplasm. In no instance did MEKK1, 2 or 4 appear associated with the mitochondria or endoplasmic reticulum. These findings were verified using additional antibodies recognizing independent epitopes of MEKK1 and 2 and a green fluorescent protein–MEKK4 fusion as described in the Materials and methods. Thus, MEKK1, 2 and 4 are found in unique subcellular locations in the cell with MEKK2 and 4 overlapping with BFA sensitive coat proteins in the trans-Golgi. The differential distribution of the MEKK proteins suggests selective roles for each in responses involving MAPK pathways. Figure 2.Subcellular localization of MEKK family members. COS cells were plated onto glass coverslips and, using type-specific MEKK antibodies, digital confocal immunofluorescence was utilized to determine the subcellular localization of the MEKK family members. In (A), (D) and (G), cells were stained with a combination of fluorescein conjugated WGA (green) to better visualize cell membrane (WGA also stains the Golgi complex in COS cells), as well as with type-specific antibodies directed against either (A) MEKK1, (D) MEKK2 or (G) MEKK4 (red). In (B), (E) and (H), cells were stained with a combination of an anti-adaptin (AP-1) antibody that identifies the Golgi complex by recognizing the Golgi adaptor complex AP-1 (green), as well as with antibodies specific for (B) MEKK1, (E) MEKK2 and (H) MEKK4 (red). Cells were treated with brefeldin A (BFA) to induce redistribution of coat proteins associated with clathrin-coated vesicles from the trans-Golgi and stained with anti-AP-1 (green) in combination with antibodies specific for (C) MEKK1, (F) MEKK2 and (I) MEKK4. In order to better identify subcellular structures (J) the Golgi complex was identified with BODIPY-ceramide, (K) the mitochondria was identified with rhodamine 123 and (L) the endoplasmic reticulum (ER) (denoted by arrow) was identified with DIOC6(3). Download figure Download PowerPoint MEKK1 and 2 are activated by EGF It has been particularly difficult to demonstrate the regulation of MEKKs in response to extracellular stimuli primarily because transiently expressed MEKKs are constitutively active and immunoprecipitation of endogenous MEKKs results in high basal activity. The high basal activity of MEKKs when recombinantly expressed and/or immunoprecipitated is similar to STE11p in Saccharomyces cerevisiae, a MAPK kinase kinase component of the pheromone response pathway (Neiman and Herskowitz, 1994). To overcome this problem we used dilutions of MEKK1 and 2 immunoprecipitations from control and EGF stimulated COS cells in an in vitro sequential protein kinase assay (Gardner et al., 1994; Blank et al., 1996). In this assay, bacterially expressed JNK kinase and JNK are added to the kinase reaction and the MEKK activation of the pathway is assayed by monitoring JNK phosphorylation of GST–c-Jun (Figure 3). In three independent experiments imager analysis of the phosphorylated substrate demonstrated EGF stimulated the activity of MEKK1 an average of 2.1-fold and MEKK2 an average of 3.9-fold. Figure 3.Regulation of MEKK1 and 2 by EGF. Representative example of at least three complex kinase assays where either endogenous MEKK1 or 2 was immunoprecipitated following stimulation with EGF. Where indicated cells were pretreated with 100 nM wortmannin (W) prior to EGF (E) stimulation. c-Jun phosphorylation was measured following addition of recombinant wild type JNKK and JNK. Samples were separated by SDS–PAGE and visualized with autoradiography. PhosphorImager analysis was utilized to determine the relative changes in MEKK1 and 2 activity. Download figure Download PowerPoint In mast cells we have also been able to demonstrate MEKK1 activation in response to crosslinking of the FcϵRI receptor (Ishizuka et al., 1996). MEKK1 and JNK activation in response to FcϵRI ligation is inhibited by wortmannin, suggesting an involvement of phosphatidylinositol 3-kinase (PI3-K) in the regulation of mast cell MEKK1. Even though EGF elicited a 4.2-fold increase in PI3-K activity which was inhibited by wortmannin (data not shown), in contrast to the results with the FcϵRI receptor, wortmannin did not inhibit either MEKK1 or 2 activation in response to EGF in COS cells (Figure 3). Thus, in COS cells, MEKK1 and 2 are activated by stimulation of the EGF receptor independent of PI3-K activation. MEKK regulation of JNK and ERK activation in response to EGF In order to define the role of MEKK1, 2, 3 and 4 in mediating activation of pathways associated with MAPK family members, JNK and ERK activation was measured in response to expression of activated MEKK family members or EGF stimulation in the presence of wortmannin or kinase-inactive mutants of each MEKK family member. Expression of MEKK1, 2, 3 and 4, where the NH2-terminal regulatory domains have been deleted (ΔMEKK1–4), results in activation of JNK (Figure 4A) to similar (∼5-fold) levels and ERK (Figure 4B) to varying degrees. Although sorbitol elicited a 3.5-fold activation, none of the ΔMEKKs were able to stimulate p38 activation (data not shown). In COS cells EGF is also capable of activating both JNK and ERK (Figure 4C and D, respectively) which, similar to the activation of MEKK1 and 2, is insensitive to the effects of wortmannin (compare Figure 3 with 4C and D) indicating PI3-K is not involved. As illustrated in Figure 4E, only expression of a kinase-inactive inhibitory mutant of MEKK1 and not the kinase-inactive inhibitory mutants of MEKK2, 3 or 4 inhibit ERK activation by EGF in COS cells. The inhibition of ERK activation by kinase-inactive MEKK1 was >80%. Immunoblotting of the epitope-tagged MEKKs indicated they were expressed at similar levels (not shown), demonstrating the selectivity of kinase-inactive MEKK1 in inhibiting ERK activation. The inhibitory effect of kinase-inactive MEKK1 on activation of the ERK pathway is most likely related to the interaction of MEKK1 with Ras (Russell et al., 1995). Finally, kinase-inactive mutants of each MEKK expressed in COS cells only modestly inhibited EGF-mediated JNK activation, suggesting that multiple MEKKs and possibly additional kinases are involved in regulating the JNK pathways in response to EGF (data not shown). Figure 4.Analysis of JNK and ERK pathways in COS cells. (A) Representative example of three experiments where endogenous JNK was precipitated from cell lysates with GST–c-Jun conjugated to Sepharose beads following transfection of either empty expression vector (−) or NH2-terminally truncated forms of specific MEKK family members (ΔMEKK1–4). Activity was measured in a kinase reaction following the addition of [γ-32P]ATP. Samples were separated by SDS–PAGE and the relative JNK activity was evaluated using PhosphorImager analysis. (B) Representative MAPK assay illustrating results from three separate experiments where either empty expression vector (−) or NH2-terminally truncated MEKK family members (ΔMEKK1–4) were expressed and endogenous ERK activity was immunoprecipitated with an anti ERK2 antibody. Relative changes in ERK activity were assayed by [γ-32P]ATP phosphorylation of substrate peptide (EGFR662–681) and determined by scintillation counting. Average relative changes in JNK (C) and ERK2 (D) activity [measured as described in (A) and (B), respectively] following EGF (E) stimulation either with or without 100 nM wortmannin (W) pretreatment. Average of three separate experiments are shown. (E) Average EGF-mediated activation of MAPK activity following expression of kinase-inactive forms of the MEKK family members (MEKK1–4KM) and HA–ERK2. Empty expression vector (pCMV5) was included as necessary to maintain equal DNA concentrations during transfection. Averages represent four separate experiments. Error bars represent the standard deviation. Download figure Download PowerPoint Kinase-inactive MEKK1 and 4 block Rac and Cdc42 activation of the JNK pathway Constitutively activated mutants of Cdc42 and Rac (Cdc42QL and RacQL) stimulate JNK activity, whereas inhibitory mutants of Cdc42 and Rac having high affinity for GDP (N17Cdc42 and N17Rac) inhibit JNK activation by EGF, indicating that these low molecular weight GTP-binding proteins are critical mediators of the effects of growth factors on JNK activation (Coso et al., 1995; for review, see Vojtek and Cooper, 1995). Figure 5 demonstrates that kinase-inactive mutants of MEKK1 and 4 block Rac and Cdc42 stimulation of the JNK pathway. In contrast, kinase-inactive mutants of MEKK2 and 3 do not affect Rac and Cdc42 stimulation of the JNK pathway. Figure 5A shows a representative experiment, whereas Figure 5B demonstrates that equal amounts of HA-tagged JNK were immunoprecipitated from each sample. Figure 5C and D show the average of four to six experiments for Cdc42 and Rac, demonstrating, respectively, the regulation of the JNK pathway and selective inhibition by kinase-inactive MEKK1 and 4. These findings strongly implicate both MEKK1 and 4 in Rac/Cdc42 regulated signal transduction pathways. Figure 5.MEKK1 and 4, but not MEKK2 and 3 contribute to JNK activation by Cdc42 and Rac. COS cells were transfected with activated forms of either Cdc42Q61L or RacQ61L along with kinase-inactive forms of each MEKK family member (MEKK1–4KM). Empty expression vector (pCMV5) was included as necessary to maintain equal DNA concentrations during transfection. JNK activity was measured following immunoprecipitation of HA-JNK. (A) Autoradiograph of a representative JNK assay. (B) Western blot of a portion of the HA–JNK immunoprecipitation demonstrating the equivalent levels of HA–JNK utilized in the assay. Average fold inductions of JNK activation from 4–6 separate experiments following stimulation with either (C) Cdc42 or (D) Rac. Error bars represent the standard deviation. Download figure Download PowerPoint MEKK1 and 4 associate with Rac1 and Cdc42 Recombinant GST fusion proteins for Rac, Cdc42 and Rho were purified from bacteria and loaded with either GDP or GTP. The recombinant proteins were combined with COS cell lysates expressing truncated forms of the MEKK proteins that encode the COOH-terminal moiety including the kinase domain to determine if the MEKK proteins interacted with the low molecular weight GTP-binding proteins. MEKK1 associated with the GTP-bound Rac and Cdc42, but not when they were loaded with GDP (Figure 6A). MEKK1 did not associate with Rho in either a GTP or GDP bound state. MEKK2 and 3 did not associate with Cdc42, Rac or Rho in either nucleotide bound state (Figure 6B and C, respectively). The full-length forms of MEKK2 and 3 were also unable to associate with Rac, Cdc42 or Rho (data not shown) providing additional evidence that these two MEKKs do not bind to the Rho family of GTP-binding proteins. Although MEKK4 bound to all three GTP-binding proteins in both the GTP and GDP bound forms, MEKK4 did not bind to GST alone (Figure 6D) or to several other GST fusion proteins (data not shown). Figure 6.MEKK1 and 4, but not MEKK2 or 3 associate with Rac and Cdc42. COS cells were transfected with mammalian expression plasmids expressing COOH-terminal kinase domains encoding either MEKK1, 2, 3 or 4. In (A), (B), (C) and (D) lysates were incubated with 10 μg of bacterially expressed GST fusions of either Cdc42, Rac, or Rho loaded with either GDP or GTPγS and conjugated to Sepharose beads. GST alone [GST(−)] was also included as a negative control for nonspecific binding. In (E) and (F), COS cell lysates recombinantly expressing either MEKK1 or 4 were incubated with 10 μg of Sf9 expressed polyhistidine (pHIS) tagged Cdc42 or Rho loaded with either GDP or GTPγS and conjugated to Sepharose beads. NTA-Ni Sepharose beads preincubated with Sf9 lysate (−) were included as a negative control for nonspecific binding. After washing, precipitates were separated by SDS–PAGE, transferred to nitrocellulose and Western blotted with type-specific antibodies for (A) and (E) MEKK1, (B) MEKK2, (C) MEKK3, and (D) and (F) MEKK4. Download figure Download PowerPoint To validate the findings with bacterially expressed GST fusions we prepared recombinant polyhistidine fusions of Cdc42 and Rho in Sf9 cells using baculovirus infection. Similar results were obtained where MEKK1 bound to Cdc42 in a GTP dependent manner and did not bind to Rho (Figure 6E). In contrast to MEKK1, MEKK4 bound to Cdc42 and Rho in both the GTP and GDP states (Figure 6F). These findings highlight the differential binding characteristics of MEKK1 and 4 to low molecular weight GTP-binding proteins and predict different potential regulatory functions for Cdc42/Rac in the control of MEKK1 and 4. MEKK1 and 4 bind directly to Cdc42 and Rac To determine if the interaction of MEKK1 and 4 with Rac and Cdc42 was direct, MEKK family members were expressed as maltose binding protein (MBP) fusions in bacteria and purified. Recombinant Rac and Cdc42 were loaded with [γ-32P]GTPγS and incubated with either MEKK1, 3 or 4 bound to amylose beads, washed, and the relative binding was determined for each MEKK–bead preparation. Rac and Cdc42 bound to MEKK1 and 4, but not to either MEKK3 or protein that encodes only MBP (Figure 7), confirming the findings in Figure 6 that MEKK1 and 4 interact with Rac and Cdc42. Interestingly, the ability of MEKK4 to bind Rho [γ-32P]GTPγS was low relative to its association with Cdc42 and Rac (not shown). The reason for this is not apparent, because the specific activity of the Rho [γ-32P]GTPγS was similar to that for Rac and Cdc42. This suggests that the interaction of Rho with MEKK4 (see Figure 6) may involve another protein and, although this binding event may be indirect, it contrasts to the direct binding of MEKK1 and 4 to Cdc42 and Rac. Figure 7.MEKK1 and 4 bind directly to Rac and Cdc42. 1 μg of GST–Cdc42 or GST–Rac was loaded with [γ-32P]GTPγS and incubated with 10 μg of bacterially expressed MBP fusions of MEKK1, 3 and 4 conjugated to amylose resin. MBP alone [MBP(−)] was used as a negative control for nonspecific binding. After washing, reactions were counted with a scintillation counter and plotted relative to control levels. Values represent at least three separate experiments. Error bars represent the standard deviation. Download figure Download PowerPoint Inhibitory mutant MEKKs do not block PAK stimulation of JNK activity The PAKs bind Cdc42 and Rac in a GTP-dependent manner (Manser et al., 1994) and mediate specific responses involving Cdc42/Rac (Zhang et al., 1995; Westwick et al., 1997). The homology between the PAKs and STE20p (Manser et al., 1994), which mediates specific responses involving Cdc42p in S.cerevisiae (Leberer et al., 1997), has prompted the hypothesis that PAKs are intermediate in the regulatory pathway Cdc42/Rac→PAK→ MEKK (Minden et al., 1995; for review, see Vojtek and Cooper, 1995). However, the association of MEKK1 and 4 with Rac and Cdc42 argues that a more likely scenario is a pathway where PAK and MEKK1/4 associate independently with Cdc42 and Rac and are parallel to one another in component signaling pathways. Consistent with previously published results (Manser et al., 1994), Figure 8A shows that recombinant PAK associates with Cdc42 and Rac in the GTP-bound form and this interaction stimulates the kinase activity of PAK (Figure 8B). Whereas kinase-inactive inhibitory mutants of MEKK1 and 4 inhibit Cdc42 and Rac activation of the JNK pathway (Figure 5), these inhibitory mutants do not block PAK stimulation of the JNK pathway. Kinase-inactive mutants of MEKK2 and 3 also did not inhibit PAK stimulation of the JNK activity (Figure 8C). In addition, we have been unable to measure any detectable phosphorylation of catalytically inactive mutants of MEKK1–4 by PAK under conditions where PAK efficiently autophosphorylated and phosphorylated specific substrates (data not shown). Therefore, these results indicate that PAK does not lie directly upstream of MEKK1, 2, 3 or 4 in a pathway leading to JNK activation. Rather, the data supports the prediction of two independent pathways involving PAKs and MEKK1/4 that regulate JNK activity. Figure 8.MEKKs do not lie downstream and mediate the effects of PAK. (A) Western blot of haemagglutinin tagged PAK. COS cells were transfected with a plasmid expressing the full-length rat βPAK gene and lysates were incubated with 20 μg of GST fusions of either Cdc42, Rac, or Rho loaded with either GDP or GTPγS and conjugated to Sepharose beads. GST alone [GST(−)] was also included as a negative control for nonspecific binding. After washing, precipitates were separated by SDS–PAGE, transferred to nitrocellulose and Western blot analysis was performed with an anti-HA antibody. (B) Kinase assay of PAK incubated in either the presence or absence of GTPγS-loaded Cdc42Q61L. Myelin basic protein was included in the reaction as a substrate. (C) Relative levels of JNK activation following transfection of the wild type PAK with kinase-inactive forms of the MEKK family members (MEKK1–4KM). pCMV5 was included to maintain equal total levels of transfected plasmid. Western blot analysis of a portion of the immunoprecipitation indicated that equivalent levels of HA–JNK were immunoprecipitated in each sample. Averages represent five separate experiments. Error bars indicate the standard deviation. Download figure Download PowerPoint Discussion In this study, we have better defined the mechanisms controlling regulation of the MEKK family members (MEKK1–4), as well as their role as growth factor-" @default.
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• W2079712059 title "MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42" @default.
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