Grb2 Interaction with MEK-Kinase 1 Is Involved in Regulation of Jun-Kinase Activities in Response to Epidermal Growth Factor*

Epidermal growth factor (EGF) receptor was shown to be involved in the activation pathway of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) cascade not only by EGF, but also by UV radiation or osmotic stress. This paper describes a specific interaction between the COOH-terminal SH3 domain of Grb2 and the NH2-terminal regulatory domain of MEKK1 in ER22 cells overexpressing the EGF receptor. This interaction results in the formation of a constitutive complex between Grb2 and MEKK1 in both proliferating and resting cells. EGF stimulation causes this complex to be rapidly and transiently recruited by Shc proteins. The subsequent release of the Grb2-MEKK1 complex from Shc proteins correlates with JNK activation. Transfection of the NH2-terminal regulatory domain of MEKK1 specifically inhibits EGF-dependent JNK activation indicating that Grb2 is involved in MEKK1 activation. Thus, adaptor proteins have a new role in the regulation of the SAPK/JNK cascade after EGF stimulation.

kinases (JNKs) (3,4), and p38/Hog1 MAP kinases (5,6). The activation of MAP kinases appears to be a key event in many cell responses to external stimuli. The JNK cascade is activated in response to a variety of cell stresses, including UV irradiation, ␥-rays, pro-inflammatory cytokines, ceramides, vasoactive peptides, protein synthesis inhibitors, heat shock, osmotic shock, and also in response to mitogens such as epidermal growth factor (EGF) and nerve growth factor. The JNK cascade leads to the activation by phosphorylation of a series of transcription factors including c-Jun, Elk-1, and ATF-2 (7). The stimulation of the JNKs (JNK1, JNK2, and JNK3) is associated with proliferation and differentiation but also with the arrest of cell growth and apoptosis. It was recently reported that stimulation of the JNKs mediates Ras transformation (8).
The involvement of small GTP-binding proteins, such as Ras proteins and the members of the Rho subfamily, in the activation of the JNK cascade is well documented in cell responses to stress or growth factors. EGF activates the JNK pathway (9), and phosphatidylinositol 3-kinase was recently reported to be a link between JNK signaling mediated by the Rho family and the Ras pathway upon EGF stimulation (10). Exposure to UV radiation or to osmotic shock was also found to induce clustering and internalization of EGF receptors and their activation by tyrosine autophosphorylation, with subsequent activation of the JNK cascade through Ras activation (11).
The JNKs are activated by dual phosphorylation on tyrosine and threonine residues by the JNK kinases (MKK4/SEK1), which are, in turn, activated by upstream kinases referred to as MEK kinases (MEKKs) (9,12). MEKKs are an expanding family of kinases (13). Mammalian MEKK1 cDNA encodes a 78-kDa protein, but several forms of MEKK1 have been found in various cell lines (50, 78, or 98 kDa) (14). Thereafter, a rat MEKK1 full-length cDNA was cloned, encoding a 195-kDa protein (15). The 98-kDa protein corresponds to the 625 COOHterminal amino acids of the full-length MEKK1 and might be produced by specific cleavage (16).
Few data describe the mechanism of transient activation of MEKK1 in response to growth factors. We have therefore studied the activation of MEKK1 in response to EGF in Chinese hamster lung fibroblasts (ER22 cells), in which the main immunoreactive form of MEKK1 is a 98-kDa protein. We found a constitutive Grb2-MEKK1 complex in the ER22 cells. In response to EGF stimulation, this complex is recruited by Shc proteins. The JNK activation occurs when the complex is released from Shc proteins.

EXPERIMENTAL PROCEDURES
Cell Culture and Transient Transfections-ER22 cells, derived from CCL39 cells by stable expression of the EGF receptor (17), were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS). Transient transfections were performed using Lipo-fectAMINE, and cells were harvested 48 h later.
cDNA Constructs and Recombinant Proteins-The mutant Grb2 Y7V cDNA was obtained from the Grb2 cDNA by PCR mutagenesis with the following primers: sense (5Ј-ATGGAAGCCATCGCCAAAGTCGACT-TCAAA-3Ј) and antisense (5Ј-AGATCTCACTATTAGGTGCAGCTC-3Ј). The Grb2⌬SH3C cDNA was obtained from the Grb2 cDNA by deletion of the region encoding the COOH-terminal domain from amino acid Q157. HA-tagged MEKK1 COOH(301-672) and VSV-tagged MEKK1  were a generous gift from Dr. J. Pouysségur (UMR134, CNRS Nice, France). MEKK1  was constructed from the MEKK1 (1-672) cDNA by PCR and subsequent sequencing. The Myc-tagged Grb2, Grb2, and Grb3-3 expression vectors have been described (18). GST-Crk and GST-* This work was supported in part by Grant 10000312-01 from La Fondation pour la Recherche Médicale, France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Transactivation Analysis-ER22 cells were transfected with the reporter plasmid Gal4-CAT, the activator plasmid encoding Gal4-cJun  , and pSV2 plasmid without a cDNA insert or with MEKK1  or MEKK1 (301-672) cDNAs. Cells were incubated for 24 h after transfection, deprived of FCS for an additional 24 h, and treated with or without 100 ng/ml EGF. CAT activity was measured using an InstantImager (Packard Instrument Corp.).
GST Fusion Protein Purification and Affinity Chromatography-GST fusion proteins (Grb2, Grb2Y7V, Grb2⌬SH3C, Grb3-3, Nck, Crk) were adsorbed onto glutathione-agarose beads equilibrated in buffer A containing 0.2% Triton X-100 and incubated with the cell lysates for 3 h at 4°C. Bound proteins were dissolved in Laemmli sample buffer and subjected to SDS-PAGE and Western blot analysis.

RESULTS AND DISCUSSION
Grb2 Binds to MEKK1 in Vitro and in Vivo-The MEKK1 (1-672) sequence (14) contains several proline-rich stretches in the NH 2 -terminal region that could interact with protein SH3 domains. We examined the interaction of MEKK1 with various GST-adaptor proteins (containing mainly SH2 and SH3 domains). Lysates from FCS-cultured ER22 cells were subjected to affinity chromatography on glutathione-agarose coupled to GST-Grb2, GST-Nck, or GST-Crk fusion proteins (Fig. 1A). Immobilized GST-Grb2 specifically retained the 98-kDa MEKK1, as revealed by Western blot analysis using antibodies directed against the COOH-terminal sequence of MEKK1. This molecular weight has already been reported for the MEKK1 in NIH or Swiss 3T3 fibroblasts (22). In contrast, the anti-MEKK1 antibodies detected no immunoreactive protein in eluates from GST-Nck and GST-Crk affinity chromatography columns. We also found a 78-kDa protein associated with Grb2 in primary cultures of rat astrocytes (results not shown). This corresponds to the molecular weight of MEKK1 in PC12 cells (14).
No immunoreactive protein was detected with antibodies against other MAP kinase kinase kinases, such as c-Raf1 or B-Raf, or with antibodies against the MAP kinase kinase (MEK1), which contains many proline-rich stretches (results not shown). In contrast, the Ras exchange factor, Sos1, was found associated with Grb2 and Crk (results not shown). These data suggest that MEKK1 interacts specifically with Grb2 in vitro.
We then examined the Grb2-MEKK1 interaction in vivo (Fig.  1B). Extracts of proliferating ER22 cells were immunoprecipitated with anti-Grb2 antibodies and analyzed by Western blotting with anti-MEKK1 antibodies (lane 1). Two major polypep-tides were detected, a 98-kDa protein corresponding to the molecular mass of the wild type MEKK1 and a 195-kDa protein having the same size as the large form of MEKK1 (15). The cell extracts were immunoprecipitated with anti-MEKK1 antibodies, and the 25-kDa Grb2 protein was detected using anti-Grb2 antibodies (lane 3). These data indicate that endogenous Grb2 and MEKK1 associate in vivo.
To confirm this interaction, ER22 cells were transiently transfected with plasmids carrying VSV-tagged MEKK1 (lane 2). Grb2 was associated with VSV-tagged MEKK1 after immunoprecipitation of Grb2 followed by Western blot analysis with anti-VSV tag antibodies. ER22 cells were also transfected with plasmids carrying Myc-tagged Grb2. Anti-MEKK1 immunoprecipitates of cell extracts, blotted with anti-Grb2 antibodies, detected two proteins, one corresponding to endogenous Grb2 and the other to Myc-Grb2 (lane 4), the latter was also detected by anti-Myc antibodies (results not shown). This confirms that MEKK1 interacts with Grb2 in ER22 cells.
Mapping of the Interaction Domains on Grb2 and on MEKK1-The functional domains of Grb2 that interact with MEKK1 were identified by affinity chromatography using mutants or variants of Grb2 ( Fig. 2A). Grb3-3 is a natural splicing isoform of Grb2 with a crippled SH2 domain that cannot interact with the phosphorylated EGF receptor (18). It interacted with MEKK1 as well as did unspliced Grb2, indicating that the SH2 domain of Grb2 is not implicated in the binding with MEKK1. Grb2Y7V is an NH 2 -terminal SH3 mutant that has lost the ability to bind dynamin. 2 It still bound to MEKK1, although less strongly than Grb2, indicating that MEKK1 does not bind to the same site as dynamin. Grb2⌬SH3C is deleted from its COOH-terminal SH3 domain and did not interact any more with MEKK1 ( Fig. 2A), although it did interact with Sos1 (result not shown). The Grb2 G203R mutant did not bind to Sos1 under our experimental conditions 2 and is known to block DNA synthesis induced by activation of the Ras pathway (23). This mutant still interacted with MEKK1 (result not shown). Therefore the Grb2-MEKK1 interaction appears to be different from canonical Grb2-Sos binding. Taken together, our experiments indicate that the COOH-terminal SH3 domain of Grb2 is needed to bind to MEKK1, whereas the NH 2 -terminal SH3 domain may act as a putative modulator.
We identified the domain of MEKK1 that interacted with Grb2 by transfecting ER22 cells with plasmids encoding VSVtagged MEKK1  or HA-tagged MEKK1 (301-672) (Fig. 2B). Tagged MEKKs were immunoprecipitated using anti-tag antibodies and blotted with anti-Grb2 antibodies. We found that Grb2 interacted in vivo with MEKK1 (1-672) but not with its COOH-terminal catalytic domain.
The identity of the MEKK1 interaction domain was confirmed by far-Western blot analysis. Recombinant polyhisti-dine-tagged MEKK1  was probed with GST or GST-Grb2 (Fig. 2C). As expected, GST-Grb2 bound to a single polypeptide corresponding to the tagged MEKK1  , whereas GST gave no detectable signal. The NH 2 -terminal domain of MEKK1  contains three proline-rich regions according to the Finger-PRINTS program. One of these proline-rich stretches matches a class I binding site for a putative SH3 domain (sequence RPKPSRP) as described by Cohen et al. (24).
Grb2 and MEKK1, a Constitutive Complex in ER22 Cells-We checked to determine whether MEKK1 associated with Grb2 in quiescent cells or in response to EGF. Fig. 3A shows that similar amounts of the complex were immunoprecipitated from quiescent, EGF-treated, and FCS-cultured cells. ER22 cells were grown to confluence in Dulbecco's modified Eagle's medium/F-12 containing 10% FCS and then in medium without serum. Cells were treated for the indicated time with 100 ng/ml EGF. A, the protein extracts were immunoprecipitated with anti-MEKK1 antibodies and the immunoprecipitated proteins were analyzed by Western blot with monoclonal anti-Grb2 antibodies. B and C, Shc proteins were immunoprecipitated with polyclonal anti-Shc antibodies (Transduction Laboratories Inc.). Association between Shc proteins and Grb2 or MEKK1 was monitored by Western blot analysis of the immunoprecipitated proteins using monoclonal anti-Grb2 antibodies (B) or anti-MEKK1 antibodies (C). D, cell extracts were prepared and analyzed for JNK activity in vitro using GST-Jun  adsorbed onto glutathioneagarose beads as a substrate. Phosphorylation products were analyzed by 10% SDS-PAGE and autoradiography.

FIG. 4. Effect of MEKK1 (1-410) transfection on EGF-induced
Jun-kinase activity. ER22 cells were cotransfected with the Gal4-CAT and the Gal4-cJun  plasmids and pSV2 plasmid without a cDNA insert or with MEKK1  or MEKK1 (301-672) cDNAs. Cells were incubated for 24 h after transfection, deprived of FCS for an additional 24 h, and treated with 100 ng/ml EGF. CAT activity was measured using an InstantImager (Packard Instrument Corp.). Values were normalized to equivalent amounts of proteins. Therefore, we conclude that the Grb2-MEKK1 association is not modulated by EGF or serum and that the complex is constitutive, as is the Grb2-Sos complex (25)(26)(27)(28).
EGF Receptor Tyrosine Phosphorylation Promotes Grb2-MEKK1-Shc Complex Formation-Shc proteins were immunoprecipitated from ER22 cells and were found to be associated with Grb2 upon EGF stimulation (Fig. 3B), as described previously (29,30). This association was transient, reaching a peak after 5-10 min of EGF treatment. Concomitantly, MEKK1 was detected in the Shc immunoprecipitates (Fig. 3C). This association was confirmed using ER22 cells transfected with plasmids carrying VSV-tagged MEKK1. Immunoprecipitation with anti-VSV antibodies followed by Western blot analysis with anti-Shc antibodies revealed p66, p52, and p46 Shc proteins (results not shown). The JNK activities in ER22 cells were stimulated by EGF and reached a peak 20 min after adding EGF (Fig. 3D), following the release of MEKK1 from Shc proteins.
Dissociation of the Grb2-MEKK1 complex from Shc, 10 min after EGF stimulation, may allow this complex to be recycled to the cytoplasm, in a way similar to the Grb2-Sos complex. Our results suggest that the activation of JNK by EGF requires a transient association of a Grb2-MEKK1 complex with Shc proteins and perhaps with the EGF receptor. This may result in a transient translocation of MEKK1 to the plasma membrane. Other protein kinases involved in JNK activation such as phosphatidylinositol 3-kinase, PAKs, and HPK1 require recruitment at the membrane for their activation (31)(32)(33). MEKK1 could interact with GTP-loaded small GTPases at the membrane. Indeed, JNK activation has been found to depend on Ras (22) and on members of the Rho superfamily (34), and an interaction between Ras-GTP and MEKK1 was documented (35). MEKK1 could also interact with partners at the membrane, such as 14-3-3 proteins, allowing caspase-mediated cleavage (36), or with its specific kinases. Diener et al. (37) reported that MEKK1 is phosphorylated by the germinal center kinase like kinase (GLK), which shares homology with the germinal center kinase (GCK), the hematopoietic progenitor kinase 1 (HPK1), and the yeast kinase Ste20.
Transfection of the NH 2 -terminal Domain of MEKK1 Inhibits EGF-dependent JNK Activation-We evaluated the role of the Grb2-MEKK1 interaction in response to EGF by examining the effect of MEKK1  transfection on JNK activity in EGFtreated ER22 cells (Fig. 4). Cells were cotransfected with the Gal4-CAT plasmid and the Gal4-cJun  plasmid with or without MEKK1 expression vectors. 48 h after transfection, starved cells were treated with EGF, and JNK activity was measured by CAT assay. As described previously (9), EGF stimulated JNK activity (ϳ15-fold). The catalytic domain of MEKK1 (301-672) increased JNK activity but the NH 2 -terminal domain of MEKK1 reduced EGF-stimulated JNK activity by 40%. This effect is specific to the JNK pathway, because transfection of the NH 2 -terminal domain of MEKK1 (1-410) did not interfere with ERK activation (results not shown).
Our results therefore indicate that there is a constitutive Grb2-MEKK1 complex in both resting and growing cells. This complex involves the COOH-terminal SH3 domain of Grb2 and the NH 2 -terminal region of MEKK1. EGF stimulation causes this complex to be recruited by Shc proteins. Our data emphasize the role of adaptors like Grb2 and Shc in the activation of MEKK1 and suggest that Grb2 is involved in several signaling pathways. The recruitment of MEKK1 at the cell membrane may promote its activation by locating it close to a suitable kinase. This mechanism is an integral part of the signal generated by the EGF receptor to activate JNKs.