Differential Inhibition of Signaling Pathways by Dominant-Negative ...

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1995, p. 6829–6837 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 15, No. 12

Differential Inhibition of Signaling Pathways by Dominant-Negative SH2/SH3 Adapter Proteins MASAMITSU TANAKA, RUCHIKA GUPTA,

AND

BRUCE J. MAYER*

Howard Hughes Medical Institute, Children’s Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 6 June 1995/Returned for modification 13 July 1995/Accepted 1 September 1995

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abltransformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominantnegative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli. termed Crk-1 and Crk-2, containing one and two SH3 domains, respectively (26, 43); and Nck (18). While each of these adapters contains only SH2 and SH3 domains, the organization of these domains (see Fig. 1) and their binding specificities (51, 57) are quite different. Crk also has very highly homologous relative, termed CRKL (54); it is not known whether genes closely related to those encoding Grb2 and Nck also exist. The adapter protein Grb2 is well known to make a complex with a Ras guanine nucleotide release protein, Sos, and transmit signals from tyrosine kinases to Ras. This has been convincingly demonstrated by genetic studies in nematodes and Drosophila melanogaster (3, 38, 50, 52). In mammalian systems, the SH2 domain of Grb2 has been shown to bind directly to the activated EGF receptor or to make a complex with phosphorylated Shc and Syp after stimulation by EGF, nerve growth factor (NGF), or platelet-derived growth factor (9, 20, 48). The ultimate effect of these Grb2-mediated complexes is to bring Sos to the plasma membrane, where it can activate Ras. Recently, the association of another SH2/SH3 adapter protein, Crk, with activated receptors has been reported. v-Crk can directly couple to the EGF receptor and NGF receptor (10), and the Crk SH2 domain binds to Shc protein phosphorylated on tyrosine residues as a result of NGF treatment (24). Nck protein has also been reported bind to Sos and interact with the Ras pathway (14). Nck becomes phosphorylated by stimulation of cells with platelet-derived growth factor and EGF and makes a complex with these receptors (2, 19, 34, 39). It is now important to sort out which of the adapter proteins might actually be involved in signal transmission from various mitogenic stimuli in vivo. To date, the only adapter that has been implicated genetically in signaling is Grb2; however, it is

The signaling pathway leading from transmembrane growth factor receptors, such as the epidermal growth factor (EGF) receptor, to the intracellular serine-threonine kinases of the mitogen-activated protein kinase (MAPK)/Erk family involves the GTP-binding protein p21ras (46, 47), which interacts with the c-Raf kinase. Raf phosphorylates and thereby activates MAPK kinase (MEK) (4, 15, 16, 22), which in turn phosphorylates Erk-1 and Erk-2 on serine/threonine and tyrosine residues (8, 11, 17, 37, 47). Furthermore, serum, 12-O-tetradecanoylphorbol-13-acetate and some classes of oncogenes, including v-src and v-abl, all depend on Raf-1 for activation of MAPK/Erk (55). It is now apparent that signal transduction is often mediated by specific protein-protein complexes. In many cases, the formation of such complexes is controlled by small modular domains termed the Src homology 2 and 3 (SH2 and SH3) domains, which are found in a wide variety of proteins (28, 40, 41). SH2 domains have been shown to bind specifically and with high affinity to tyrosine-phosphorylated proteins and are thought to mediate the association of signaling proteins in response to tyrosine phosphorylation (1, 23, 25, 32). SH3 domains bind to specific proline-rich sites on target proteins (30, 40, 44). The SH2/SH3 adapters are a family of small proteins consisting almost entirely of SH2 and SH3 domains. The presently known members of the family are Grb2/Sem5/Drk/ASH (3, 21, 27, 38); Crk, which exists in two alternatively spliced forms, * Corresponding author. Mailing address: Howard Hughes Medical Institute, Children’s Hospital, 320 Longwood Ave., Boston, MA 02115. Phone: (617) 355-7916. Fax: (617) 730-0506. Electronic mail address: [email protected]. 6829

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not known whether the relatively simple genetic model organisms Caenorhabditis elegans and D. melanogaster possess homologs of the genes encoding Nck and Crk. Both Crk and Nck can malignantly transform cells when overexpressed (2, 19, 26), which Grb2 cannot do (21), highlighting potential differences in their biological activities. It is possible that different mitogenic stimuli use different adapters or that Crk and Nck have very different functions in vivo compared with Grb2. The resolution of these questions is especially important because interactions mediated by SH2 and SH3 domains are attractive targets for intervention with small-molecule drugs. The aim of this report is to examine the extent to which these SH2/SH3 adapters contribute to Erk activation by different mitogenic stimuli and whether different adaptor proteins are redundant or have distinct functions in these signaling pathways. We have used oncogenic Abl and the EGF receptor as models of the nonreceptor and receptor class of tyrosine kinases, respectively. Abl is a nonreceptor tyrosine kinase that when activated by mutation (for example, deletion of its SH3 domain) can induce malignant transformation (56). In this study, we have used various dominant-negative (DN) SH2/SH3 adapters and examined their abilities to block the signaling pathways leading to Erk activation. Because the adapters function as molecular cross-linkers, mutation of one of their protein-binding domains would be predicted to block signaling by forming complexes that are unable to couple to other signaling components. For example, SH2 mutants would be expected to bind to and sequester SH3 domain-binding effector molecules such as Sos, whereas SH3 mutants would bind to and block signaling from tyrosine-phosphorylated SH2 domain-binding sites. We found that DN Grb2 and Crk-1 mutants could effectively block Erk activation by oncogenic Abl and that a combination of SH3 mutants of Grb2 and Crk-1 had striking synergistic effects in inhibiting Erk activation. Nck mutants had almost no effect on Erk activation by Abl. By contrast, only Grb2 mutants had a significant effect on Erk activation induced by EGF. Our results demonstrate that there are differences in the biological activities of the different adapters, strongly suggesting that they are not merely redundant. Our results also demonstrate that mitogenic stimulation of the Ras pathway by Abl is significantly different from stimulation by EGF, implicating tyrosine-phosphorylated sites that can bind to Crk in activation of Ras in Abl-transformed cells. MATERIALS AND METHODS Cell lines and plasmids. 293-T human embryonal kidney cells (expressing simian virus 40 T antigen) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum. pEBB is derived from pEF-BOS, an expression vector driven by the human elongation factor 1-a promoter (35). pEBB has a multiple cloning site which replaces the BstXI-NotI stuffer fragment of pEF-BOS. pEBG is a glutathione S-transferase (GST) fusion vector derived by inserting a PCR-generated BglII-BamHI GST fragment from pGEX-2T (Pharmacia) into the BamHI site of pEBB. The entire coding region of Erk-1 cDNA was cloned into the BamHI site of pEBG as previously reported (49). pGDN-G consists of an SH3 domain-deleted abl gene which was constructed by PCR using wild-type murine c-abl cDNA and cloned into the pGDN retroviral expression vector as described previously (29); it is referred to in this work as DSH3-Abl. N17 H-Ras is a DN H-Ras allele (containing a serine-toasparagine mutation at position 17) expressed in pZIP-neo (6). Construction of DN SH2/SH3 adapters. To produce DN mutants, the conserved arginine of the FLVRES sequence in the SH2 domain was changed to lysine, or the first (absolutely conserved) tryptophan of the characteristic tryptophan doublet of the SH3 domain was changed to lysine. In all cases examined, these changes eliminated detectable ligand binding of the mutated domain without affecting the binding activity of unmutated domains. This was assayed for the SH3 domains of Abl and Crk by elimination of binding to the Abl proline-rich region (45) and for the SH2 domains by elimination of binding to purified tyrosine-phosphorylated proteins in a filter-binding assay (33) (data not shown). The amino acid substitutions were produced by an overlap extension using PCR (12) with mutant oligonucleotides. For mutants with multiple domains mutated,

MOL. CELL. BIOL. DNA from clones already containing mutations was used for a second (or third) round of PCR mutagenesis. All of the mutated clones were amplified with a BamHI site at the 59 end and a NotI site at the 39 end. The NckW38,143,229K clone was made by ligation of BstEII-digested fragments from W38,143K, and W229K. All mutants were cloned into pEBB plasmid vector cleaved with BamHI and NotI, and resulting clones were sequenced in regions of primer binding. In all cases, two independent PCR clones were characterized and had identical properties. Transfection and in vitro kinase assay. For Erk kinase assay, 3 3 106 293 cells were plated in 100-mm-diameter tissue culture dishes 24 h prior to transfection. Transfections were carried out by a calcium phosphate coprecipitation method with concurrent treatment with 25 mM chloroquine essentially as described previously (42). For all transfections, the amounts of reporter plasmid (pEBGErk) and activator plasmid (expressing activated Abl or Ras), if used, were held constant and the amount of DN adaptor plasmid was varied. For EGF stimulation, 106 293 cells were plated in 60-mm-diameter culture dishes 24 h prior to transfection and treated with EGF (Upstate Biotechnology Inc.) at 100 ng/ml for 15 min prior to lysis. At 48 h posttransfection, cells were lysed in 1 ml of ice-cold Triton lysis buffer (25 mM Tris [pH 7.4], 150 mM NaCl, 5 mM Na2EDTA, 1% Triton X-100, 10 mM sodium pyrophosphate, 10 mM b-glycerophosphate, 1 mM Na3VO4, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 mg of aprotinin per ml). Lysates were cleared by centrifugation for 10 min, and protein concentrations were assayed by the Bradford method (Bio-Rad). To purify GST-Erk protein, equal amount of cell proteins were incubated with glutathione-agarose beads (Molecular Probes Inc.) for 30 min at 48C and then washed three times in lysis buffer and once in kinase buffer (50 mM HEPES [N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid; pH 7.4], 10 mM MgCl2). For GST-Erk kinase assays, beads were incubated in 15 ml of kinase buffer containing 10 mCi of [g-32P]ATP, 100 mM ATP, and 0.2 mg of myelin basic protein (MBP; Sigma) per ml at 308C for 10 min. Reactions were stopped by the addition of 5 ml of 53 Laemmli sample buffer and boiled for 3 min. Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE); the bands corresponding to MBP were excised and counted, and the result was expressed as the percentage of counts in control transfections without adaptor cotransfection. If the value was greater than or equal to the control value, it was expressed as 100%. While the amount of GST-Erk determined by Coomassie blue staining is quite high relative to the amount of substrate, its specific activity is relatively low and the assays are therefore in the linear range under these conditions (data not shown). It is likely that only a small fraction of the purified GST-Erk is activated. In some experiments, there were significant differences (up to two- to threefold) in the amounts of GST-Erk in different cell lysates. In these cases, the amount of lysate bound to glutathione-agarose was adjusted (on the basis of the counts incorporated by autophosphorylation into GST-Erk, which correlated with amount of GST-Erk seen by Coomassie blue staining) so that equal amounts of GST-Erk were assayed. No consistent relationship was observed between the GST-Erk protein levels and transfection of any adapter or activator plasmid.

RESULTS Inhibition of Erk-1 activation by oncogenic Abl, using DN SH2/SH3 adapters. To analyze whether SH2/SH3 adapters could inhibit the Abl pathway leading to Erk activation, various DN SH2/SH3 adapters of Grb2, Crk, and Nck were used (Fig. 1). The mutations that were introduced abolish detectable ligand binding to the mutated domains without affecting the binding activity of unmutated domains (data not shown). When expressed at high levels in cells, the mutant adapters would be expected to exert dominant inhibitory effects on signaling by binding to and sequestering tyrosine-phosphorylated sites (in the case of SH3 mutants) or SH3 domain-binding effector molecules (in the case of SH2 mutants). Activation of Erk-1 kinase fused to GST, which is encoded by the pEBGErk reporter plasmid, was used to assay activation of the Ras/ Raf signaling cascade. Plasmids encoding the DN adapters were cotransfected into 293 cells along with pEBG-Erk and a plasmid encoding a transforming, SH3 domain-deleted Abl protein. Transfected cells were subsequently lysed, GST-Erk protein was purified, and Erk activity was measured by in vitro kinase assay using MBP as the substrate. The effects of DN mutants of Crk-1, Crk-2, Grb2, and Nck on Erk activation were compared (Fig. 2). The basal Erk activity without stimulation was very low and almost undetectable. When activated by cotransfection with the transforming Abl plasmid (DSH3-Abl), Erk activity was significantly in-

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EFFECTS OF DN SH2/SH3 ADAPTERS ON SIGNALING PATHWAYS

FIG. 1. Schematic representation of wild-type SH2/SH3 adapters and potential DN mutants. Proteins are depicted to scale; SH2 and SH3 domains are represented by shaded boxes. Crk-1 and Crk-2 are the two forms of wild-type (wt) Crk protein generated by alternative splicing. Mutant domains in which binding activity has been ablated are indicated by 3’s. On the right, the amino acid number of each arginine (R) or tryptophan (W) residue which was substituted by lysine (K) is indicated.

creased (compare lanes 1 and 2). Cotransfection of a plasmid encoding a Ras DN mutant (N17 H-Ras) resulted in complete inhibition of Erk activation by DSH3-Abl (lane 14). This result demonstrates that the activation of Erk-1 by Abl is completely

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Ras dependent. Among DN SH2/SH3 adapters, activation by DSH3-Abl was most effectively inhibited by Grb2 with mutations in either its SH2 domain (lane 8) or both SH3 domains (lane 11) and by Crk-1 with an SH3 domain mutation (lane 4). The Crk-1 SH2 mutant and Crk-2 mutants were less effective, while Nck mutants had no detectable effect on the Erk kinase activity. When higher amounts of DNA were transfected, the Nck mutants showed slight inhibitory activity, but this was always much less than in the case of the Grb2 and Crk mutants and never exceeded 50% inhibition (data not shown). These experiments were repeated at least three times with similar results. The protein expression of transfected DN adapters was confirmed by immunoblot analysis using specific antibodies against Crk, Grb2, and Nck. By comparing with purified adapter protein standards, we determined that all adapter proteins were expressed at roughly the same level in the cell lysates. Effect of combinations of DN adapters. Because the effects of Grb2 and Crk-1 mutants were greater than those of other mutants, we focused on these two adapters and examined whether combinations of DN Grb2 and Crk-1 adapters could inhibit Erk activation more effectively than single mutants. These experiments address whether Grb2 and Crk mutants inhibit the same interactions (that is, are redundant) or if they inhibit distinct signaling pathways, each of which can lead to Ras activation. Increasing amounts of the Crk or Grb2 mutants, alone or in combination, were compared for the ability to inhibit activation of Erk-1 by Abl. The efficacy of pairwise combinations of 1, 2, 4, and 6 mg of each mutant was directly compared with that of 2, 4, 8, and 12 mg of each mutant alone. Immunoblotting analysis indicated that the expressed protein level of each adapter was in proportion to the amount of plasmid DNA transfected (data not shown). Representative results are shown in Fig. 3. For example, 2 mg of any single DN adapter had little effect, while the combination of 1 mg of the Crk-1 SH3 mutant plus 1 mg of the Grb2 SH3 mutant strongly inhibited Erk activation (Fig. 3A, lane 10), demonstrating a synergistic effect. Similarly, the combination of 4 mg each of the Crk-1 SH3 mutant and the Grb2 SH3 mutant (Fig. 3B, lane 10) inhibited Erk activity much more effectively than 8 mg of

FIG. 2. Effects of DN SH2/SH3 adapters on Erk-1 activation by oncogenic Abl. Four micrograms of pEBG-Erk was cotransfected into 293 cells in 100-mm-diameter tissue culture dishes with or without 4 mg of plasmid encoding SH3 domain-deleted Abl (DSH3-Abl) and DN SH2/SH3 adapters as indicated above the lanes. GST-Erk protein was purified from cell lysates, and Erk activity was measured by in vitro kinase assay. Proteins were separated by SDS-PAGE. Amount of GST-Erk protein was normalized as detailed in Materials and Methods. (A) Autoradiogram; (B) Coomassie blue-stained gel. The number below each lane indicates the relative amount of Erk kinase activity as a percentage of the activity present in cells lacking DN adapters (lane 2). Horizontal bars on the left are molecular size markers representing 68, 43, 29, and 18 kDa (from the top). The migration positions of GST-Erk and MBP are indicated.

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FIG. 3. Inhibition of Erk-1 activation by Abl with combinations of DN Grb2 and Crk-1 mutants. (A) Two micrograms of single mutants (lanes 2 to 5) and 1 mg each of two mutants of Grb2 and Crk-1 (lane 6 to 11) were transfected to 293 cells along with 4 mg of pEBG-Erk and DSH3-Abl. In vitro kinase assay of Erk-1 was performed. (B) Erk activity when 8 mg of single mutants (lane 2 to 5) or 4 mg each of Grb2 and Crk-1 mutants (lane 6 to 11) were cotransfected. The percentage of control activity is given under each lane. The migration position of MBP is indicated.

either these mutants alone (lanes 3 and 5). The amount of Abl protein detected by immunoblotting in cells transfected with this combination was indistinguishable from that seen in cells transfected with individual mutants (data not shown), and so the synergistic effect cannot be due to effects on Abl transcription or stability. All combinations of Crk-1 and Grb2 mutants were compared, and the results are summarized in Fig. 4. By far the most effective combination was the Crk-1 SH3 mutant plus the Grb2 SH3 mutant (Fig. 4E). Over a range of DNA amounts, this combination reproducibly gave approximately threefoldgreater inhibition than the same amount of any single mutant. The amount of plasmid transfected was decreased to determine the point at which Erk activity was half-maximal (Fig. 4E, inset). Fifty percent inhibition was achieved by the combination of Crk-1 SH3 and Grb2 SH3 mutants at a DNA amount 5.4 times less than that required for the Crk-1 SH3 mutant alone and 3.6 times less than that required for the Grb2 SH3 mutant alone. Although some other combinations of Grb2 and Crk-1 mutants, for example, Crk-1 SH2 plus Crk-1 SH3 (Fig. 4A), Crk-1 SH3 plus Grb2 SH2 (Fig. 4D), and Grb2 SH2 plus Grb2 SH3 (Fig. 4F), showed slight synergistic effects, the degree was much less than with the Crk-1 SH3–Grb2 SH3 combination. Because the DN mutants were constructed by PCR, two independent clones were characterized for all constructs to control for undetected PCR-induced mutations. This experiment was repeated twice with two independent clones, and similar results were obtained. In contrast, when the effects of DN mutants of Grb2 and Crk-1 on Erk-1 activation by H-Ras were examined, the DN adapters had no effect (Fig. 5). Erk-1 activation was not affected even by combinations of these DN adapters. This result demonstrates that the effect on Erk activation by Abl is not due to nonspecific toxicity, since Erk could still be activated by Ras.

It also demonstrates that, as expected, the DN adapters block signaling at a position in the pathway upstream from Ras. Inhibition of EGF-induced Erk-1 activation. We were interested in examining whether signaling from a mitogenic growth factor would show the same profile of inhibition by DN adapters as in the case of activation by an oncogenic nonreceptor tyrosine kinase such as Abl. We therefore examined whether DN SH2/SH3 adapters could inhibit Erk-1 activation by EGF (Fig. 6A). When 293 cells were treated with EGF, the Erk kinase activity was significantly activated (compare lanes 1 and 2). Clearly, Grb2 with a mutation in either its SH2 domain or any one of its SH3 domains inhibited the activation of Erk very effectively, almost to the same extent as N17 H-Ras. On the other hand, DN mutants of Crk-1, Crk-2, and Nck had little or no effect (at most 25% inhibition) when 4 mg of plasmid was transfected in 60-mm-diameter dishes (equivalent to 10 mg in the previous experiments, at which amount both Crk-1 SH2 and Crk-1 SH3 mutants inhibited Abl-induced activation of Erk-1 by at least 70% [Fig. 4]). When the combined effects of the Grb2 and Crk-1 mutants were examined as described above, in contrast to the case of Abl activation, none of the combinations of Grb2 and Crk-1 mutants gave a synergistic effect (Fig. 6B). It is interesting that Crk-2 SH3 mutants reproducibly gave a slight activation of Erk activity upon EGF treatment compared with controls (Fig. 6A, lanes 6 and 7). Wild-type Crk-1 behaved similarly (data not shown), and so it is possible that the presence of the intact SH2 domain and one of the two SH3 domains in the Crk-2 mutants increased coupling to downstream effectors. The failure of Crk-1 DN mutants to inhibit EGF-induced signaling suggests, however, that such coupling is not necessary for signal transmission by the EGF receptor.

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FIG. 4. Synergistic effects of combinations of Grb2 and Crk-1 mutants on Erk-1 activation by Abl. Two, 4, 8, and 12 mg of DN Grb2 and Crk-1 mutants, alone or in combination, were cotransfected with 4 mg of pEBG-Erk and DSH3-Abl. MBP phosphorylation was quantitated as in Fig. 2 and 3. Each panel presents the inhibition curve for a combination of two mutants compared with those for each mutant alone. The data are from a single set of transfections; essentially identical results were obtained in independent experiments. The activity present in extract of Abl-stimulated cells without DN adapters was set to 100% as described in the legend to Fig. 2. The panel E inset shows transfection of 0.2, 0.7, 1.4, 2, and 4 mg of mutant plasmids, alone or in combination, with 4 mg of pEBG-Erk and DSH3-Abl; these transfections were done in a separate experiment.

DISCUSSION In this study, we have used a series of mutants of the SH2/ SH3 adapter proteins Crk-1, Crk-2, Grb2, and Nck to probe signaling pathways leading from tyrosine kinases to Erk activation. DN adapters with mutations in their SH2 domains cannot bind to tyrosine-phosphorylated sites but retain the ability to bind to SH3 domain-binding effector proteins; such mutants should block signaling mediated by any proteins that can bind to the SH3 domain(s). On the other hand, DN adapters with SH3 domain mutations block signaling through SH2 domain-binding sites, because they can bind to tyrosine-phosphorylated sites but are unable to bind to downstream effector proteins. Using such mutants, we can address whether different adapter proteins are redundant or have unique functions in signaling by comparing the abilities of different mutants and combinations of mutants to block signaling; we can also address whether different tyrosine kinases lead to Erk activation via the same or different pathways. We find that mutants of both Grb2 and Crk-1 effectively suppressed Erk activation by oncogenic Abl and that the combination of the Grb2 SH3 mutant plus the Crk-1 SH3 mutant had a dramatic synergistic effect on blocking Erk activation. On the other hand, only Grb2 mutants were able to block the Erk activation induced by EGF. Because the cells, plasmids, and transfection system were identical in the two sets of experiments, these results strongly suggest that signaling via the EGF receptor is fundamentally different than in the case of the Abl nonreceptor tyrosine kinase; for EGF receptor signaling, Ras activation is specifically mediated by proteins that bind Grb2, whereas when Abl is the stimulus, proteins that bind Crk are

also implicated in Ras activation. It is important to remember that DN mutants such as those used here are quite different from genetic null mutants. The ability of a DN variant to block a biological function does not necessarily imply that the specific protein is involved in this function in all cells (the protein may not be available for interaction because of factors such as expression level or subcellular localization). A biological effect does necessarily imply, however, that proteins that interact with the mutant are involved. There are two other implications of these studies. First, because Nck mutants were ineffective at blocking Erk activation in response to either Abl or EGF, our results suggest that Nck and the sites that bind to it are not required for or involved in Ras activation in response to mitogenic signals. Second, Crk-2 DN mutants were in general less effective at inhibiting signaling than the corresponding mutants of Crk-1, which lacks the C-terminal SH3 of Crk-2; in particular, the Crk-2 SH3-N mutant was less effective than the Crk-1 SH3 mutant (Fig. 2). There are several plausible explanations for this difference, but one interesting possibility is that the less potent activity of Crk-2 is due to tyrosine phosphorylation at a site not present in Crk-1, which has been proposed to negatively regulate its binding activity (7, 31). Analysis of the effects of combinations of different mutant adapters can illuminate whether they are functioning on overlapping of distinct pathways. Considering the effect of combinations of Grb2 and Crk-1 mutants on Erk activation by Abl, four simplified models can be constructed (Fig. 7). If Grb2 and Crk-1 bind to the same tyrosine-phosphorylated sites and the same SH3 domain-binding proteins, then none of the combi-

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FIG. 5. DN Grb2 and Crk-1 mutants do not inhibit Erk activation by H-Ras. The in vitro kinase assay of Erk-1 when 4 mg of single mutants (lanes 3 to 6) or 2 mg each of two mutants of Grb2 and Crk-1 (lanes 7 to 12) were transfected into 293 cells along with 4 mg of pEBG-Erk and pZIPneo-H-Ras (A) or when 8 mg of single mutants or 4 mg each of two mutants were transfected (B). The percentage of Erk-1 kinase activity relative to the control is given below each lane. The migration position of MBP is indicated.

nations of DN mutants should give synergistic effects; the most that could be expected would be additive effects (Fig. 7A). In contrast, if Grb2 and Crk-1 bind to completely different sets of tyrosine-phosphorylated sites and effectors (that is, they bind nonoverlapping sets of ligands, each of which can lead to Ras activation), then combinations of both SH2 and SH3 mutants might give synergistic effects (Fig. 7B). Similarly, if Grb2 and

Crk-1 bind to different tyrosine-phosphorylated sites but the same effectors, only the combination of SH3 mutants would give a synergistic effect (Fig. 7C), while if they coupled to the same tyrosine-phosphorylated site but different effectors, only the combination of SH2 mutants might give a synergistic effect (Fig. 7D). Our actual result, that only the combination of the

FIG. 6. Inhibition of EGF-stimulated Erk-1 activation with DN SH2/SH3 adapters. (A) Two micrograms of pEBG-Erk was transfected to 293 cells in 60-mmdiameter tissue culture dishes with or without 4 mg of DN mutants of Crk, Grb2, and Nck; 48 h later, transfected cells were incubated with EGF at 100 ng/ml for 15 min, and in vitro kinase assay of GST-Erk was performed. The number below each lane indicates the percentage of Erk-1 kinase activity compared with the control without DN adapter (lane 2). (B) In vitro Erk-1 kinase assay when 2 mg of single mutants (lanes 2 to 5) or 1 mg each of two mutants of Grb2 and Crk-1 (lanes 6 to 11) were transfected. The position of MBP is indicated.

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FIG. 7. Diagram of possible relationships of Crk and Grb2 signaling pathways leading from oncogenic Abl tyrosine kinase to Ras activation. The SH2 domains of Crk and Grb2 adapter proteins bind to tyrosine-phosphorylated (P-Tyr) sites, and the SH3 domains bind to proline-rich regions of effector molecules. The predicted inhibitory effects of combined DN adapters on Erk activation via the Ras signaling pathway are summarized at the bottom in each case.

Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a significant synergistic effect, is most consistent with the model in Fig. 7C. This result suggests that in Abl-transformed cells, two classes of tyrosine-phosphorylated sites, those that can bind the Grb2 SH2 domain and those that bind the Crk SH2 domain, can lead to Ras activation. The SH2 domains of Grb2 and Crk have different binding specificities for phosphotyrosine-containing peptides (51); our results show that phosphorylation of both classes of sites can lead to Ras activation. Evaluation of the roles of SH3 domain-binding effectors is more complicated. It has been reported that two guanine nucleotide release factors, Sos and C3G, can bind to the SH3 domains of Grb2 and Crk (24, 53). In our data, the combination of SH2 mutants of Crk and Grb2 was not significantly more effective at inhibiting the Erk activation than each mutant alone. This result suggests that in the activated Abl pathway leading to Ras activation, the pivotal effector is either Sos or C3G or, alternatively, the Crk and Grb2 SH3 domains bind equally well to Sos and C3G. However, in EGF-treated cells (see below), the Grb2 SH2 mutant is much more effective than the Crk SH2 mutant, demonstrating that their SH3 domain-binding specificities cannot be identical. Structural and binding studies suggest that the Grb2 SH3 domains prefer binding sites of the PxxPxR type, whereas the Crk SH3 domains prefer PxxPxK sites (45, 57); the former are found in Sos, while the latter are found in C3G. Further experiments using DN Sos and C3G might be useful to clarify these ambiguities. In both Abl-initiated and EGF-initiated signaling, however, the Grb2 SH2 mutant is more effective than the Crk SH2 mutant at inhibiting Erk activation, consistent with Sos (or another Grb2 SH3 domain-specific binding protein) being the most significant activator of Ras in 293 cells. One interesting way to explain the potent effect of Crk mutants on Abl-induced signaling is by a direct effect of Crk on Abl kinase activity. In fact, it is known that Crk protein can bind via its SH3 domain to a proline-rich region in the C terminus of Abl (7, 44) and serve in vitro as a processivity factor to facilitate hyperphosphorylation of proteins containing

Crk SH2 domain-binding sites (31). If this were also the case in vivo, many Abl substrates would therefore be expected to bind tightly to the Crk SH2 domain, and a Crk SH3 mutant would effectively block downstream signaling from such sites. Furthermore, a plausible explanation for the modest ability of the Crk SH2 mutant to block Abl-induced signaling that we observe is that it acts to abrogate binding of endogenous Crk to Abl, preventing efficient phosphorylation of Crk SH2 domainbinding sites by Abl. Consistent with this view, there are qualitative and quantitative differences in the patterns of tyrosinephosphorylated proteins when the Crk-1 SH2 mutant is coexpressed in cells with activated Abl (data not shown). The data from our DN adapter studies are therefore entirely consistent with the in vitro data suggesting a direct role for Crk in the activity of Abl. In contrast to the results with Abl or EGF, none of the DN SH2/SH3 adapters could inhibit the Erk activation by H-Ras. Together with the result that DN H-Ras could completely inhibit the Erk activation by Abl and EGF, these results demonstrate that, as expected, the SH2/SH3 adapters function upstream of Ras. The ability of the Ras N17 mutant to block both Abl- and EGF-induced Erk activation also demonstrates that in our system, there is no significant Ras-independent activation of Erk-1; this is important because tyrosine kinases have been shown in some cases to activate Raf in a Ras-independent fashion (5, 13, 36). Our results showed that even when large amounts of plasmids were transfected, none of the DN mutants of Crk and Nck could effectively inhibit the Erk activation induced by EGF. In previous reports, SH2 domains of Crk and Nck have been shown to bind to activated EGF receptor, assayed in vitro by precipitation with Crk or Nck SH2 domains immobilized on Sepharose beads (10, 19), and Nck has also been implicated in Ras-dependent signaling by the platelet-derived growth factor receptor (14), suggesting that these adapters might function in a manner analogous to that of Grb2 in receptor-mediated signaling. The results of this study, however, along with our observation by immunoblot analysis that in 293 cells, endoge-

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nous Grb2 protein is more abundant than Crk and Nck (data not shown), strongly suggest that Grb2 is the adapter that is critical for activation of the Ras/Raf pathway by EGF in vivo. Of course, it remains possible that Crk and Nck perform this function in other cell types or for other receptors. The DN SH2/SH3 adapter proteins used in this study were useful tools with which to examine in detail the roles of the SH2 and SH3 domains in signaling pathways from tyrosine kinases to Ras and MAPK. The confusing overabundance of results from in vitro studies demonstrating association of SH2 domain-containing proteins with tyrosine-phosphorylated proteins, and of SH3 domain-containing proteins with proline-rich targets, makes it imperative to begin to sort out which of these interactions are biologically relevant for individual signaling pathways. This study is a first step in that direction. Our results suggest, for example, that there are molecules selectively phosphorylated by oncogenic Abl, but not by the EGF receptor, that bind to the SH2 domain of Crk and whose phosphorylation leads to activation of Ras. Studies such as we have outlined here will also be important in efforts to use small-molecule inhibitors of tyrosine kinase-initiated signaling in human disease. For example, our results suggest that simultaneously blocking both Crk SH2 domain-ligand interactions and Grb2 SH2 domain-ligand interactions would be more effective at blocking mitogenic signals from activated Abl than blocking either interaction alone. ACKNOWLEDGMENTS We thank Larry Feig, David Foster, Hidesaburo Hanafusa, Joseph Schlessinger, and Len Zon for kindly providing plasmids used in this study, Andrea Musacchio for critically reading the manuscript, and Erica Heinrich and Erica Marieb for excellent technical assistance. B.J.M. is an Assistant Investigator of the Howard Hughes Medical Institute. REFERENCES 1. Anderson, D., C. A. Koch, L. Grey, C. Ellis, M. F. Moran, and T. Pawson. 1990. Binding of SH2 domains of phospholipase Cg1, GAP, and src to activated growth factor receptors. Science 250:979–982. 2. Chou, M. M., J. E. Fajardo, and H. Hanafusa. 1992. The SH2- and SH3containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels. Mol. Cell. Biol. 12:5834–5842. 3. Clark, S. G., M. J. Stern, and H. R. Horvitz. 1992. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature (London) 356:340–344. 4. Dent, P., W. Haster, T. A. J. Haystead, L. A. Vincent, T. M. Roberts, and T. W. Sturgill. 1992. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH3T3 cells and in vivo. Science 257:1404–1407. 5. Fabian, J. R., A. B. Vojtek, J. A. Cooper, and D. K. Morrison. 1994. A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function. Proc. Natl. Acad. Sci. USA 91:5982–5986. 6. Feig, L. A., and G. M. Cooper. 1988. Inhibition of NIH 3T3 cell proliferation by a mutant Ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8:3235–3243. 7. Feller, S. M., B. Knudsen, and H. Hanafusa. 1994. c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 13:2341–2351. 8. Gomez, N., and P. Cohen. 1991. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature (London) 353: 170–173. 9. Hashimoto, Y., K. Matsuoka, T. Takenawa, K. Muroya, S. Hattori, and S. Nakamura. 1994. Different interactions of Grb2/Ash molecule with the NGF and EGF receptors in rat pheochromocytoma PC12 cells. Oncogene 9:869– 875. 10. Hempstead, B., R. B. Birge, J. E. Fajardo, R. Glassman, D. Mahadeo, R. Kraemer, and H. Hanafusa. 1994. Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor and epidermal growth factor-dependent pathways. Mol. Cell. Biol. 14:1964–1971. 11. Her, J. H., S. Lakhani, K. Zu, J. Vila, P. Dent, T. W. Sturgill, and M. J. Weber. 1993. Dual phosphorylation and autophosphorylation in mitogenactivated protein (MAP) kinase activation. Biochem. J. 296:25–31. 12. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59.

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