Hans H.Grunickel and Axel UlIrich2. Department of Molecular Biology, Max-Planck-Institute for. Biochemistry, Am Klopferspitz 18A, 82152 Martinsried, Germany ...
The EMBO Journal vol.15 no. 1 pp.73-82, 1996
Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase Cy affinity and signal activation Axel Obermeier, Inge Tinhofer1, Hans H.Grunickel and Axel UlIrich2 Department of Molecular Biology, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18A, 82152 Martinsried, Germany and 'institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz-Preglstrasse 3, A-6020 Innsbruck, Austria 2Corresponding author
The exchange of nerve growth factor receptor/lrk and epidermal growth factor receptor (EGFR) phospholipase Cy (PLCy) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLCy affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLCy binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLCy affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations. Keywords: affinity/cell transformation/EGF receptor/ PLCy/Trk
Introduction Hormones, growth factors and differentiation factors, such as insulin, epidermal growth factor (EGF) and nerve
growth factor (NGF), regulate diverse cell functions of multicellular organisms. Cells respond to these factors by alterations in their morphology, metabolism, proliferation rate or kinetic behavior, and by initiating processes such as differentiation or apoptosis (reviewed in Schlessinger and Ullrich, 1992). These polypeptide factors employ plasma membrane-spanning receptors with tyrosine kinase activity to translate their respective messages across the cell surface into an intracellular signal. Upon extracellular binding of the appropriate ligand, these receptors dimerize. This is followed by the activation of their intracellular kinase and autophosphorylation of the tyrosine residues (Ullrich and Schlessinger, 1990), thereby acquiring the potential to interact with a repertoire of signaling molecules that bind to specific phosphotyrosine residues via their ( Oxford University Press
Src homology 2 (SH2) domains (reviewed in Koch et al., 1991; Schlessinger and Ullrich, 1992). Subsequently, these proteins may become phosphorylated and activated enzymatically, as in the case of phospholipase Cy (PLCy, Nishibe et al., 1990; Kim et al., 1991; Yang et al., 1994) or, as suggested for phosphatidylinositol 3'-kinase (PI3 '-K), association-induced conformational changes may result in the modulation of an enzymatic function (Backer et al., 1992; Van Horn et al., 1994). Moreover, for some proteins such as PLCy, P13'-K or the Grb2bound Ras guanine nucleotide exchanger Sos, recruitment to the inner surface of the plasma membrane is critical for juxtaposition to their membrane-anchored phospholipid or protein substrates and thus activation of the correspond-
ing signaling pathways.
Numerous SH2-containing proteins have been identified and shown to interact with activated receptor tyrosine kinases (RTKs), and the concept that the specific recruitment of subsets of these signal transducers by different RTKs represents a critical parameter for signal definition is generally accepted. However, the sets of SH2-containing proteins utilized by individual RTKs overlap widely, and no effectors have been found so far that are strictly specific for a given receptor or a certain biological response. One RTK can induce distinct or even opposite effects in different cellular environments. For example, neurotrophin-activated Trk family receptors induce the neuronal differentiation of certain neurons and PC 12 cells but elicit enhanced proliferation and transformation when expressed in fibroblasts (Cordon-Cardo et al., 1991; Glass et al., 1991). This finding led to the conclusion that the cell type- and cell stage-specific expression of RTKs and SH2-containing proteins is critical for signal definition. Conversely, in one and the same cell, certain receptors may induce opposite biological effects without apparent differences in biochemically detectable actions. This is true for the NGF receptor (NGFR) and the EGF receptor (EGFR) in PC 12 cells, which under physiological conditions mediate differentiation and proliferation, respectively. Here, distinct responses of the cell appear to be defined by differences in the duration of the ras-rafmitogen-activated protein (MAP) kinase kinase-MAP kinase signal, with extended kinetics favoring differentiation (Qiu and Green, 1992; Dikic et al., 1994; Traverse et al., 1994). This scenario suggests the role of negative regulatory elements such as phosphotyrosine phosphatases as decisive in signal definition. Another level of distinction between receptors of the tyrosine kinase family is the affinity of their interaction with SH2-containing primary signal transducers. These affinities are believed to be defined by the amino acids immediately flanking phosphotyrosines (pY) in their cytoplasmic domain, which are specifically recognized by nonconserved residues in SH2 domains (Koch et al., 1991; 73
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Fig. 1. Schematic representation of PLCy binding sites on (A) EGFR and Trk and (B) the receptors EGFR, ETR and their exchange mutants EGFR.X and ETR.X. (A) The cytoplasmic portions of EGFR and Trk/ ETR are shown. Boxes denote the tyrosine kinase domains. (B) The horizontal bar indicates the plasma membrane. Below this, the cytoplasmic portions of the receptors are drawn with the tyrosine kinase domain indicated as a box in each case. The PLCy binding sites of EGFR and Trk shown in (A) are symbolized by (0) and (0), respectively (not drawn to scale).
Bibbins et al., 1993). To investigate the relevance of RTK/ SH2-protein interaction parameters for signal definition, we exchanged the ±5 pY-flanking amino acids of the EGFR and NGFR PLCy binding sites, which exhibit strikingly different affinities for this protein (Obermeier et al., 1993a). By this approach, we demonstrated that PLCy affinities are in fact determined by few pY-flanking amino acids. In NIH 3T3 cells, these affinity changes parallel altered biochemical and overall response characteristics of the receptor-mediated signals, demonstrating the critical importance of biophysical parameters in receptor-substrate interactions for signal definition.
Results Exchange of PLCy affinities between EGFR and Trk/ETR Of the parameters that have been proposed to play a role in RTK signal definition, the affinity for SH2 domains of signal-transducing proteins appears to be of critical importance. To investigate the significance of this fundamental biological problem, we employed the receptors for NGF and EGF, which display pronounced differences in their affinities to PLCy. The remarkably high affinity of the activated NGFR/Trk is thought to be defined by a few amino acid residues flanking tyrosine 785, QAPPVYLDVLG, which are different from the site identified in the EGFR to bind PLCy (VDADEYLIPQQ) that surrounds tyrosine 992 (Figure 1A). To investigate the significance of characteristically distinct substrate binding properties 74
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Fig. 2. PLCy coprecipitation by different receptors. 293 cells transiently expressing the indicated receptors were left unstimulated (-) or stimulated (+) with EGF (100 ng/ml) prior to lysis. Receptors were precipitated with mAb 108. Receptor lysates were mixed with equal aliquots of lysate from 293 cells overexpressing PLCy before precipitation (lanes 9-15). Precipitates were subjected to SDS-PAGE and immunoblotted with anti-PLCy mAb (UBI) to visualize quantities of coprecipitated PLCy from endogenous (lanes 1-7) or overexpressed levels (lanes 9-15). As a control, lysates containing the same amounts of PLCy that were present in the corresponding association experiments were subjected to anti-PLCy immunoprecipitation with an excess of antibody (lanes 8 and 16). The amounts and concentrations of total cellular proteins, as well as the amounts of the expressed receptors, were equal in all precipitations, as confirmed by protein determination and Coomassie staining (results not shown).
for the biological response of a cell, we exchanged the sequences thought to comprise the entire PLCy binding site of each receptor and analyzed the biochemical and biological consequences of the resulting exchange (X) mutants. Instead of Trk, we employed in our studies, for experimental reasons, the chimeric receptor ETR, with EGFR extracellular and Trk transmembrane and cytoplasmic signaling domains, which we demonstrated previously to be fully functional and capable of transmitting an NGF signal when stimulated by EGF (Obermeier et al., 1993a,b). Thus, the exchange mutant EGFR.X carried the Trk residues 780-790 in place of the EGFR amino acids 987-997; in ETR, Trk residues 780-790 were replaced by the EGFR sequence 987-997, to yield ETR.X (Figure lB). To confirm the functionality of the mutants and the transfer of PLCy binding properties, receptors were transiently expressed in 293 cells and immunoprecipitated from cell lysates with the monoclonal antibody (mAb) 108 (Honegger et al., 1989), which was directed against the EGFR extracellular domain. The quantity of coprecipitated endogenous PLCy was visualized by the anti-PLCy immunoblotting of anti-receptor precipitates. As shown in Figure 2 (lanes 1-7), EGFR.X exhibited the high affinity for PLCy characteristic of Trk (lane 6), while ETR.X had acquired the low affinity characteristic of the EGFR (lane 3). For a clear detection of ETR.X- and EGFRcoprecipitated PLCy (Figure 2, lanes 12 and 13), it was necessary to employ the lysate from PLCy-overexpressing 293 cells in the association experiments, demonstrating the EGFR-like low affinity of ETR.X. In contrast, the amount of ETR-associated PLCy was only somewhat increased under conditions of PLCy excess (lane 9), indicating that, because of its high affinity, the receptor PLCy binding site was almost saturated at the endogenous substrate concentration. The moderate increase in EGFR.X-associated PLCy in the presence of high amounts of this substrate (lane 15) was possibly caused by the presence of secondary, low affinity binding sites in the EGFR, such as Y 1068 or Y 1173 (Rotin et al., 1992). This
PLCy affinity in signaling by EGFR and Trk
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+ from internal stores. While both the 3T3/pLXSN and 3T3/ ET-YF control cells did not exhibit an IP3 response to EGF stimulation, ETR.X mediated a weak (-30%) and transient elevation of IP3 (Figure 4), comparable with that achieved by EGFR activation. Consistent with the presence of the Trk PLCy binding site, EGFR.X displayed a strong and persistent induction of intracellular IP3, which was even more pronounced than that triggered by the ETR control, reaching 3.5 and 2.0 times basal levels, respectively (Figure 4). Thus, the high binding affinity of ETR and EGFR.X correlated with the strong activation of this second messenger-producing RTK substrate. Consistent with this, an EGF-inducible increase in cytosolic Ca>+ concentration, as a consequence of IP3 action on its receptor, was weaker in cells expressing ETR.X compared with ETR and more pronounced in cells expressing EGFR.X relative to EGFR (Figure 5). No significant change in cytosolic Ca>+ concentrations was measured after the addition of EGF to 3T3/pLXSN and 3T3/ET-YF cells. The lack of a quantitative correlation between cellular IP3 level increases and [Ca>+] peaks exerted in NIH 3T3 cell transfectants suggests an involvement of factors other than IP3 in Ca>+ release from internal stores and/or the existence of a negative feedback effect on IP -activated Ca>+ channels by Ca>+ and IP3 itself, as has been reported recently (Hajnoczky and Thomas, 1994). The data shown in Figure 5 were obtained in Ca2+-free medium and thus represent the Ca>+ release from internal stores only. In the presence of extracellular Ca>+ (1 mM) the results were essentially the same, with the exception that, in general, the peak maxima were higher and the elevated cytosolic Ca>+ concentrations were sustained for a longer period (data not shown). Taken together, these measurements clearly indicated that an increased RTK affinity for PLCy (EGFR-o EGFR.X) results in a stronger and more sustained IP3 signal and a more pronounced ICa2+] peak, whereas a decreased PLCy affinity (ETR--ETR.X) results in a weaker Ca>+ peak and a weaker and more transient IP3 signal. The fact that the PLCy binding-incompetent mutant ET-YF cannot induce any IP3 or Ca>+ signal upon EGF stimulation demonstrates that the observed effects are directly caused by the action of PLCy.
experiments.
To verify that the expressed receptors carried the desired PLCy binding sites, mAb 108 precipitates from 3T3 cell lines were immunoblotted with the anti-Trk ATC antiserum (Obermeier et al., 1 993a) raised against the Trk C-terminal
Receptor dephosphorylation Because of the report of an inhibitory effect of calcium ionophore treatment on RTK autophosphorylation (Gandino et al., 1991), we determined the receptor wild-
75
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Fig. 4. 1P3 formation. Quiescent cell lines werle tr-eated wsith EGF (100)( ne/tsll) for) the inidicalted titacs or left uIntrealted: (control). The 1P1 levels were mealsured a1s described in Malterials aInd methods. Filled bar.s alwayls corrlesponid to the tirst-tiientionled cell line. Dalta represetit the maICIs __+SEM (t a 6)] of an EGE-indiuced increase1P2 in inrelaRtion to the initial IP; (controlG levels aRrbitraril set to ContrXol levels ranged between 0(1 200)(plX:SN. FiT-YF. ETR.X.2. FGF R.4. 100)) 2(1(1 e.p.m./15 cells (ETR. ETR.2. EGFR.F.TR.X.3. EFR.X.I. FGFR.X atiXd
type and mutant dephosphorylation kinetics after stimulation with EGF for 5. 20( 60 or 240 min prior to cell lysis and precipitation with mAb 108. After separation by SDSPAGE, lysates were analyzed by immunoblot detection with anti-pY mAb SE2 (Fendly et (il., 1990). ET-YF and ETR.X mutant receptors displayed, in comparison with ETR, a slightly attenuated time course of dephosphoi-ylation (Figure 6). After 60 min, the receptor pY content had decreased from the 5 min value to 48 and 71 % in 3T3/ ETR. I and 3T3/ETR.2 cells, respectively, compared with 91, 90 and 110% in 3T3/ET-YF, 3T3/ETR.X.2 and 3T3/ ETR.X.3 cells, respectively. A dramatic difference in the dephosphorylation kinetics was obvious between EGFR and EGFR.X. While EGFR autophosphorylation remained equal or even increased up to 240 min in relation to the level reached after 5 min of EGF stimulation, EGFR.X was rapidly downregulated (Figure 6). A determination of the receptor pY state in cell lysates did not allow us to distinguish between actual dephosphorylation and degradation events, nor did it permit an estimate of what proportion of the receptors remained in the plasma membrane from the point of EGF addition or what proportion represented newly expressed or recycled receptors. Nevertheless, it appears that in overexpressing NIH 3T3 cells, the total amount of autophosphorylated and thereby signaling-competent receptors was more rapidly decreased with increasing PLCy affinity and was better sustained when PLCy affinity was decreased or lost.
MAP kinase activation The activation of MAP kinases p44ERKt and p42FK2 is implicated in Ras-dependent mitogenic signaling (reviewed by Marx, 1993). To determine the effects of receptor/PLCy affinity changes on MAP kinase activation, serum-starved cells were stimulated with EGF (20 ng/ml) for 3, 20 and 120 min, followed by the addition of SDS sample buffer and the separationi of equal amounts of total cellular protein on an SDS gel containing myelin basic protein (MBP; 0.5 mg/ml), a substrate of MAP kinases. Proteins in the gel were subjected to an in-gel kinase assay with [y--32PJATP, and phosphorylated MBP was visualized by autoradiography (Figure 7; Kamesita and Fujisawa, 1989; Lee et cil.. 1993).
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The EGF stimulation of 3T3/pLXSN control cells induced a transient MAP kinase activation with a peak after 3 min. In compaiison, 3T3/ETR. I and 3T3/ETR.2 cells exhibited an only slightly increased MBP phosphorylation/activity after 3 min of EGF treatment. In both cases, however, MAP kinase activity was sustained for longer than in control cells. Unexpectedly, compared with ETR-expressing cells, EGF-induced MBP phosphorylation was significantly weaker in ET-YF- and ETR.X-expressing NIH 3T3 cells, with values even lower than in control cells at the 3 min time point. In contrast, EGFR and EGFR.X mediated strong MAP kinase activation substantially above control cell levels, which for the latter was consistently mnore sustained in both cell lines. In summary, in the ETR systemn, mutant receptors ETR.X and ET-YF, with lower or no affinity for PLCy, respectively, exerted an inhibitory effect on MAP kinase activation. Analogously in the EGFR system, an increase in PLCy affinity correlated with a more sustained MAP kinase activation.
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Fig. 8. l3HIThymidine incorporation. Subconfluent quiescent cells incubated or not (0) with EGF (1. 10 or 100 ng/ml) or FCS (10%) for 16 h and labeled with 3H]thymidine for the last 4 h. Levels of [3H]thymidine incorporated into DNA are expressed as c.p.m. Data represent the means of two out of four independent experiments (each performed in duplicate) +SEM (indicated by the error bars). were
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determine the rate of DNA synthesis in subconfluent cells of NIH 3T3 lines. Cells expressing ET-YF or ETR.X displayed an EGF-independent, constitutively high rate of DNA synthesis, characteristic of transformed cells. In contrast, [3H]thymidine incorporation in 3T3/ETR cells was clearly EGF-inducible, with an optimal EGF concentration of 10 ng/ml (Figure 8A). EGFR-expressing cell lines displayed an EGF-dependent rate of DNA synthesis, with the maximum at 100 ng EGF/ml, the highest EGF concentration tested. Meanwhile, EGFR.X-expressing cells showed a more sensitive thymidine incorporation response to EGF, which was maximal at 10 ng/ml (Figure 8B). Similar results were obtained when the measurements were conducted after cells had reached confluency (data not shown), indicating that the mitogenic response mediated by a receptor is paralleled by its transforming potential.
Cell transformation To determine the transforming potential of ETR.X and EGFR.X mutants, 105 NIH 3T3 cells were plated onto 6 cm dishes together with 103 or I04 cells of each receptoroverexpressing NIH 3T3 cell line. After cells had grown to confluency, serum concentrations were reduced from 10 to 4%, EGF (10 ng/ml) was added, and 2 weeks later cell foci were scored. Cells expressing ET-YF or ETR.X formed foci more frequently than ETR-expressing cells, and 3T3/EGFR.X lines produced fewer foci than 3T3/ EGFR lines (Figure 9). The formation of foci requires the loss of the cell-cell contact inhibition, a characteristic attribute of transformed cells. Thus, our data are consistent 77
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