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Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR¹

Institute of Immunology, University of Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany; and
^* Institute of Clinical Chemistry and Laboratory Medicine, University of Münster, 48129 Münster, Germany

³Correspondence: Institute of Immunology, University of Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany. E-mail: thomasd{at}uni-wh.de

SPECIFIC AIM

The aim of this study was to explore the underlying molecular mechanisms of EGF-induced EGFR homo- and EGFR/c-erbB-2 heterodimer signaling resulting in a different timing of the kinetics of PLC-1-dependent signal transduction. We point out that the EGF-mediated induction of cell migration via PLC-1 signaling depends particularly on tyrosine-phosphorylated c-erbB-2 residue 1248.

PRINCIPAL FINDINGS

1. The PLC-1 signal transduction cascade is regulated differently by EGFR homo- and EGFR/c-erbB-2 heterodimers
We recently showed that EGF-treated, EGFR/c-erbB-2 double-positive cell lines [MDA-MB-468-HER2 (MDA-HER2)] displayed a different PIP₂ turnover from solely EGFR-overexpressing cells [MDA-MB-468-NEO (MDA-NEO)] that likely is due to a differently regulated PLC-1 kinetic in these cells. Therefore, the PLC-1-dependent signal transduction of both cell lines was analyzed in detail. MDA-NEO cells revealed long-term PLC-1 tyrosine phosphorylation (Fig. 1 A) concomitant with sustained levels of IP₃ and DAG (Fig. 1B ). In accordance with the sustained IP₃ levels, we observed sinusoidal calcium oscillations (mean frequency: 5.6±2.8 min^-1) in this cell line (Fig. 1C ). In contrast to MDA-NEO cells, MDA-HER2 cells displayed a short but rapid increase in IP₃ and DAG after EGF application (Fig. 1B ). IP₃ levels fell to initial values 300 and 600 s after EGF treatment whereas elevated levels of DAG were still detectable (Fig. 1B ), consistent with the PLC-1 tyrosine phosphorylation in MDA-HER2 cells (Fig. 1A ). Due to short-term IP₃ turnover, MDA-HER2 cells displayed baseline calcium spiking (mean frequency: 2.5±1.4 min^-1) after EGF stimulation (Fig. 1C ).

View larger version (41K): [in this window] [in a new window]   Figure 1. PLC-1 activation mediated by EGF-induced EGFR homo- and EGFR/c-erbB-2 heterodimers. MDA-NEO and MDA-HER2 cells were stimulated with 100 ng/mL EGF for 0, 30, 60, 120, 300, and 600 s. A) Upper row: Western blot analysis of tyrosine-phosphorylated PLC-1. Lower row: same blot but reprobed for total PLC-1. B) Time course of IP3 (solid line) and DAG (dashed line) turnover of EGF-treated MDA-NEO (diaf) and MDA-HER2 (squlf) cells. C) Time course of calcium oscillations of EGF-treated MDA-NEO (diaf) and MDA-HER2 cells (squlf).

2. EGFR kinase activity is required for induction of the PLC-1-dependent signal transduction in EGFR/c-erbB-2-positive breast cancer cells
To prove whether the observed different time frames of PLC-1 activation mediated by EGFR homo- and EGFR/c-erbB-2 heterodimers were attributed exclusively to erbB receptor signaling, we analyzed PLC-1 activation of both cell lines in the presence of the specific EGFR tyrosine kinase inhibitor TAG1478. Since the mode of calcium oscillation is determined by the kinetics of PLC-1-activation, the mode of the calcium signaling was used as a read-out. In the presence of 30 nM TAG1478, the frequency of calcium oscillations of both cell lines was inhibited by nearly 90% (MDA-NEO: 100 ng/mL EGF: 5.6±2.8 min^-1 vs. 100 ng/mL EGF+30 nM/mL TAG1478: 0.8±1.0 min^-1; MDA-HER2: 100 ng/mL EGF: 2.5±1.4 min^-1 vs. 100 ng/mL EGF+30 nM/mL TAG1478: 0.3±0.4 min^-1). These results indicate that PLC-1 activation depends on EGFR in either homo- or heterodimer signaling. Unexpectedly, the blockade of PLC-1 using the PLC-1 inhibitor U73122 yielded different results. A moderate concentration of 2 µM U73122 (IC₅₀ concentration) sufficiently blocked PLC-1 activity, indicated by a completely inhibited calcium signaling in MDA-HER2 cells. In contrast to MDA-HER2 cells, MDA-NEO cells still displayed unchanged sinusoidal calcium oscillations in the presence of 2 µM U73122. Since the adaptor protein PLC-1 cannot be activated merely by tyrosine phosphorylation but also independently of tyrosine phosphorylation by lipid compounds such as arachidonic acid or phosphatidylinositol-3,4,5-bisphosphate, we conclude that in MDA-NEO cells additional signal transduction pathways participate in PLC-1 activation.

3. Modulation of the kinetics of PLC-1-activation by EGFR/c-erbB-2 heterodimer signaling depends on c-erbB-2 tyrosine residue 1248
The aforementioned data indicate that the time frame of PLC-1 activation is directed by c-erbB-2 in EGFR/c-erbB-2 heterodimers. To substantiate this assumption, we generated an expression plasmid coding for a c-erbB-2 receptor containing a point mutation at amino acid position 1248 (Y1248F), which was transfected into the MDA-MB-468 cell line to establish the stable cell line MDA-MB-468-PM (MDA-PM). Neither PLC-1 tyrosine phosphorylation, production of IP₃ and DAG, nor calcium oscillations could be observed in this cell line after stimulation with 100 ng/mL EGF. This clearly demonstrates that the PLC-1 signal transduction pathway is completely abrogated and that c-erbB-2 tyrosine residue 1248 is required for subsequent PLC-1 activation.

4. EGFR/c-erbB-2-expressing breast cancer cells respond to EGF by a subtle actin reorganization and enhanced migratory activity due to c-erbB-2-modulated PLC-1 activity
To study how the kinetics of the PLC-1 signal transduction regulated by c-erbB-2 contributes to cell migration, we investigated the reorganization of the actin cytoskeleton as well as the migratory behavior within a 3-dimensional collagen lattice of all MDA cell lines. Prolonged EGF-induced PLC-1 tyrosine phosphorylation in MDA-NEO cells resulted in an increased average degree of actin polymerization in EGF-treated MDA-NEO cells (Fig. 2 ,upper row) consistent with no increased migratory activity regarding either the quantity of migrating cells (displacement) or the velocity or the distance migrated. In contrast, EGF-stimulated MDA-HER2 cells displaying a short-term PLC-1 tyrosine phosphorylation showed a coordinated actin polymerization promoting lamellipodia formation (Fig. 2 ; middle row, white arrowheads) consistent with a significantly increased locomotory activity ranging from +80 ± 18% (displacement), +149 ± 38% (velocity) to +288 ± 93% (distance migrated). In accordance with the U73122 dose-dependent inhibition of calcium oscillations observed in the MDA-NEO and MDA-HER2 cell line, analogous results were obtained for the EGF-induced actin reorganization and migration of each cell. Neither actin reorganization nor migratory activity was affected by 2 µM U73122 whereas actin rearrangement, lamellipodia formation, and induction of migration of MDA-HER2 cells were completely blocked by 2 µM U73122. Due to an impaired PLC-1-dependent signal transduction in MDA-PM cells, neither actin reorganization (Fig. 2 , lower row) nor induction of cell migration was observed after EGF stimulation. These results clearly show that activation of PLC-1 leading to induction and maintenance of cell migration is attributed to c-erbB-2 phosphotyrosine 1248 in EGFR/c-erbB-2 heterodimer signaling.

View larger version (68K): [in this window] [in a new window]   Figure 2. Treatment with EGF results in F-actin reorganization in MDA-NEO and MDA-HER2 but not in MDA-PM cells. MDA cells were seeded in chamber slides and treated with 100 ng/mL EGF for 0, 30, 60, 120, 300, and 600 s. Bar = 10 µm.

CONCLUSIONS AND SIGNIFICANCE

ErbB receptors mediate growth stimulatory signals and trigger proliferation, differentiation, and migration in several physiological processes including embryogenesis, organogenesis, tissue regeneration, wound healing, and metastasis. Here we clearly point out the importance of a c-erbB-2-dependent EGFR/c-erbB-2 heterodimer signaling regulating a distinct timing of PLC-1-dependent signal transduction, resulting in actin reorganization and lamellipodia formation, thereby inducing cell migration. Incubation of those MDA-HER2 cells expressing EGFR/c-erbB-2 heterodimers with the PLC-1-specific inhibitor U73122 totally abrogated IP3 production, calcium oscillation, actin reorganization, and thus cell migration.

The pivotal role of the c-erbB-2-dependent EGFR/c-erbB-2 heterodimer signaling for PLC-1-dependent cell migration was further substantiated by generating the MDA-PM cell line expressing the c-erbB-2-Y1248F mutated receptor. In these cells, no actin reorganization was seen and no cell migration occurred due to partially impaired, PLC-1-dependent signal transduction, since the activation of actin-modifying enzymes requires PLC-1-mediated PIP₂ hydrolysis. This suggests that the site-specific phosphorylation of c-erbB-2 tyrosine residue 1248 is decisive for modulation of the PLC-1-dependent signal transduction, thereby initiating and maintaining cell migration.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the presented results. Expression of c-erbB-2 or c-erbB-2-Y1248F, respectively, results in formation of the appropriate heterodimer after EGF stimulation. EGFR/c-erbB-2 heterodimer signaling modulates the time course of PLC-1 activation, thereby inducing and maintaining cell migration. In contrast, the PLC-1 signal transduction cascade is completely abrogated in MDA-PM cells, clearly indicating the pivotal role of c-erbB-2 in modulating the time frame of PLC-1 activation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0096fje; to cite this article, use FASEB J. (September 19, 2002) 10.1096/fj.02-0096fje

² Both authors contributed equally to this work.

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