Christian Wallasch

EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.

Cross-communication between different signalling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. The transactivation of epidermal growth factor receptor (EGFR)-dependent signalling pathways upon stimulation of G-protein-coupled receptors (GPCRs), which are critical for the mitogenic activity of ligands such as lysophosphatidic acid, endothelin, thrombin, bombesin and carbachol, provides evidence for such an interconnected communication network. Here we show that EGFR transactivation upon GPCR stimulation involves proHB-EGF and a metalloproteinase activity that is rapidly induced upon GPCR–ligand interaction. We show that inhibition of proHB-EGF processing blocks GPCR-induced EGFR transactivation and downstream signals. The pathophysiological significance of this mechanism is demonstrated by inhibition of constitutive EGFR activity upon treatment of PC3 prostate carcinoma cells with the metalloproteinase inhibitor batimastat. Together, our results establish a new mechanistic concept for cross-communication among different signalling systems.

Signal characteristics of G protein-transactivated EGF receptor

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen‐activated protein kinase (MAPK) in response to G protein‐coupled receptor (GPCR) agonists in Rat‐1 fibroblasts. This cross‐talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS‐7 cells. Transient expression of either Gq‐ or Gi‐coupled receptors in COS‐7 cells allowed GPCR agonist‐induced EGFR transactivation, and lysophosphatidic acid (LPA)‐generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq‐ and Gi‐coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3‐kinase did not affect GPCR‐induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA‐ and EGF‐induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq‐ and Gi‐coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor‐derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG‐induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3‐kinase in transfected 293 and mammary carcinoma‐derived MCF‐7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG‐dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG‐induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.

Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

ABSTRACT The class II phosphoinositide 3-kinases (PI3K) PI3K-C2α and PI3K-C2β are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2α and PI3K-C2β represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2α and PI3K-C2β immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca2+ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2α is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2β was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2β with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2β occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2β was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3′ phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

Distinct Calcium-dependent Pathways of Epidermal Growth Factor Receptor Transactivation and PYK2 Tyrosine Phosphorylation in PC12 Cells

Recently, we have demonstrated that in PC12 cells activation of the Ras/extracellular signal-regulated kinase pathway in response to membrane depolarization or bradykinin is mediated by calcium-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we address the question whether Ca2+-calmodulin-dependent protein kinase (CaM kinase) has a role in the EGFR transactivation signal. Using compounds that selectively interfere with either CaM kinase activity or calmodulin function, we show that KCl-mediated membrane depolarization-triggered, but not bradykinin-mediated signals involve CaM kinase function upstream of the EGFR. Although both depolarization-induced calcium influx and bradykinin stimulation of PC12 cells were found to induce c-fos transcription through EGFR activation, the former signal is CaM kinase-dependent and the latter was shown to be independent. As PYK2 is also activated upon elevation of intracellular calcium, we investigated the potential involvement of this cytoplasmic tyrosine kinase in EGFR transactivation. Interestingly, we observed that inhibition of CaM kinase activity in PC12 cells abrogated tyrosine phosphorylation of PYK2 upon KCl but not bradykinin treatment. Nevertheless, PYK2 activation in response to both stimuli appeared to be mediated by pathways parallel to EGFR transactivation. Our data demonstrate the existence of two distinct calcium-dependent mechanisms leading either to EGFR-mediated extracellular signal-regulated activation or to PYK2 tyrosine phosphorylation. Both pathways either in concert or independently might contribute to the definition of biological responses in neuronal cell types.

SHP-2, SH2-containing protein tyrosine phosphatase-2.

SHP-2 is an ubiquitously expressed cytosolic protein tyrosine phosphatase composed of two amino-terminal SH2 domains, a central phosphatase domain and a carboxy-terminal tail. Upon activation of cells with different stimuli, SHP-2 is recruited to the plasma membrane where it can associate with a number of tyrosine phosphorylated molecules, including receptor tyrosine kinases (e.g. growth factor receptors), multisite adapter proteins and cell adhesion molecules. SHP-2 is thought to function as a positive mediator of signals generated by activated membrane receptor complexes although the number and diversity of binding partners and substrates identified thus far suggests that it may have other functions. It is likely that several negative regulatory influences exist but that these are obscured by its positive function making the investigation of the inhibitory effects of this phosphatase difficult. The positive regulatory role of SHP-2 in signal cascades leading to cell growth suggests involvement in tumorigenesis, raising the possibility that SHP-2 may be a target in the treatment of some forms of cancer.

Distinct characteristics of heregulin signals mediated by HER3 or HER4.

Members of the epidermal growth‐factor‐receptor tyrosine‐kinase (EGFR) family play important roles both in normal growth regulation/cell differentiation and in the genesis and progression of human neoplasia. In the present study, we analysed distinct heregulin (HRG) signals mediated by the HRG receptors HER3 and HER4. In overexpression cell systems, we demonstrate that HRG‐induced transformation by “kinase‐impaired” HER3 is dependent on coexpression of kinase active HER2. In cells coexpressing HER2 and HER4, however, both kinases significantly contribute to the HRG‐induced mitogenic stimulus. In addition, we show that HER3 is no substrate of HRG‐activated HER4. Analysis of EGFR crosstalk in a panel of human carcinoma cell lines revealed mainly HRG‐induced activation of HER2/HER3, whereas HER4 activation is also detectable to various extents. Evidence for HRG‐induced activation of HER3 and/or HER4 indicates relevance of cell‐specific expression patterns of these high‐ and low‐affinity HRG receptors in the modulation of a ligand‐induced stimulus. Specific signal modulation and definition can be demonstrated further by distinct time courses of mitogen‐activated protein (MAP) kinase (MAPK) activation, which are induced by distinct HRG isotypes via differential binding to HER2/HER3 versus HER2/HER4. In concert, these mechanisms of signal modulation may be decisive for the diverse biological activities of HRG in different cell types. J. Cell. Physiol. 173:187–195, 1997.

HER2/neu: a target for breast cancer therapy.

The receptor tyrosine kinase HER2 and its rat homologue neu were independently identified as close relatives of erbB, the gene encoding the epidermal growth factor receptor (EGFR). Genomic analysis of primary tumors revealed HER2 gene amplification and overexpression in breast and ovarian adenocarcinomas and demonstrated strong correlation with poor prognosis. Since its validation as the first human oncogene, HER2 has been intensively investigated as a target for therapeutic intervention. Nevertheless, it is still poorly understood how HER2 overexpression and enhanced cellular signaling contributes to the development of human cancer. Here we summarize the signaling characteristics of HER2 in regard to its function in tumorigenesis and address recent advances towards the pharmacological intervention in HER2 signaling and anti-cancer therapy.