No abstract

Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr 694 ) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr 694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr 694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF-and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases.The ErbB family of receptor tyrosine kinases, consisting of the epidermal growth factor (EGF) 1 receptor ErbB1, ErbB2/ Neu, ErbB3, and ErbB4, binds a large group of ligands, the EGF-related peptides (1, 2). Upon ligand-induced dimerization, the receptors autophosphorylate on specific tyrosine residues in their cytoplasmic tails. These residues provide docking sites for phosphotyrosine binding, cytoplasmic signaling molecules that activate numerous intracellular signaling pathways. In some cases, for example the mitogen-activated protein (MAP) kinase pathway, the important intermediates in the signaling cascade have been well described (3). Receptor tyrosine kinasemediated activation of signal transducer and activators of transcription (Stat) proteins, however, is less well understood.Stats were initially identified as a family of transcription factors activated by engagement of interferon (IFN) and cytokine receptors, which lack intrinsic kinase activity and depend upon the receptor-associated Janus kinases (Jaks) to transduce signals. Cytokine receptor oligomerization brings the Jaks into juxtaposition, leading to their cross-phosphorylation and activation. Jaks in turn phosphorylate receptors on tyrosine residues, thereby providing...

Patients with high-grade serous ovarian cancer (HGSC) frequently receive platinum-based chemotherapeutics, such as cisplatin. Cisplatin binds to DNA and induces DNA-damage culminating in mitochondria-mediated apoptosis. Interestingly, mitochondrial DNA is critically affected by cisplatin but its relevance in cell death induction is scarcely investigated. We find that cisplatin sensitive HGSC cell lines contain higher mitochondrial content and higher levels of mitochondrial ROS (mtROS) than cells resistant to cisplatin induced cell death. In clonal sub-lines from OVCAR-3 mitochondrial content and basal oxygen consumption rate correlate with sensitivity to cisplatin induced apoptosis. Mitochondria are in two ways pivotal for cisplatin sensitivity because not only knock-down of BAX and BAK but also the ROS scavenger glutathione diminish cisplatin induced apoptosis. Mitochondrial ROS correlates with mitochondrial content and reduction of mitochondrial biogenesis by knock-down of transcription factors PGC1α or TFAM attenuates both mtROS induction and cisplatin induced apoptosis. Increasing mitochondrial ROS by inhibition or knock-down of the ROS-protective uncoupling protein UCP2 enhances cisplatin induced apoptosis. Similarly, enhancing ROS by high-dose ascorbic acid or H2O2 augments cisplatin induced apoptosis. In summary, mitochondrial content and the resulting mitochondrial capacity to produce ROS critically determine HGSC cell sensitivity to cisplatin induced apoptosis. In line with this observation, data from the human protein atlas (www.proteinatlas.org) indicates that high expression of mitochondrial marker proteins (TFAM and TIMM23) is a favorable prognostic factor in ovarian cancer patients. Thus, we propose mitochondrial content as a biomarker for the response to platinum-based therapies. Functionally, this might be exploited by increasing mitochondrial content or mitochondrial ROS production to enhance sensitivity to cisplatin based anti-cancer therapies.

The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGFactivated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.The ErbB family of receptor tyrosine kinases has four members: epidermal growth factor (EGF) receptor (ErbB-1), ErbB-2, ErbB-3, and ErbB-4. The ErbB receptors are expressed in epithelial, mesenchymal, and neuronal tissue and play fundamental roles during development. Two of the family members, ErbB-1 and ErbB-2, are involved in the development of many types of human cancer (reviewed in references 29 and 44).A large family of growth factors, the EGF-related peptides, serve as ligands for ErbB receptors (42,44). The ligands fall into three groups: EGF, amphiregulin (AR), and transforming growth factor ␣, which bind ErbB-1; betacellulin, epiregulin, and heparin binding EGF-like growth factor, which bind both ErbB-1 and ErbB-4; and Neu differentiation factors (NDFs) or heregulins, which are ligands for ErbB-3 and ErbB-4.Ligand binding promotes ErbB receptor homo-and heterodimerization. Although no direct ligand for ErbB-2 has been identified, it appears to be the preferred heterodimerization partner of all ErbB proteins (21,30,48). Despite the lack of an ErbB-2-specific ligand, homodimerization of this receptor can be achieved by mutating a single amino acid residue in the transmembrane domain (1), leading to constitutive ErbB-2 dimerization and activation. Alternatively, antibody binding to the extracellul...

ErbB (also termed HER) receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin, in which they are involved in the control of diverse biological processes such as proliferation, differentiation, migration and apoptosis. Furthermore, their deregulated expression has been implicated in many types of human cancers and is associated with poor clinical prognosis. Owing to the importance of ErbB proteins in both development and cellular transformation, a lot of attention has been drawn to the intracellular signals initiated by the engagement of this family of receptor tyrosine kinases. This review will focus on the membrane proximal events triggered by the ErbB receptor network and will address questions of how receptor heterodimerization may contribute to signal specification and diversification.