STAT Activation by Epidermal Growth Factor (EGF) and Amphiregulin

Abstract

The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF-and AR-induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1-independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation.The activated epidermal growth factor (EGF) 1 receptor mediates a number of important biological responses in mammalian cells including the stimulation of cell division, migration, and differentiation (1). Six different polypeptide ligands, which derive from distinct genes, are capable of binding to the extracellular domain of the EGF receptor. These ligands include EGF, transforming growth factor-␣, amphiregulin (AR), heparin binding EGF-like growth factor, epiregulin, and betacellulin (2). All of these growth factors contain a characteristic EGF-like domain, which is defined by 6 evenly spaced cysteine residues that generate 3 loops through the formation of disulfide bonds. In the case of AR it has been demonstrated that heparan sulfate proteoglycans are necessary for activation of the EGF receptor and a cellular response (3). Upon ligand binding, the intrinsic tyrosine kinase activity of the EGF receptor is augmented, resulting in autophosphorylation as well as the phosphorylation of specific intracellular substrates (4). Among the target proteins that become phosphorylated in response to EGF are mitogen-activated protein kinase, the adapter protein SHC, the GTPase-activating protein of p21 ras , phospholipase C-␥, the phosphotyrosine phosphatase 1D, as well as the non-receptor tyrosine kinase Jak1 (5) and members of the STAT (signal transducers and activators of transcription) family of transcription factors (6 -8). Several reports have shown that the intrinsic receptor kinase activity is necessary for activation of a subset of these proteins, whereas others can be activated also via a kinase-inactive EGF receptor (9 -11). Additional mutations have allowed for the identification of receptor domains that are needed for cell motility, mitogenesis, and activation of mitogen-activated protein kinase or phospholipase C-␥ (12, 13). In the case of the STAT proteins it has been sugges...

Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma

“…Deletion of EGFR carboxyterminal tyrosine residues that conform to the consensus YXXQ led to the loss of STAT activation by this receptor in NIH3T3 cells (Co er and Kruijer, 1995). On the other hand an EGFR mutant lacking all carboxyterminal tyrosine phosphorylation sites is still able to induce tyrosine phosphorylation and DNA binding of STAT1, 3 and 5 in NR6 ®broblasts (David et al, 1996). Therefore, although Xmrk is permanently tyrosine phosphorylated in PSM cells and thus might provide substrate docking sites, phospho- tyrosines may not be required for STAT activation by RTKs of the EGFR family.…”

“…Therefore, although Xmrk is permanently tyrosine phosphorylated in PSM cells and thus might provide substrate docking sites, phospho- tyrosines may not be required for STAT activation by RTKs of the EGFR family. One factor that seems to be essential for STAT activation by RTKs is an intact kinase domain, since a kinase de®cient EGFR mutant fails to stimulate DNA binding of STATs (Leaman et al, 1996;David et al, 1996). Inhibition of Xmrk by the tyrosine kinase inhibitor AG555 resulted in a loss of constitutive STAT5 tyrosine phosphorylation in ®sh melanoma cells, indicating that this phosphorylation is depent on an active Xmrk kinase either in a direct or indirect way.…”

“…Injection of EGF into mice caused tyrosine phosphorylation and speci®c DNA-binding of STAT5 in liver (Ru -Jamison et al, 1995). Both, EGF and PDGF mediated STAT5 tyrosine phosphorylation was detected in ®broblasts expressing the respective receptors (David et al, 1996;Vignais et al, 1996). However, apart from its activation through RTKs, STAT5 is playing its major role in signal transduction of the prolactin receptor and other cytokine receptors such as the IL-2-, IL-3-, the growth hormone and the erythropoietin (Epo) receptor in a variety of di erent cell types Ga en et al, 1995;Lin et al, 1996;Pallard et al, 1995;Xu et al, 1996).…”

STAT activation by the PDGF receptor requires juxtamembrane phosphorylation sites but not Src tyrosine kinase activation

“…Another tyrosine kinase receptor, the EGF receptor, directly binds STATs but the signi®cance is again unclear. The EGF receptor directly interacts in vitro with the STAT1 SH2 domain, in a phosphorylation-dependent manner, yet all known phosphorylation sites in the EGF receptor are dispensable for EGF-induced STAT activation in vivo (David et al, 1996). This suggests that STAT activation does not require STAT binding to the EGF receptor.…”

“…Thus for growth factor signaling, either JAKs are not involved or are redundant. Direct interaction of STAT proteins with receptor tyrosine kinases has been suggested, implying direct phosphorylation of STATs by the receptors; however proof of direct interactions in vivo is lacking (Fu and Zhang, 1993;Novak et al, 1996;David et al, 1996;Chen et al, 1997).…”