Abstract
We report a mechanism by which the adapter protein Gene 33 (also called RALT and MIG6) regulates epidermal growth factor receptor (EGFR) signaling. We find that Gene 33 inhibits EGFR autophosphorylation and specifically blunts epidermal growth factor (EGF)-induced activation and/or phosphorylation of Ras, ERK, JNK, Akt/PKB, and retinoblastoma protein. The Ack homology domain of Gene 33, which contains the previously identified EGFR binding domain, is both necessary and sufficient for this inhibition of EGFR autophosphorylation. The endogenous Gene 33 polypeptide is induced by EGF, platelet-derived growth factor, serum, and dexamethasone (Dex) in Rat 2 rat fibroblasts. Dex induces Gene 33 expression and inhibits EGFR phosphorylation and EGF signaling. RNA interference-mediated silencing of Gene 33 significantly reverses this effect. Overexpression of Gene 33 completely blocks EGF-induced protein and DNA synthesis in Rat 2 cells, whereas gene 33 RNA interference substantially enhances EGF-induced protein and DNA synthesis in Rat 2 cells. Our results indicate that Gene 33 is a physiological feedback inhibitor of the EGFR, functioning to inhibit EGFR phosphorylation and all events induced by EGFR activation. Our results also indicate a role for Gene 33 in the suppression, by Dex, of EGF signaling pathways. We propose that Gene 33 may function in the cross-talk between EGF signaling and other mitogenic and/or stress signaling pathways.
Highlights
Feedback inhibition and cross-talk among signal transduction networks are regulatory mechanisms critical to the coordination of complex biological activities
We report a mechanism by which the adapter protein Gene 33 regulates epidermal growth factor receptor (EGFR) signaling
We find that Gene 33 inhibits EGFR autophosphorylation and blunts epidermal growth factor (EGF)-induced activation and/or phosphorylation of Ras, extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase (JNK), Akt/PKB, and retinoblastoma protein
Summary
Feedback inhibition and cross-talk among signal transduction networks are regulatory mechanisms critical to the coordination of complex biological activities. It has been well documented that the EGFR is subject to ligand-induced down-regulation mediated by receptor internalization and subsequent degradation by lysosome and the ubiquitin/proteasome systems [14, 15] Another negative regulator of EGFR signaling is the activated CDC42associated kinase (Ack), which is believed to facilitate EGFR factor; ERK, extracellular signal-regulated kinase; GST, glutathione Stransferase; HEK, human embryonic kidney; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; Rb, retinoblastoma protein; RNAi, RNA interference; siRNA, small interfering RNA; Ack, activated CDC42-associated kinase; MOI, multiplicity of infection. Dex induces Gene 33 expression and inhibits EGF-induced EGFR autophosphorylation through a mechanism that requires in part Gene 33 These results indicate that Gene 33 is a specific inhibitor of EGFR signaling at the receptor level and that Gene 33 mediates the cross-talk between Dex and EGFR signaling
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