Herstatin, an Autoinhibitor of the Human Epidermal Growth Factor Receptor 2 Tyrosine Kinase, Modulates Epidermal Growth Factor Signaling Pathways Resulting in Growth Arrest

Quincey A Justman,Gail M Clinton

doi:10.1074/jbc.m111359200

Abstract

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.

Highlights

The ErbB family of receptor tyrosine kinases includes the prototypical EGFR,1 human epidermal growth factor receptor (HER)-2, HER-3, and HER-4
EGFR3T3 cells were transfected with control or herstatin expression plasmid, and clonal populations were isolated by selection with hygromycin B
Whereas several inhibitors of the EGFR have been investigated, herstatin is distinguished by its novel structure, consisting of subdomains I and II of the HER-2 extracellular ligand binding domain (ECD) and an intron encoded-C-terminal domain [32]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Generation of Stable Herstatin EGFR3T3 Clones— EGFR3T3 cells were derived from NIH3T3 cells by transfection with human EGFR in mammalian expression vector pCDNA3.1 (Invitrogen). Receptor Internalization Assays—Cells were grown to 70% confluence, serum-starved for 20 h in 0.5% FBS, washed twice in ice-cold PBS, and incubated with EGF (Upstate Biotechnology) at 100 ng/ml in cold DMEM for 2 h at 4 °C. Immune complexes were bound to 25 ␮l of protein G-Sepharose (Amersham Biosciences) by co-incubation for 40 min at 4 °C, centrifuged, and washed three times with 1 ml of ice-cold MTG (EGFR) or PBS (Grb). Cells were grown to 70% confluence, serum-starved for 24 h, and incubated at 4 °C for 2 h with 175 pM 125I-EGF (NEN) and different amounts of unlabeled EGF (total EGF concentrations ranged from 175 pM to 10 nM) in binding buffer (DMEM plus 50 mM Hepes, pH 7.4, and 5% (w/v) bovine serum albumin). Cells were washed and extracted in 0.1 N NaOH plus 0.1% (w/v) SDS, and bound 125I-EGF was quantified by gamma counting

RESULTS

Herstatin Blocks EGF Activation of Akt and Growth

DISCUSSION