C65
The ovarian TSG OPCML interacts with the ErbB family of receptor tyrosine kinases, abrogating growth factor-induced autophosphorylation and downstream signaling
Arthur McKie, Imoh Okon, Dmitry Shaposhnikov, Hani Gabra
Ovarian Cancer Action Research Centre, Imperial College London, London, UK
We previously identified the OPCML gene on 11q25 being consistently inactivated through epigenetic silencing in >80% of epithelial ovarian carcinomas (EOC). This GPI-anchored cell surface protein slows in vitro proliferation and suppresses tumorigenicity in xenograft models, thus fulfilling the criterion as a functional tumour suppressor in EOC [1]. To identify the functional role of this protein, we initially undertook a candidate approach to identify OPCML-interacting proteins. We demonstrated by quantitative RT-PCR that the endogenous expression of OPCML is acutely upregulated in a number of ovarian cancer cell lines upon stimulation with epidermal growth factor (EGF). OPCML mRNA levels exhibit a very rapid increase in response to EGF stimulation (20-fold up regulation at 30 minutes), suggesting an immediate early response, consistent with a feedback inhibitory role as described by Citri and Yarden [2]. In pull-down experiments using a commercial OPCML antibody (R&D Systems), we demonstrate co-immunoprecipitation (co-IP) of the two ErbB proteins; EGFR/HER1 and ErbB2/HER2 with OPCML upon Western blotting. We have demonstrated OPCML-specific abrogation of EGFR/ErbB2 auto-phosphorylation upon EGF stimulation (at Y1068 and Y1173 on EGFR, Y1248 on ErbB2). OPCML-specific reduction in activation of downstream components of MAPK pathway, namely phospho-ERK1&2 is also observed. We have further utilized an EGFR phospho-tyrosine array membrane from Panomics to demonstrate reduced binding of EGFR from stimulated OPCML-expressing cells to the Abl1 SH2 domain. Although precise role of Abl1 in EGFR signaling has not been established, it has been implicated in inhibiting Cbl2-mediated EGFR endocytosis [3].
References
[1] Sellar GC, Watt KP et al. (2003) Nat Genet 34, 337-343.
[2] Citri A and Yarden Y (2006) Nat Rev Mol Cell Biol 7, 505-516.
[3] Tanos B and Pendergast AM (2006) J Biol Chem 281, 32714-32723.
