Abstract Background Protein tyrosine phosphatases (PTPs) are known to be responsible for the negative regulation of many receptor tyrosine kinases by serving as antagonists to tyrosine kinase signaling. Several PTPs have been identified as candidate regulators of EGFR phosphorylation such as PTP1B, RPTP-sigma, RPTP-kappa, and PTPRJ (DEP-1). Here, we focused on PTPRO, a member of R3 family. The Drosophila ortholog of this family, Ptp4E/Ptp10D, negatively regulates EGFR signalling. Gene expression analysis of 688 primary tumours led us to identify PTPRO as a gene with strongly reduced expression in colorectal cancers with poor prognosis in particular the ones belonging to the Braf mutant subgroup. This suggests that PTPRO could act as a tumour suppressor by negative regulation of EGFR in colorectal cancer. Material and methods HEK293 cells were transiently transfected with wild-type (WT) PTPRO and with its catalytic mutant (C/S) and stimulated with EGF. After cell lysis, extracts were analyzed by SDS-PAGE and probed by western blotting with an anti-phosphotyrosine antibody (pY99, 4G10). The total level of EGFR is detected with anti-EGFR antibody. EGFR phosphorylation antibody arrays are used to identify the specific tyrosine phosphorylation sites. Results and discussion Upon expression of the PTPRO (C/S) mutant, we observe a delay in EGF induced EGFR degradation. Our preliminary data suggests that PTPRO might play a role in EGFR trafficking through the endocytic pathway and its catalytic activity is required for efficient endocytic progression of EGFR. Ongoing experiments will answer to this question. WT-PTPRO over-expression specifically decreases the Tyr 845 phosphorylation of EGFR, a known Src family tyrosine kinase (SFK) phosphorylation site. SFKs also regulate receptor turnover both at the level of endocytosis and cbl-mediated ubiquitination by phosphorylation. However, underlying mechanism how PTPRO can modulate EGFR signalling through the regulation of endocytic machinery is not known yet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1218. doi:1538-7445.AM2012-1218