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048 TYROSINE PHOSPHORYLATION MODULATES DETACHMENT-INDUCED ACTIVATION OF NF-kappaB SIGNALING IN INTESTINAL EPITHELIAL CELLS D Pink1, S Rong Yan1, AW Stadnyk1,2 Detachment of intestinal epithelial cells (IEC) results in a specific type of apoptosis anoikis. Delays in the signals leading to anoikis improve cell survival and increase oncogenic potential. Using rat IEC-18, we investigated tyrosine phosphorylation events contributing to NF-kappaB activation, which we previously showed to be important in IEC survival during anoikis. Detachment resulted in immediate and global tyrosine dephosphorylation, followed by gradual rephosphorylation. Addition of tyrosine phosphatase inhibitors pervanadate (PV, 10µM) or phenylarsine oxide (PAO, 0.5µM) blocked detachment-induced tyrosine dephosphorylation. PAO increased cell death after 8 hr detachment whereas PV had no effect, yet PAO, but not PV, decreased levels of phosphorylated IKKalpha and IkappaBalpha, and of nuclear translocated p65. Additionally PAO caused a time-dependent decay of IKKalpha. Thus, detachment of IEC leads to PAO-sensitive, tyrosine-phosphorylation-dependent NF-kappaB activation despite IKKalpha, IkappaBalpha, and p65/p50 activation by serine and threonine phosphorylation. Therefore we investigated the tyrosine-specific phosphatases, SHP-1 and SHP-2 as potential regulatory molecules as both have been implicated in NF-kappaB signaling and apoptosis. Expression of SHP-1 and SHP-2 protein levels were detected in IEC, but were not changed following detachment. However SHP-1 enzyme activity was significantly increased after detachment, and susceptible to PAO inhibition. In contrast, SHP-2 activity was not altered by detachment, but was PAO-sensitive. Immunoprecipitation experiments revealed that SHP-1 is associated with IKKalpha and IKBalpha.
1Departments of Pediatrics, 2Microbiology and Immunology, The Dalhousie Inflammation Group, Dalhousie University, Halifax, Nova Scotia
Our data reveal that detachment-induced activation of NF-kappaB in IEC is regulated by tyrosine phosphatases; more specifically a role for the PAO sensitive phospho-tyrosine phosphatase SHP-1 has been implicated. SHP-1 has been linked with different forms of cancer, hence understanding the role of SHP-1 in anoikis could lead to potential therapies.
This work was supported by NSERC