Purpose of EGFR in Akt phosphorylation induced by neurotensin EGF induced a marked phosphorylation of Akt in HCT116 cells, indicating activation on the phosphoinosi tide three kinase pathway Neurotensin also stimulated phosphorylation of Akt, although not as strongly as EGF The result of neurotensin on Akt to start with appeared soon after three min, whereas ERK phosphor ylation was evident currently at 1 min Moreover, in contrast to the data indicating a PKC mediated activation of ERK, neurotensin induced phosphorylation of Akt was not impacted by inhibition of PKC and was not mimicked by TPA We upcoming examined the means of neurotensin to induce tyrosine phosphorylation of EGFR in HCT116 cells. Fig ure 5A exhibits that treating the cells with neurotensin or EGF resulted in phosphorylation in the EGFR.
Despite the fact that the result of neurotensin was obviously less than that of EGF, the phosphorylation induced by each these ago nists was blocked CC292 by pretreatment with the EGFR tyro sine kinase inhibitor gefitinib In addition, we uncovered that neurotensin stimulated phosphorylation of Shc which can be an adaptor protein that binds to, and is phosphorylated by, active RTKs Taken together, these results suggest the EGFR is often transactivated by neurotensin in HCT116 cells. Pretreatment with gefitinib strongly attenuated neuro tensin induced phosphorylation of Akt in HCT116 cells In these experiments, TGFa was made use of because the EGFR ligand, as well as impact of TGFa on Akt phos phorylation was pletely abolished by gefitinib.
Neu rotensin also induced Akt phosphorylation in HT29 and Panc one cells Whereas this effect was abol ished by pretreatment with gefitinib in HT29 cells neither gefitinib nor the PKC inhibitor GF109203X inhibited neurotensin VX702 stimulated Akt phos phorylation in Panc 1 cells Neurotensin induced transactivation within the EGFR is partly mediated by shedding of extracellular ligands Evidence from countless cell sorts indicates that transactiva tion from the EGFR induced by GPCRs could possibly be mediated from the activation of cell surface proteinases, resulting in subsequent shedding of EGFR ligands or by intra cellular mechanisms involving kinases just like Src and Pyk2 To discover more the mechanism of the gefitinib sensitive Akt phosphorylation induced by neuro tensin, we examined the result of cetuximab, an antibody which binds on the extracellular domain of the EGFR and therefore blocks the skill of ligand induced activation. As expected, EGF stimulated phosphorylation of the two Shc and Akt was pletely inhibited by cetuximab Cetuximab pretreatment also blocked neurotensin stimulated Shc phosphorylation, suggesting the involve ment of a ligand dependent mechanism.