Neurotensin Phosphorylates GSK-3A/B through the Activation of PKC in Human Colon Cancer Cells

Neurotensin (NT), a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR)] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3) regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3A/B in the human colon cancer cell line HT29, HCT116, or SW480, which possesses highaffinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of mitogen-activated protein kinase/extracellular signalregulated kinase (MEK1) or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3b phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCa and PKCb1) or downregulation of endogenous PKCa or PKCb1 blocked NT-mediated GSK-3b (but not GSK-3a) phosphorylation. Moreover, a selective PKCb inhibitor, LY379196, reduced NTmediated GSK-3b (but not GSK-3a) phosphorylation, suggesting a role for PKCb1 in the NT-mediated phosphorylation of GSK-3b and an undefined kinase in the NT-mediated phosphorylation of GSK-3a. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth. Neoplasia (2006) 8, 781–787