Characterization of Akt overexpression in MiaPaCa-2 cells: prohibitin is an Akt substrate both in vitro and in cells.

Abstract

Akt (PKB) is a serine/threonine protein kinase that plays an important role in the transduction of signals affecting apoptosis, cell proliferation and survival. The Akt gene is frequently hyperactivated in tumors and has been shown to be amplified in a number of types of human cancers. Furthermore, Akt activity is elevated in cell lines with the mutated PTEN tumor suppressor gene. These studies establish Akt as an attractive target for cancer therapy. To determine the roles of Akt1, Akt2 and Akt3 in signal transduction, constitutively active Akt1, Akt2 and Akt3 was ectopically overexpressed in human pancreatic MiaPaCa-2 cells. The three Akt stable clones were characterized to determine their effects on transformation and proliferation. Compared to a vector control, the three Akt clones were able to drive cellular proliferation even in reduced serum conditions. Furthermore, in soft-agar assays, the Akt clones showed an 25-38% increase in colony formation in 2% serum. Our results indicate that all three forms of Akt may have protective effects within the cell depending on the type of apoptotic stimuli. Using 2D-PAGE comparisons between parental and Akt overexpressing cells, we attempted to determine novel targets of Akt phosphorylation. In this study, we identified prohibitin as a substrate for Akt both in vitro and in vivo. These studies suggest that Akt may regulate the cellular function of prohibitin via its phosphorylation.