AKT IS A DIRECT TARGET OF THE PHOSPHATIDYLINOSITOL 3-KINASE - ACTIVATION BY GROWTH-FACTORS, V-SRC AND V-HA-RAS, IN SF9 AND MAMMALIAN-CELLS

The Akt protooncogene encodes a serine-threonine protein kinase which is activated by growth factor-generated signals that are transduced vi a the phosphatidylinositol 3'-kinase (PI3-K). Earlier studies suggeste d that the activation of Akt by PB-K may be mediated by the binding of D-3-phosphorylated phosphoinositides to the Akt pleckstrin homology ( PH) domain. On the basis of these studies, it was hypothesized that Ab t is a direct PB-K target. To test this hypothesis, we reconstituted t he pathway of Akt activation in baculovirus-infected Sf9 cells. The re sults showed that Akt, which is normally catalytically inactive in the se cells, was activated when coexpressed with the activated PI3-K. Mor eover, they showed that activated forms of c-Ha-ras (v-Ha-ras) and c-s rc (v-src or srcY527F), two molecules that transduce growth factor-gen erated signals, also activate Akt in a PI3-K-dependent manner in Sf9 a s well as NIH 3T3 cells, The activation of Akt by both growth factors and v-ras and v-src (or srcY527F) depends on the integrity of the Akt PH domain and carboxyl-terminal tail. These results show that Akt acti vation via the PB-B: can be faithfully reproduced in baculovirus-infec ted Sf9 cells, The same results support the hypothesis that Akt is a d irect target of the PI3-K and identify cytoplasmic signaling molecules that may contribute to the transduction of PI3-K/Akt activation signa ls.