AKT ACTIVATION BY GROWTH-FACTORS IS A MULTIPLE-STEP PROCESS - THE ROLE OF THE PH DOMAIN

The protein kinase encoded by the Akt proto-oncogene is activated by p hospholipid binding, membrane translocation and phosphorylation. To ad dress the relative roles of these mechanisms of Akt activation, we hav e employed a combination of genetic and pharmacological approaches, Tr ansient transfection of NIH3T3 cells with wild-type Akt, pleckstrin ho mology (PH) domain mutants, generated on the basis of a PH domain stru ctural model, and phosphorylation site Akt mutants provided evidence f or a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by cons titutive phosphorylation of threonine 450 (T450) and perhaps serine 12 4 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translo cation step; and (3) a PI3-K-dependent step, characterized by phosphor ylation at T308 and S473, that occurs in the cell membrane and is requ ired for activation, When forced to translocate to the membrane, wild- type Akt and PH domain Akt mutants that are defective in the first ste p become constitutively active, suggesting that the purpose of this st ep is to prepare the protein for membrane translocation, Both growth f actor stimulation and forced membrane translocation, however, failed t o activate a T308A mutant, This, combined with the finding that T308D/ S473D double mutant is constitutively active, suggests that the purpos e of the three-step process of Akt activation is the phosphorylation o f the protein at T308 and S473, The proposed model provides a framewor k for a comprehensive understanding of the temporal and spatial requir ements for Akt activation by growth factors.