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Akt activation by growth factors is a multiple-step process: the role of the PH domain
A Bellacosa1, T O Chan, N N Ahmed
1Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Abstract:
The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.
Insights
Akt protein activation involves three sequential steps: PH domain interaction, PI3-K-dependent membrane translocation, and phosphorylation at T308/S473. This process is crucial for growth factor signaling.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The Akt proto-oncogene encodes a protein kinase central to cell signaling pathways.
- Akt activation is known to involve phospholipid binding, membrane translocation, and phosphorylation.
- The precise sequence and interplay of these activation mechanisms require further elucidation.
Purpose of the Study:
- To delineate the sequential steps and relative importance of different mechanisms in Akt protein activation.
- To investigate the roles of the pleckstrin homology (PH) domain and specific phosphorylation sites in Akt activation.
- To develop a comprehensive model for growth factor-induced Akt activation.
Main Methods:
- Utilized transient transfection of NIH3T3 cells with wild-type Akt, PH domain mutants, and phosphorylation site mutants.
- Employed genetic and pharmacological approaches to study Akt activation.
- Analyzed the effects of forced membrane translocation and growth factor stimulation on various Akt mutants.
Main Results:
- Proposed a three-step model for Akt activation: PH domain-dependent priming, PI3-K-dependent membrane translocation, and PI3-K-dependent phosphorylation at T308 and S473.
- Demonstrated that PH domain mutants defective in the first step become constitutively active upon forced membrane translocation.
- Showed that mutations at T308 prevent activation, highlighting the critical role of T308 and S473 phosphorylation in the final activation step.
Conclusions:
- The initial PH domain-dependent step primes Akt for subsequent membrane translocation.
- Membrane translocation and subsequent phosphorylation at T308 and S473 are essential for full Akt activation.
- The proposed model clarifies the temporal and spatial requirements for Akt activation by growth factors, emphasizing the phosphorylation events at T308 and S473.