PI3K signaling – navigating upstream and downstream of Akt

Growth factors and hormones engage receptors on the surface of cells to initiate intracellular responses essential for tissues to respond to changes in their environment. The engagement of these receptors leads to a signaling cascade in which a number of phosphorylated lipids are produced in the plasma membrane, which serve as signaling molecules for the initiation of downstream pathways that drive the physiological response. This pathway is frequently hyperactivated in human cancers and overgrowth disorders, as well as inactivated in metabolic diseases such as diabetes. Akt is a lipid-activated protein kinase that responds to one of these lipid signaling molecules, phosphoinositide-3,4,5-trisphosphate (PIP3). By phosphorylating downstream effector molecules following its activation by PIP3, Akt controls essential processes such as growth, proliferation, differentiation, and metabolism. Whilst we now understand how Akt activity is restricted to membranes enriched in PIP3, the precise mechanisms by which Akt is activated and inactivated are not known. Akt is activated by another protein kinase, 3-phosphoinositide dependent kinase 1 (PDK1), which also responds to PIP3. In contrast to Akt, PDK1 activates itself by the process of autophosphorylation, though the mechanism is not currently understood. We will investigate precisely how PDK1 is activated in response to growth factors and how it is then able to activate its downstream substrates, including Akt. We will address the precise mechanism by which PIP3 activates Akt itself, and then, finally, we will focus on a phosphatase, the PH domain-containing leucine rich phosphatase (PHLPP) that has been proposed to terminate Akt signaling by removing one of its activating phosphates. We will try to understand how PHLPP has specificity for Akt and the conditions under which it operates on Akt. The proposal will exploit a wide range of structural, biophysical, and biochemical techniques to obtain new insights into the biochemistry of Akt, PDK1, and PHLPP. Whilst PDK1 dimerization and autoactivation have already been well established, the molecular understanding of the mechanism is conspicuously absent. Since PDK1 is often referred to as a master kinase due to its role in activating a number of downstream signaling pathways, and Akt is the central effector of growth factor signaling, the successful execution of this proposal is expected to provide important insights into this critical signaling pathway. These insights will have implications not only for basic research scientists, but also for oncologists and pharmaceutical developers.