Understanding the pleckstrin homology (PH) domain peculiar mechanism in akt translocation, phosphorylation, and activation

The protein kinase B (PKB, also designated Akt), is unarguably a crucial player in cell proliferation, survival, metabolism, angiogenesis, and apoptosis. Akt plays a pivotal role in the Ras-PI3K-Akt-mTOR signaling pathway. Akt recruitment to the plasma membrane is enabled by its Pleckstrin homology (PH) domain, which interacts with signaling membrane lipid, PIP3, or PIP2. The interplay between the PH domain and PIP3 results in conformational changes that facilitate phosphorylations of Thr308 in the kinase domain, and Ser473 in the C-terminal regulatory domain by PDK1 and mTORC2 complex, respectively. Enormous Akt activation mechanisms have been proposed by various studies, which all seem to result in PH domain peculiar role in translocating Akt to the plasma membrane, and to implication of Calmodulin (CaM) intermolecularly interacting with the PH domain in breast cancer as established by previous NMR studies. However, the exact mechanism of how CaM interacts with the PH domain at the atomic level remains unclear. Also, in Akt autoinhibition state or a "PH-in" conformer, the PH domain intramolecularly interact with the kinase domain to prevent phosphorylation of a functional residue in the kinase domain. Several residues in the PH-kinase allosteric interface maintain the PH-kinase domain autoinhibition, and mutations of critical residues in the PH-kinase domain interface have the proclivity to disrupt the interface, resulting in a "PH-out" conformer state. A "PH-out" conformer of Akt is required for the PH domain to interact with membrane lipids, an event critical for Akt activation. Furthermore, phosphorylation of Ser473 in the C-terminal tail that forms electrostatic interaction with Arg 144 in the PH-kinase linker leads to conformational rearrangements in the PH domain activating Akt. However, the structure of full-length Akt autoinhibited state is yet to be crystalized to understand the PH and kinase domain autoinhibition. Here, this dissertation purpose is two-fold: using modeling and molecular dynamics (MD) simulations to figure out how CaM interacts with the PH domain to recruit Akt to the plasma membrane and how the PH domain intramolecularly interact with the kinase domain at the atomic level with emphasis on the interfacial residues that play a crucial role in the PH-kinase domain autoinhibition. In the dissertation's first part, CaM-PH domain complexes were modeled and subjected to all atoms MD simulations. The simulation results show that CaM-PH domain interactions are thermodynamically stable and involve a 𝝱-strand, rather than an 𝛂-helix interaction, both agreeing with the NMR data and that electrostatic and hydrophobic interactions are critical to maintaining CaM-PH complex. The PH domain interacts with CaM lobes; however, multiple modes are possible, and the involvement of IP4, polar head of PIP3 attenuates CaM-PH domain interaction, implicating the release mechanism at the plasma membrane. In the second part of the dissertation, iv we modeled full-length Akt (480 residues) in the inactive state to explore the intramolecular interaction between the PH and kinase domains and identify crucial interfacial residues that maintain the PH-kinase intact interface. Further, Asp 323, an important interface residue was mutated to His to discern its effect on PH-kinase allosteric interface. The results show that the mutation led to substantial displacement of ATP from the ATP binding pocket in Akt and led to kinase domain adopting a more open conformation. Additionally, the RMSD and RMSF profiles depict that D323H leads to an increase in conformational changes, and we deduce that although the mutation is approximately 21Å away from ATP binding site, perhaps there is an allosteric communication between these two functional sites in Akt. The modeled structures of full-length autoinhibited Akt state are best representations that would guide pharmaceutical chemists to develop Akt allosteric inhibitors, which require an intact PH-kinase interface, and ATP-competitive inhibitors that do not require such intact PH-kinase interface but might rely on intact kinase domain N- and C- lobes interface for their cellular activity. The development of allosteric and ATP-competitive inhibitors is crucial for targeting Akt since it is frequently activated in tumor cells, necessitating its regulation. The dissertation results have functional implications in developing inhibitors that would target CaM-PH domain complex and allosteric and ATP-competitive inhibitors that bind to the PH-kinase domain interface and ATP binding pocket in Akt, respectively. Finally, these results add to the already existing incredible wealth of information on the Akt kinase activation mechanism, which all seem to result from loosening the PH-kinase domain autoinhibition.