The structural determinants of PH domain-mediated regulation of Akt revealed by segmental labeling

Abstract

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.

Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation

“…The PIP 3 -Binding Site Is Sequestered in Autoinhibited Akt. While structures of Akt in complex with various allosteric inhibitors have provided evidence for an autoinhibitory interaction between the PH and kinase domains (20,21,23,32), the extent to which these inhibitor-bound complexes reflect the physiological conformation and regulation of Akt is still controversial. In order to resolve these issues, we set out to determine the structure of Akt1 without the use of inhibitors.…”

Targeting the PI3K/AKT/mTOR Signaling Pathway in Lung Cancer: An Update Regarding Potential Drugs and Natural Products

“…AKT is activated in response to the binding of PIP3 to the PH domain of AKT, which leads to conformational changes in AKT (PH-out conformation) and translocation to the plasma membrane. The PH-out conformation exposes the CAT and regulatory domains, resulting in phosphorylation at two main residues: threonine residues in the activation loop of the CAT domain (Thr308 in AKT1, Thr309 in AKT2 and Thr305 in AKT3) and serine residues in the HM domain (Ser473 in AKT1, Ser474 in AKT2 and Ser472 in AKT2) [ 150 , 151 ]. Moreover, ATP occupies the ATP binding site located in the CAT domain to decelerate the dephosphorylation of AKT, leading to its full activation [ 152 ].…”

Akt Isoforms: A Family Affair in Breast Cancer

“…Cdk2/cyclin A also phosphorylated Akt2 at S478 and synergized with S474 phosphorylation at the HM site to allosterically activate Akt2 [ 41 ]. Recently, it was reported that amino acids 44-46 (DVD) at the PH domain affect C-terminal phosphorylation [ 42 ]. Triple mutation of DVD (Asp Val Asp) to GPG (Gly Pro Gly) affected phosphorylation of Akt at S473 and T308 but not at S477 and S479.…”