Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis

Abstract

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.

Autoinhibition can identify rare driver mutations and advise pharmacology

“…mTOR complex 2 (mTORC2) phosphorylation at pSer473 at the C-terminal promotes its interaction with the N-lobe of the kinase domain and Arg144 in the PHkinase linker, relieving the PH-kinase domain autoinhibiting interaction. 97 An E17K mutation in the PH domain causes constitutive activation of the kinase domain, 98 a scenario in line with other membrane recruitments for catalysis and signaling ( Figure 2A). 103 Gly insertions in the PH-kinase domain linker indicate that linker flexibility stabilizes the PH-kinase domains autoinhibited interaction.…”

Control of Akt activity and substrate phosphorylation in cells

“…This “PIF pocket” mechanism is common for many AGC kinases and was also proposed for Akt. Indeed, the in vitro activity of Akt monophosphorylated on T308 is only a fraction of the maximal, and phosphorylation of S473 in the HM or presence of the peptides mimicking the latter was often reported to increase Akt in vitro activity ten‐ to hundred‐fold …”

“…Crystal structures of the unphosphorylated and T308‐phosphorylated kinase domain lacking the HM are nearly identical, suggesting that engagement of the PIF pocket could stabilize the active conformation. However, recent studies using protein semisynthesis and genetic code expansion demonstrated that while phosphorylation of S473 increased activity of T308‐phosphorylated Akt in vitro , the former site is essentially dispensable for the activity in cells . Indeed, mutation of S473 or genetic ablation of mTORC2 components have only minor effect on cellular Akt phosphorylation and activity …”

“…Residues surrounding S477 (but not T479) partially resemble a Cdk2 consensus sequence, and its phosphorylation preceded that of S473, prompting the authors to suggest that S477 primes Akt to phosphorylation by mTORC2. An alternative model proposed that S477 and T479 interact with the PH‐KD linker, helping destabilize the autoinhibitory interface (see in the following), though the authors' own data argue against this model, showing that phosphorylation of S477 and T479 increased K M for ATP to millimolar (3.7 mM) range. The exact role of the newly discovered C‐terminal phosphorylation sites in Akt regulation as well as the structural insight into their role in the context of full‐length Akt remain to be established.…”

“…In line with this, PI3K inhibition or mutations in the PH domain interfering with lipid binding all prevent Akt activation and phosphorylation of its substrates. However, deletion of the PH domain also dramatically increased Akt activity in vitro , suggesting that it could also act as an allosteric site.…”