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.
Highlights
Akt1 is a Ser/Thr kinase that is a critical node in cell signaling and connects growth factor receptor activation to cell growth and metabolic regulation (Manning and Toker, 2017; Liao and Hung, 2010; Fruman et al, 2017)
These results are consistent with the insect cell expression experiments described above and suggest that the intramolecular pleckstrin homology (PH) domain-kinase domain interactions are altered in a way that interferes with PDK1-catalyzed phosphorylation of the activation loop
Our understanding of the molecular basis of Akt regulation by its PH domain have primarily derived from Akt crystal structures that either lack the PH domain or contain an allosteric inhibitor, HD-exchange studies have been performed on S473D Akt (Lucicet al., 2018)
Summary
Akt (termed Akt in the present work) is a Ser/Thr kinase that is a critical node in cell signaling and connects growth factor receptor activation to cell growth and metabolic regulation (Manning and Toker, 2017; Liao and Hung, 2010; Fruman et al, 2017). It has been suggested that PIP3 activates Akt not by membrane recruitment but by dislodging the PH domain from the kinase domain independent of promoting Akt phosphorylation by upstream kinases (Ebner et al, 2017; Lucicet al., 2018) In contrast to these studies, this allosteric mechanism of Akt activation by PIP3 was not confirmed by our group where neither soluble nor vesicle-embedded PIP3 stimulated. We have used a combination of enzymatic analysis of linker-altered Akts and solution NMR analysis of segmentally isotopically labeled semisynthetic Akt forms to characterize the structural features of the PH domain within the full-length protein in differentially phosphorylated states These structural measurements combined with biochemical data highlight regions in the PH domain that appear to show distinct changes in the context of C-terminal phosphorylation and allosteric drug inhibition
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