Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis

Cell. 2018 Aug 9;174(4):897-907.e14. doi: 10.1016/j.cell.2018.07.003. Epub 2018 Aug 2.

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.

Keywords: X-ray crystal structure; bisubstrate analog; expressed protein ligation; kinase; mass spectrometry; peptide; phosphorylation; photocrosslinking.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Crystallography, X-Ray
  • Enzyme Activation
  • HCT116 Cells
  • Humans
  • Phosphorylation
  • Pleckstrin Homology Domains
  • Protein Binding
  • Protein Biosynthesis*
  • Protein Conformation
  • Protein Processing, Post-Translational*
  • Proto-Oncogene Proteins c-akt / chemistry
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Serine / chemistry
  • Serine / metabolism*
  • Signal Transduction
  • Threonine / chemistry
  • Threonine / metabolism*

Substances

  • Threonine
  • Serine
  • AKT1 protein, human
  • Proto-Oncogene Proteins c-akt