Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry.
Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival.
Section of Mass Spectrometry, Laboratory of Membrane Biophysics and Biochemistry, NIAAA, National Institutes of Health, Bethesda, Maryland 20892-9410, USA.
Bill X
BX
Huang
Hee-Yong
HY
Kim
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D008239:Lysine; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D003432:Cross-Linking Reagents; D051057:Proto-Oncogene Proteins c-akt; D008239:Lysine