Editors' ChoiceCell Biology

Cycling Akt Activity

Sci. Signal.  29 Apr 2014:
Vol. 7, Issue 323, pp. ec116
DOI: 10.1126/scisignal.2005422

Cells need to carefully control the activity of kinases, especially those that are involved in regulating cell proliferation and survival, such as Akt (also known as protein kinase B). Liu et al. found that phosphorylation of Akt (an indication of activity) fluctuated with the cell cycle, peaking at the same time as cyclin A, which starts to accumulate in S phase and peaks in G2. Akt1 had four putative cyclin A–binding motifs, and mutation of two of these motifs substantially reduced coimmunoprecipitation of Akt1 with cyclin A and also reduced its phosphorylation. In vitro kinase assays and mass spectrometry analysis revealed that cyclin A associated with cyclin-dependent kinase 2 (Cdk2) phosphorylated Ser477 and Thr479 in the C-terminal tail of Akt1, which are near the activating phosphorylation site Ser473. Surprisingly, these two sites did not conform to the consensus motif for cyclin A/Cdk2 phosphorylation, and their location near the C terminus may provide the structural flexibility that is typically supplied by the proline residue in the consensus motif. Analysis of phosphorylation of Akt1 in cells by immunoblotting or immunofluorescence confirmed that these sites were phosphorylated in a manner that fluctuated with the cell cycle and was dependent on cyclin A/Cdk2 activity. Two other kinases, mechanistic target of rapamycin complex 2 (mTORC2) and DNA-dependent protein kinase (DNAPK) also stimulated phosphorylation of Ser477 and Thr479, suggesting that these residues also contribute to the activity of Akt in response to growth factor stimulation and DNA damage, respectively. Phosphorylation of Ser473 and Thr308 was undetectable in cells in which Akt1 was reconstituted with a mutant that could not be phosphorylated at the C-terminal residues (Akt1-S477A/T479A) and was enhanced in cells reconstituted with the phosphorylation-mimetic mutant Akt1-S477D/T479E, suggesting that phosphorylation of the C-terminal residues was necessary for phosphorylation of the two other sites. Cells expressing the phosphomimetic Akt1-S477D/T479E exhibited increased tumor growth in mouse xenografts. Brain-specific knockout of cyclin A reduced Ser477 and Thr479 phosphorylation in the olfactory bulb and increased markers of apoptosis, suggesting that these cell cycle–regulated phosphorylation events may be critical for cell survival.

P. Liu, M. Begley, W. Michowski, H. Inuzuka, M. Ginzberg, D. Gao, P. Tsou, W. Gan, A. Papa, B. M. Kim, L. Wan, A. Singh, B. Zhai, M. Yuan, Z. Wang, S. P. Gygi, T. H. Lee, K.-P. Lu, A. Toker, P. P. Pandolfi, J. M. Asara, M. W. Kirschner, P. Sicinski, L. Cantley, W. Wei, Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. Nature 508, 541–545 (2014). [PubMed]