Multifaceted Regulation of Akt by Diverse C-Terminal Post-translational Modifications

Antonieta L. Salguero, Maggie Chen, Aaron T. Balana, Nam Chu, Hanjie Jiang, Brad A. Palanski, Hwan Bae, Katharine M. Wright, Sara Nathan, Heng Zhu, Sandra B. Gabelli, Matthew R. Pratt, Philip A. Cole

Research output: Contribution to journalArticlepeer-review


Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt’s kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.

Original languageEnglish (US)
Pages (from-to)68-76
Number of pages9
JournalACS chemical biology
Issue number1
StatePublished - Jan 21 2022

ASJC Scopus subject areas

  • Molecular Medicine
  • Biochemistry


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