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

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Signaling

Background:

• Akt (Protein Kinase B) is a crucial regulator of cell growth and metabolism, targeted in cancer therapy.
• Akt activation is primarily mediated by phosphorylation at Thr308 and Ser473, involving PDK1 and mTORC2.
• The functional impact of other C-terminal tail post-translational modifications (PTMs) on Akt remains largely uncharacterized.

Purpose of the Study:

• To investigate the regulatory roles of Akt phosphorylation at Tyr474 and O-GlcNAcylation at Ser473.
• To dissect the enzymatic functions of these specific PTMs on Akt activity and substrate specificity.

Main Methods:

• Utilized expressed protein ligation to generate semisynthetic Akt proteins with phosphoTyr474 and O-GlcNAcSer473.
• Performed kinase assays with peptide and protein substrates to assess Akt activity.
• Employed human protein microarrays for global substrate specificity analysis of modified Akt forms.

Main Results:

• O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 partially increase Akt kinase activity.
• Both modified Akt forms exhibit high similarity in protein substrates phosphorylated.
• Validated PPM1H (protein phosphatase) and NEDD4L (E3 ubiquitin ligase) as novel Akt substrates.

Conclusions:

• Phosphorylation at Tyr474 and O-GlcNAcylation at Ser473 represent novel regulatory mechanisms for Akt.
• These PTMs contribute to Akt activation and influence its substrate targeting.
• Identified PPM1H and NEDD4L as key substrates, providing new insights into Akt signaling pathways.