O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important
types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition
to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay
between these two modifications and the biological significance remain unclear, largely due to the technique challenges.
Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent Ophosphate
immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and
phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its
phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations
at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1.
The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed
cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between OGlcNAcylations
and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for
Akt signaling.