Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates Akt signaling.
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 O-phosphate 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 O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.
Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Shuai
S
Wang
Xun
X
Huang
Danni
D
Sun
Xianliang
X
Xin
Qiuming
Q
Pan
Shuying
S
Peng
Zhongjie
Z
Liang
Cheng
C
Luo
Yiming
Y
Yang
Hualiang
H
Jiang
Min
M
Huang
Wengang
W
Chai
Jian
J
Ding
Meiyu
M
Geng
D002460:Cell Line; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D051057:Proto-Oncogene Proteins c-akt