Figure 4. Illustration of the multiple phosphorylation of LS456 by Akt.

The data suggest addition of multiple phosphate groups to one residue of the diserine molecular probe, LS456. After insulin binding to its cell membrane receptors, PI3K was activated to phosphorylate PIP2 into PIP3, which then binds Akt. mTOR complex 2 phosphorylates Akt on the hydrophobic motif ⁴⁷³Ser, and the activated Akt phosphorylates LS456 stepwise to form the mono-, pyro-, and tri-phosphoserine sequentially. (a) Insulin-mediated phosphorylation of LS456. (b) De-phosphorylation of mono- and multiply-phosphorylated LS456 after insulin removal. Phosphatidylinositol 3-kinase (PI3K), phosphatidylinositol-3,4,5-trisphosphate (PIP3), mTOR complex 2 (mTOR C2).