Phosphorylation-Dependent Inhibition of Akt1

Abstract

Akt1 is a proto-oncogene that is over active in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal autoinhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in sitespecifically phosphorylated forms, the contribution of each activating phosphorylation site to autoinhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1 T308 and showed 4-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

Neuronal activity‐induced SUMOylation of Akt1 by PIAS3 is required for long‐term potentiation of synaptic transmission

“…MK‐801 also blocked the binding of Akt1‐PIAS1 (Figure S1B). The enzymatic activity of Akt1 is reported to be fully stimulated by both T308 and S473 phosphorylation 25,26 . We found that Bicuculline treatment increased Akt1 T308 phosphorylation, while relatively stable levels of S473 phosphorylation were maintained (Figure 1A).…”

“…The enzymatic activity of Akt1 is reported to be fully stimulated by both T308 and S473 phosphorylation. 25,26 We found that Bicuculline treatment increased Akt1 T308 phosphorylation, while relatively stable levels of S473 phosphorylation were maintained (Figure 1A). These data implicate that NMDA receptordependent neuronal activity upregulates Akt1 SUMO1ylation mainly by PIAS3 and facilitates Akt1 activity by accelerating T308 phosphorylation.…”

“…The brains isolated from Sprague-Dawley rats (postnatal days 21-30) of both sexes were incubated in ice-cold artificial cerebrospinal fluid (ACSF; 290-300 mOsm, pH 7. 26. Hippocampal slices (400μm) were prepared using a Vibroslicer from the middle of the hippocampus, incubated with ACSF saturated with 95% O 2 and 5% CO 2 at 28℃ for 1 hour, and ultimately subjected to electrophysiological recordings.…”