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Abstract
Protein kinase B (Akt1) is a proto-oncogene that is overactive 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 auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition 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 pAkt1T308 and showed four-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.
Highlights
Protein kinase B (Akt) is a human serine–threonine kinase and a member of the AGC family of protein kinases [1,2]
The approach combines in vivo enzymatic phosphorylation with genetic code expansion to produce Akt1 variants containing either or both pThr308 and pSer473 (Figure 1)
Thr308 was site- phosphorylated by co-expression of the upstream kinase phosphoinositide-dependent kinase 1 (PDK1) in E. coli
Summary
Protein kinase B (Akt) is a human serine–threonine kinase and a member of the AGC family of protein kinases [1,2]. The pathway regulated by Akt is the most commonly activated signaling pathway in human cancers [3]. There are three AKT genes in humans, encoding the isozymes Akt, Akt, and Akt. The Akt isozyme has well-established roles in many human cancers. Overactive Akt is a hallmark of diverse human malignancies [3,4] and linked to reduced survival outcomes [5,6]. Akt is as a leading drug target in cancer [7,8]. Over 300 clinical trials have been completed or are under way that involve targeting the Akt signaling pathway [9,10]
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