Abstract
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.
Highlights
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival
In the cell mTORC2 is recognized as the major upstream kinase for Ser-473 [11, 16], incorporation of phosphate at Ser-473 is challenging in the test tube
Previous approaches relied on phosphorylation of Ser-473 by MAPKAPK-2, which was pre-activated by p38 in the presence of phosphatidylinositol [3,4,5]-trisphosphate and lipid vesicles [17]
Summary
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. We used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. Activated AKT1 phosphorylates downstream targets that regulate many cellular processes including cell survival and apoptosis [5]. We recently developed an approach to produce AKT1 variants with site-specific phosphorylation at either or both key regulatory sites (Thr-308 and Ser-473) [8, 9]. Substrates with Arg residues at position Ϫ5 and Ϫ3 were found to be specific to AKT, whereas other AGC family kinases, such as the S6 kinase (S6K1), prefer Lys at Ϫ3 and Ϫ5 positions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
