Electronic Thesis and Dissertation Repository


Doctor of Philosophy




O'Donoghue, Patrick.

2nd Supervisor

Li, Shawn S.-C.

3rd Supervisor

Junop, Murray.


The proto-oncogene Akt/protein kinase B plays a pivotal role in cell growth and survival. Phosphorylation of Akt at Thr308 and Ser473 activates the kinase following growth factor stimulation. Delineating specific role of each activation site in Akt1 on kinase activation, inhibition and substrate selection remain elusive.

We designed a unique set of tools, relying on genetic code expansion with phosphoserine and in vivo enzymatic phosphorylation, to produce differentially phosphorylated Akt1 variants. We found that having both sites phosphorylated increased the apparent catalytic rate of the enzyme by 1500-fold relative to the unphosphorylated enzyme. This increment was mainly due to the phosphorylation of Thr308 but not Ser473 which was confirmed via live cell imaging. We further found that the traditional use of phosphomimetics was unable to mimic the effect of p-Thr308 in the test tube and in cells.

Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. Using the same strategy, 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 Akt1 phospho-variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. The robustly active PH domain deleted enzyme variants were used in enzyme inhibition and substrate selectivity studies. We found that phosphorylation at Ser473 provided resistance to chemical inhibition by the Akt inhibitor Akti-1/2.

Finally, we used both defined and randomized peptide libraries to map the substrate selectivity of singly and doubly phosphorylated Akt1 variants. The data revealed that each phospho-form of Akt1 has distinct substrate requirements. Surprisingly, phosphorylation of Ser473 in the context of a pAkt1T308 enzyme led to increased activity on some, but not all Akt1 substrates. We also verified a new Akt1 target as the terminal nucleotidyltransferase (germline development 2) Gld2.

In conclusion, the site-specifically phosphorylated Akt1 variants that we produced enabled in characterizing phosphorylation-dependent activity, inhibition and substrate selectivity of the oncogenic kinase Akt1. Since phosphorylation status of Akt1 is used as a cancer biomarker, these variants can act as indispensable tools in further characterizing downstream oncogenic pathways and screening potential drug candidates.

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