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Date of Award
The P13K/Akt pathway is a critical mediator of growth factor signaling involving many cellular functions. The deregulation of this pathway has been shown to be involved in the development of various cancers. One of the main targets of this pathway is FoxO3a, a transcription factor whose target genes are involved in important cellular processes such as apoptosis, cell cycle control, and glucose metabolism. FoxO3a is regulated by various post translational modifications including acetylation, ubiquitination and phosphorylation. The transcription factor is directly phosphorylated by Akt on 3 residues: Threonine 32, Serine 253 and Serine 315. Phosphorylation by Akt generates binding sites for 14-3-3, a protein which regulates FoxO3a DNA binding, transcriptional activity, and nuclear localization. Once phosphorylated, FoxO3a accumulates in the cytoplasm where it is subsequently degraded. Our goal is to further elucidate the functional significance of the Akt-FoxO3a interaction and the mechanism of FoxO3a's regulation by Akt and 14-3-3. Our data supports the possibility that Akt increases the steady-state levels of FoxO3a, a function which is associated with Akt binding to FoxO3a. This has been demonstrated using the inactive Akt K179M mutant variant which is incapable of binding to FoxO3a. In addition, our data demonstrates that the RXRXXS/T motifs (Akt phosphorylation site recognition motifs) present in FoxO3a are important for Akt's ability to phosphorylate FoxO3a but not for its ability to bind FoxO3a or increase the steady-state levels of the protein. By mutating a residue located within an important region for FoxO3a-14-3-3 binding (P34A), we demonstrated that 14-3-3 binding to FoxO3a plays a role in protecting the phosphorylated FoxO3a pool from dephosphorylation. Our data supports a model where Akt, when inactive, serves as a positive regulator of FoxO3a by stabilizing the protein and 14-3-3, through binding primarily at the T32 site, plays a role in protecting FoxO3a from dephosphorylation.
Upon testing P34A FoxO3a's transcriptional activity via luciferase reporter assays, we observed that it had activity similar to the current constitutively active triple mutant model, 3XA (T32, S253, S315A). Using HepG2 cells to study activity (due to the ability to inactive Akt activity by serum starvation and removing insulin) we show that the P34A mutant is active under conditions which are suppressive to wild type FoxO3a, signifying that this mutant is a constitutive active FoxO3a variant. We also tested P34A FoxO3a activity in the prostate cancer cell line, PC3. These cells have high levels of active Akt due to loss of the PTEN tumor suppressor and therefore make an ideal model for studying FoxO3a. As was seen with the insulin stimulated HepG2 cells, P34A FoxO3a activity was higher than both over expression of wild type FoxO3a as well as the triple mutant, 3XA FoxO3a. PC3 stable cells lines were generated of FoxO3a variants (wild type, 3XA, P34A and DNA B.M.). The stable cell lines with active FoxO3a variants underwent apoptosis at the initial passages, demonstrating the detrimental effects active FoxO3a has on PC3 cell viability. Overall, this data suggest that active FoxO3a may be capable of inducing apoptosis in prostate cancer cells that rely on high PI3K/Akt pathway activity for their survival.
Dobson, Melissa Elise, "Regulatory And Functional Aspects Of Foxo3a Transcription Factor And Their Implications In Prostate Cancer" (2010). Wayne State University Dissertations. Paper 59.