The forkhead box (Fox) family of proteins is a large family of transcription factors with diverse physiological functions ranging from craniopharyngeal development to language acquisition and hearing. The evolutionary conservation of these proteins from yeast to humans and diverse biological functions highlight the importance of these proteins in developmental processes. All members of the Fox family share a conserved 110 amino acid DNA-binding domain that is referred to as the ‘forkhead box’ or ‘winged helix’ domain. It is comprised of three a-helices, three p-sheets and two loops, resembling the shape of butterfly wings and giving forkhead proteins an alternate name of ‘winged helix proteins’. DNA binding of these transcription factors relies on interaction between the highly conserved third helix in the forkhead box and DNA bases within the major groove of DNA. Residues in the two loops of the forkhead box, which display greater amino acid sequence variability, make additional contacts with the DNA-binding element, and these interactions contribute to the binding site selectivity of the different forkhead proteins. Over 100 forkhead genes have been identified to date, and in humans, this family of transcription factors has been subdivided into 19 subgroups (FoxA-FoxS) based on sequence similarity.
Members of the Forkhead O (FoxO) subfamily of transcription factors, which is the focus of this dissertation, share the characteristic of being regulated by the PI3K/AKT signaling pathway in response to growth factor and insulin stimulation. The FoxO family of transcription factors plays an important role in diverse physiologic processes, including induction of cell cycle arrest, stress resistance, apoptosis and differentiation. Four members of the FoxO family, FoxO1, FoxO3, FoxO4, and a more recently identified FoxO6, are important downstream targets of the evolutionarily conserved PI3K/AKT pathway that transduces survival signals in response to insulin and growth factor stimulation. FoxO1, FoxO3 and FoxO4 ex
FoxO function is regulated by the PI3K/AKT pathway. PI3K kinase is a lipid kinase that transduces survival signals in response to growth factor stimulation via phosphorylation of phosphoinositides. Specifically, PI3K catalyzes the conversion of phosphatidylinositol 4,5 bisphosphate (PIP2) to phosphatidylinositol 3,4,5 trisphosphate (PIP3), resulting in an increase in PIP3 at the cell membrane. Accumulation of these second messengers leads to the recruitment and activation of the serine/threonine kinase AKT (also known as protein kinase B (PKB)) and SGK (serum and glucocorticoid inducible kinase). AKT activation requires phosphorylation on Thr308 by PDK-1, and Ser473 by mTORC2. Following activation, AKT triggers a spectrum of physiologic cellular responses, ranging from cell proliferation and survival, to cell growth, metabolism and longevity, via its many downstream regulators, including the Bcl2 family of proteins, caspases, NF/cB, cell cycle regulators, mTORCI and the FoxO family of transcription factors. In the presence of growth factors, such as IGF1, insulin, interleukin 3, erythropoietin or epidermal growth factor, AKT and SGK directly phosphorylate FoxO1, FoxO3 and FoxO4 at three conserved serine and threonine residues in the nucleus. Phosphorylation of FoxO results in nuclear exclusion and subsequent transcriptional inactivation mediated by proteosomal degradation. AKT activity is negatively regulated by the dual specificity lipid and protein phosphatase PTEN that catalyzes the conversion of PIP3 to PIP2, thereby inhibiting PI3K signaling. Thus, the PI3K/AKT/FoxO pathway is a tightly regulated signal transduction pathway that plays a central role in mediating diverse cellular physiologic processes.