Regulation of the function of transcription factors by phosphorylation and ubiquitination
Sequence-specific DNA-binding factors are transcription factors that recognize specific enhancer sequences and direct differential gene expression. The motivation for the work described in this thesis is to understand how some of these transcription factors are regulated. The FKHR and NF-κB/Rel families of transcription factors are the two classes of proteins addressed in this thesis. Although both families of transcription factors are regulated by changes in their subcellular localization, the mechanisms underlying their regulation are distinct.
FKHR family members are localized in the nucleus of unstimulated cells. Growth factors such as insulin and PDGF signal the activation of Akt protein kinase and lead to the phosphorylation of Akt substrates including the FKHR family of transcription factors. Phosphorylation of FKHR family members by Akt leads to their nuclear exclusion and consequently the impairment of their abilities to activate transcription. Work described in this thesis describe studies that support the existence for such a mechanism for the regulation of FKHR by Akt and provides evidence for FKHR as a downstream target of Akt in both metabolic and cell survival signaling pathways.
The NF-κB/Rel family of transcription factors are sequestered in the cytoplasm of unstimulated cells by the IκB family of inhibitor proteins. Cytokines such as TNF-α and IL-1 stimulate the activation of a multisubunit IκB kinase (IKK) complex that phosphorylates IκB. Phosphorylation of IκB triggers its degradation by the proteasome and frees NF-κB to enter the nucleus and activate gene transcription. Studies described in this thesis provide evidence for a role of ubiquitination in the regulation of IKK function. IKKγ, an essential IKK subunit, is ubiquitinated in a fashion that does not appear to target it for degradation by the proteasome. This ubiquitination is enhanced by TNF-α treatment of cells and is dependent on an intact zinc finger motif found in IKKγ. Zinc finger mutants of IKKγ are defective in restoring IKK and NF-κB activation by TNF-α in an IKKγ deficient cell line, suggesting that ubiquitination of IKKγ is critical to IKK activation.
0379: Cellular biology