**3.2 The AIB1 life cycle**

The stability and half-life of the AIB1 protein, once translated, is regulated by a series of phosphorylation and ubiquitination events (see **Figure 1**). There is a balance achieved between multi-mono-ubiquitination and long chain poly-ubiquitination that creates a phosphorylation dependent 'time clock' for the stability of AIB1. GSK3 phosphorylates AIB1 at S505 and S509 in the absence of AKT signaling to promote its multi-mono- or poly-ubiquitination by SCFFbw7<sup>α</sup> at K723 and K726. Amplified AKT signaling inactivates GSK3. Multi-(mono)ubiquitination of AIB1 then promotes hormone dependent activity through interaction with the estrogen receptor, whereas poly-ubiquitination promotes degradation of AIB1 [33].

An alternative variety of phosphatases regulate proteasome mediated turnover by binding and dephosphorylating AIB1. PDXP, PP1, and PP2A dephosphorylate AIB1 at S101 and S102, and negatively regulate Estrogen Receptor binding and cooperation. PP1 specifically stabilizes AIB1 by preventing proteasomal turnover by dephosphorylating the N-terminal C-region degron at the S101 and S102 residues. Though more stable, decreased affinity for the estrogen receptor significantly reduced AIB1 mediated cell growth in estrogen dependent cell lines [34]. Regulation of AIB1 protein levels is thus regulated by a complex series of phosphorylation and ubiquitination events in response to cell signaling and stimulation. The phosphorylation and ubiquitination sites may be unique to the pathways promoting turnover, and thus further study is warranted to better understand the signaling cascades that control AIB1 levels.

**53**

*The AIB1/NCOA3/SRC-3 Oncogene*

nuclear receptors.

*DOI: http://dx.doi.org/10.5772/intechopen.80925*

**3.3 AIB1 post-translational modifications modulate activity**

**4. AIB1 potentiates hormone receptor signaling**

Estrogen, progesterone and androgen receptors (ER, PR, AR) are nuclear steroid receptors, which play a major role in sexual development and reproduction. Upon binding to their ligands, these receptors dimerize and translocate to the nucleus. They act as transcription factors by binding directly to unique DNA sequences termed response elements. Subsequently, histone modifying enzymes and transcriptional co-regulators are recruited to activate target gene transcription. AIB1 has been shown to directly interact with ER, PR and AR via its LXXLL motifs [39, 40]. There are two classes of estrogen receptors; ERα and ERβ. AIB1 binds and enhances ERα receptor-stimulated gene transcription in a ligand-dependent manner [6]. Upon binding to ERα, AIB1 recruits chromatin-remodeling histone acetyl-transferases enzymes and thus increases ERα transcriptional activity. On the other hand, AIB1 can regulate ERα protein levels when bound to estradiol (E2). AIB1 recruits ubiquitin-proteasome complex to the ligand bound ERα leading to its degradation. Studies have shown that when AIB1 levels are reduced, ERα levels are stabilized [41]. In AIB1−/− mice, delays in puberty and mammary gland development as well as aberrant reproductive functions have been reported [5]. Similar observations are seen in both PR−/− and ER−/− mice emphasizing the essential role of AIB1 in ER and PR dependent functions. In breast cancer, AIB1 potentiates the development of hormone-dependent tumors and contributes to antiestrogen resistance [42, 43]. Lacking the inhibitory domain, AIB1Δ4 isoform has been shown to potentiate ER and PR transcription activity to a much greater extent than AIB1 [20]. In breast tumor samples, the association between AIB1 level and both ER and PR levels has not been clearly determined. One study showed that amplification of AIB1 in breast cancer correlates with high expression of ER and PR [44]. Yet another study showed that overexpression of AIB1 in breast cancer samples was associated

AIB1 is phosphorylated at different serine and threonine residues throughout the different domain structures by a variety of kinases. As a result, AIB1 is responsive to many different upstream signaling cascades, contributing to its oncogenic nature. JNK, p38, ERK, IKK, and PKA can all phosphorylate AIB1 at different residues to promote interaction with CBP and subsequently activate transcription (see **Figure 1**). Phosphorylation sites within AIB1 have been well profiled as mediators of certain protein-protein interactions. Six phosphorylation sites were originally described as essential for interaction with the estrogen and androgen receptors (T24, S505, S543, S857, S860, and S867). Conversely, phosphorylation at only T24 and S867 was required for TNF mediated NFκB interaction and activity [35]. These phosphorylation events in response to estradiol, TNFα, and upstream IGF signaling thus activate AIB1 to interact with partners and potentiate transcription [35–37]. Additionally, we have found that Abl kinase phosphorylates AIB1 at Y1357 in response to IGF, EGF and estradiol stimulation, which results in AIB1 interaction with essential chromatin modifying enzymes. This phosphorylation event is critical for AIB1's coactivator function [38]. Phosphorylation of AIB1 is thus a critical step in activation of the protein and is mediated by a variety of upstream signals that converge on the oncogenic coactivator. Some phosphorylation sites are required for all described activity, yet much work needs to be done to better understand what regulates the selectivity of AIB1 to bind with specific transcription factors and

*Oncogenes and Carcinogenesis*

experimentation.

protein interactions.

**3.2 The AIB1 life cycle**

**3.1 Transcriptional regulation of AIB1**

**3. Modifications and PTMs regulate AIB1 activity**

Post-translational modifications (PTM) are critical to modulating the stability and activity of AIB1. Within the 165 kDa protein, there are multiple phosphorylation and ubiquitination sites, some of which have been well described by recent

AIB1 protein level is regulated by multiple processes, with levels primarily peaking during the cell cycle. AIB1 autoregulates its own expression and is recruited to its own promoter in complex with E2F1 (see Section 6.3). AIB1 transcription is responsive to cell cycle cues mediated by Rb hypophosphorylation and resulting activation of E2F; thus, AIB1 levels increase during G1, and attenuate during S phase (when comparing relative levels during the cell cycle) [29]. Downregulation of AIB1 protein levels is, in part, mediated by the FoxG1 tumor suppressor, which acts by interacting with AIB1 and disrupting the interaction with E2F1 on its own promoter (FoxG1 additionally interrupts AIB1's activity with other transcription factors, such as NFκB, AP-1, and the Estrogen Receptor) [30]. Tight control of the AIB1 activity is regulated by its own positive feedback and tempered by inhibitory

AIB1 is also targeted by microRNA that regulates its expression. miR-17-5p targets at least two sites on the AIB1 mRNA, and miR-20b also binds to AIB1 mRNA. The two miRs are negatively correlated with AIB1 expression and loss of miR expression is associated with taxol resistance in breast cancer [31, 32]. These miRs, in addition to down regulating AIB1, interact with multiple other proteins to differentially regulate their gene expression. As a result, they are implicated in the progression or suppression of several cancers, in a context dependent manner.

The stability and half-life of the AIB1 protein, once translated, is regulated by a series of phosphorylation and ubiquitination events (see **Figure 1**). There is a balance achieved between multi-mono-ubiquitination and long chain poly-ubiquitination that creates a phosphorylation dependent 'time clock' for the stability of AIB1. GSK3 phosphorylates AIB1 at S505 and S509 in the absence of AKT signaling

Amplified AKT signaling inactivates GSK3. Multi-(mono)ubiquitination of AIB1 then promotes hormone dependent activity through interaction with the estrogen

An alternative variety of phosphatases regulate proteasome mediated turnover by binding and dephosphorylating AIB1. PDXP, PP1, and PP2A dephosphorylate AIB1 at S101 and S102, and negatively regulate Estrogen Receptor binding and cooperation. PP1 specifically stabilizes AIB1 by preventing proteasomal turnover by dephosphorylating the N-terminal C-region degron at the S101 and S102 residues. Though more stable, decreased affinity for the estrogen receptor significantly reduced AIB1 mediated cell growth in estrogen dependent cell lines [34]. Regulation of AIB1 protein levels is thus regulated by a complex series of phosphorylation and ubiquitination events in response to cell signaling and stimulation. The phosphorylation and ubiquitination sites may be unique to the pathways promoting turnover, and thus further study is warranted to better understand the signaling cascades that

receptor, whereas poly-ubiquitination promotes degradation of AIB1 [33].

at K723 and K726.

to promote its multi-mono- or poly-ubiquitination by SCFFbw7<sup>α</sup>

**52**

control AIB1 levels.
