**4.2 Post-translational modifications and signaling**

In MCF-7 cells, treatment of epidermal growth factor (EGF) leads to phosphorylation of ERRα and enhances its transcriptional activity [69]. In this study, PKCd was found capable of phosphorylating ERRα on the DBD, resulting in its enrichment at the ERRE containing promoters [69]. Screening breast cancer samples for expression of ER and ERR isoforms identified ERRα as the potential biomarker for poor prognosis for ER- and ErbB2 high expressing tumors [36]. In MCF-7 cells, overexpression of ErbB2 leads to hyperphosphorylation of ERRα and increased transcriptional activity [70]. This ErbB2 induced phosphorylation is readily inhibited by anti-ErbB2 as well as U0126 and LY294002, inhibitors for MAP kinase and AKT, two major signaling kinases downstream of ErbB2 signaling pathway. *In vitro*, both MAPK and AKT were found to phosphorylate AKT. Multiple phospho-sites are found throughout the protein for MAPK and phospho-sites for AKT are also predicted based on these in vitro kinase studies. However, no phospho-site has been identified thus far for each of the kinases.

Being the downstream signal induced by insulin signal, the PI3K/AKT signal plays major roles in regulating glucose metabolism, including glycolysis, gluconeogenesis as well as the TCA cycle and mitochondrial functions [71] (**Figure 3**). In hepatocytes and livers where PI3K/AKT signal is induced due to loss of negative regulator phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression, upregulation of ERRα as well as OXPHOS are observed [72]. Activation of PI3K/AKT leads to increased oxygen consumption (OCR) as well as induction of mitochondrial biogenesis [72, 73], whereas inhibiting ERRα activity blocks the induction of

#### **Figure 3.**

*PI3K/AKT signalling regulates ERRα. Activation and phosphorylation of insulin receptors results in recruitment of PI3K and the subsequent conversion of phosphatidylinositol (3,4)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). PTEN, a negative regulator of the PI3K/AKT pathway, converts PIP3 back to PIP2. Following binding to PIP3, the serine/threonine kinase AKT becomes fully activated via phosphorylation at Thr308 and Ser473 by 3-phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2), respectively. Activated AKT then phosphorylates various downstream substrates, including forkhead box O (FOXO) transcription factors, glycogen synthase kinase-3 (GSK3α/β), and tuberous sclerosis complex-2 (TSC2), a critical negative regulator of mTORC1 signaling. Activated AKT also phosphorylates CREB at Ser133, leading to an increase in PGC-1α and ERRα expression.*

mitochondrial function by PI3K/AKT signal [72]. Phosphorylation of CREB by AKT is thought to play a role in the regulation of ERRα by AKT in these cells, though direct phosphorylation of ERRα by AKT cannot be ruled out.

Due to the lack of endogenous ligands identified, ERRα is thought to be regulated primarily via transactivation and by upstream signaling pathways. However, not much has been elucidated for post-translation modification of ERRs beyond the reported phosphorylation of ERRα associated with breast cancer cell growth and survival. One addition modification reported is sumoylation at lysine 14 (Lys14), which suppresses its transcriptional activity with unexplored mechanisms [74]. It was found that this sumoylation of ERRα is dependent on its phosphorylation at serine 19 (Ser19).

The discovery of the roles ERR play in iPS have led to studies exploring how ERR signals crosstalk with those regulating pluripotency. These studies led to the discovery that ERRα physically interacts with β-catenin and lymphoid enhanced-binding factor-1 (LEF-1), with an overlap among genes previously demonstrated to be regulated by either β-catenin or ERRα [75]. A reduction of migratory capacity of breast, prostate, and colon cancer cell lines was observed following silencing of either β-catenin or ERRα with siRNAs, and this effect was further enhanced when the expression of both proteins was reduced simultaneously. The increased migratory capacity of cancer cells was suggested to occur as a result of the ERRα/β-catenin-dependent induction of Wnt 11, an activator of noncanonical Wnt signaling pathway [75]. Furthermore, ERRα

*Transcriptional Regulation by ERR and Its Role in NAFLD Pathogenesis DOI: http://dx.doi.org/10.5772/intechopen.109089*

is also reported to regulate osteoblast differentiation via the Wnt/β-catenin signaling pathway. In C3H10T1/2 cells overexpression of ERRα with PGC-1α or overexpression of Wnt3a, a significant overlap in gene expression is observed. These results suggest that the expression of ERRα and PGC-1α causes similar gene changes within the Wnt pathway as activation by Wnt3a alone [51].
