Of Akt1 Phosphorylation Of The Transcription Factor Heb Ser <sup> 559 </ Sup>-bit Point And Regulation Of Its Activity

AKT1, also known as PKB, is a key molecular in PI3K/AKT signaling pathway. It regulates multiple cellular functions, including intermediary metabolism, cell proliferation, differentiation, apoptosis and migration. In our previous study, using a TAP-MS-based approach, we isolated AKT1 complex and identified several novel binding proteins of AKT1 in human hepatocellular carcinoma line HepG2.HEB is one of the identified interactions, and it belongs to bHLH transcription factors family which are involved in cell proliferation and differentiation. In this study, the interaction between AKT1 and HEB and the regulation of HEB activity by AKT1 were investigated.The protein-protein interaction between AKT1 and HEB was confirmed by co-IP and GST-pull down assays. AKT1 can phosphoralate HEB in vitro and in vivo, suggesting that HEB is a novel phosphorylated substrate of AKT1. The activation of PI3K signaling pathway increased the phosphorylation of HEB, and the specific inhibitor of PI3K suppressed the phosphorylation of HEB by insulin and HGF, indicating that the phosphorylation of HEB depends on PI3K/AKT pathway activation. Bioinformation analysis of HEB sequence revealed that the Ser⁵⁵⁹ site is highly conserved in most of the animal species and the flanking sequence surrounding Ser⁵⁵⁹ containing a putative AKT1 phosphorylationmotif (RGRTSS). So we speculate this site can be phosphorylated by AKT1. We constructed a mutant of HEB whose phosphorylation site is mutated, and confirmed Ser⁵⁵⁹ is the phosphorylation site of HEB by AKT1. Mutation of Ser⁵⁵⁹ doesn't change the nuclear localization of HEB by immunofluorescence assay, but led to abolishement of the activation of p21 expression induced by wild type HEB (HEBwt). Also, the arrest of G0-G1 phase was attenuated and the suppression of cell growth was released by HEBmt. HEB and E47 usually form heterodimers to regulate the transcription of targets, and we found that HEBmt failed to form hetero-oligomers with E47 to transactivate the promoter activity of SRG3. In conclusion, HEB is a novel phosphorylated substrate of AKT1, and the phosphoralation of the Ser⁵⁵⁹ site by AKT1 is essential for the biological function and activity of HEB.