Identification And Functional Analysis Of Novel Substrates Of Protein Kinase AKT

AKT, a serine/threonine kinase, plays a key role in the downstream of PI3K signal pathway. AKT appears to play important roles in the regulation of cell survival, proliferation and growth. AKT is activated in cycling stem cells, but remains inactivated in quiescent stem cells; AKT-related factors such as PTEN and PML are associated with the sternness maintence of stem cells and cancer stem cells. These remind us to consider that AKT is involved in the regulation of embryonic stem cells (ESCs) or cancer stem cells (CSCs).The "core" transcriptional factors, including Oct4, Klf4and Nanog, are of great importance to the sternness maintainence of ESCs. SATB1is a chromatin organizer and global gene regulator, which palys as a differetiation factor in stem cells. It is demonstrated that SATB1counteracts the roles of pluripotency factors during the onset of cell differentiation. Intriguingly, one common feature shared by SATB1, Oct4and Klf4is that they all have a consensus AKT phosphorylation motif (RxRxxS/T), which raises the possibility that AKT is a master signaling molecule to modulate the antagonizing status between SATB1and pluripotency factors.In this study, we focused on a number of pluripotency/differentiation-regulating factors that have potential and conserved AKT phosphorylation motifs. We identified several novel substrates of AKT by using in vitro kinase assays, including Oct4, Klf4, Bmi-1, Mbd3, Twist1, Fbxw7and SATB1. To indentify the functional role of AKT in sternness, we choose SATB1, Oct4and Klf4for further analysis. We confirmed that AKT phosphrylates SATB1at serine47in a PI3K-dependent manner; AKT also protects SATB1from apoptotic cleavage. Meanwhile, AKT phosphorylates Oct4at threonine228and Klf4at threonine399, and accelerates their ubiquitination and degradation. Moreover, PI3K/AKT signaling enhances the binding of SATB1to Sox2, thereby probably impairs the formation of Oct4/Sox2regulatory complex. During retinoic acid (RA)-induced differentiation of mouse F9embryonal carcinoma cells (ECCs), the AKT activation profile as well as its substrate spectrum is strikingly correlated with the down-regulation of Oct4, Klf4and Nanog, which suggests AKT activation is coupled to the onset of differentiation. We established SATB1or Serine47-mutants stable expressing F9celllines for further studies. Accordingly, AKT-mediated phosphorylation is crucial for the capability of SATB1to repress Nanog expression and to activate transcription of Bcl2and Nestin genes. The results suggest that AKT signal is involved in the differentiation of ECCs through coordinated phosphorylations of pluripotency/differentiation factors.Here, we demonstrate that Akt phosphorylates SATB1, thereby keeping it intact and maintaining its inhibitory effects on the expression of Nanog. On the other hand, Akt phosphorylates pluripotency factors Oct4and Klf4, promoting their degradation via the ubiquitin-proteasome system. Moreover, SATB1binding to Sox2also relies on PI3K/Akt signaling, which probably disrupts complex formation of pluripotency factors as a consequence of Sox2sequestration. Taken together, we conclude that Akt-mediated phosphorylation would destroy the balance of pluripotency/differentiation factors, ultimately favoring a differentiation process of embryonal carcinoma cells. Our study favors a novel model for exploring the process of tumorigenesis and cancer metastasis. Finally, our data implys that treatments targetting for AKT should be effective in halting the metastasis of cancer.