Abstract
ABSTRACTThe phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.
Highlights
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays a central role in mediating extracellular stimuli that controls diverse functions including cell proliferation, growth, survival, and metabolism (Manning and Cantley, 2007; Vanhaesebroeck and Alessi, 2000)
We previously reported Akt isoform-specific knockdown in mouse embryonic fibroblasts (MEFs) using lentiviral short hairpin RNA (shRNA) constructs: shAkt1, shAkt2, and shAkt3 (Tang et al, 2014)
We reported here for the first time a cell survival and proliferation mechanism in embryonic stem cells (ESCs) that is Akt3, but not Akt1- or Akt2, dependent
Summary
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays a central role in mediating extracellular stimuli that controls diverse functions including cell proliferation, growth, survival, and metabolism (Manning and Cantley, 2007; Vanhaesebroeck and Alessi, 2000). Activated Akt phosphorylates its downstream targets to regulate various cellular events This process can be reverted by phosphatase. There are three Akt isoforms encoded by different genes, namely Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, which all contain conserved peptide sequences between mice and humans (Bellacosa et al, 1991; Brodbeck et al, 1999; Cheng et al, 1992) These three isoforms share a similar N-terminal Pleckstrinhomolog domain, a central serine-threonine kinase domain, and a Cterm hydrophobic motif-containing regulatory region characteristic of the protein kinase-A, -G, and -C families (Hanada et al, 2004; Manning and Cantley, 2007; Yang et al, 2004). The specific roles of each Akt isoform in different cell types are largely undefined (Chin and Toker, 2011)
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