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Abstract
Self-renewal of murine embryonic stem cells (mESCs) is controlled through the activity of leukemia inhibitory factor (LIF) by a mechanism requiring the activation of STAT3. Also critical for mESC self-renewal is the phosphotidylinositol 3-kinase (PI3K) pathway which signals through Akt and under self-renewing conditions is proposed to inhibit glycogen synthase kinase 3 (Gsk3) through the phosphorylation of serine 9 (S9). Several key studies have demonstrated that inhibition of Gsk3 activity is required to maintain mESCs in a pluripotent, self-renewing state. Recently, it was demonstrated that the proto-oncogene c-MYC, a direct target of LIF/STAT3 signaling, is essential for maintenance of mESC self-renewal. Mouse ESC differentiation relies upon the threonine 58 (T58) dependent phosphorylation and degradation of c-myc. T58 dependent degradation correlates with the activation of Gsk3, a kinase known to promote c-myc instability through T58-dependent phosphorylation. We demonstrate that Gsk3, normally inactive and cytoplasmic during self-renewal, accumulates in the nucleus an active form during early mESC differentiation. The activation and nuclear accumulation of Gsk3 was shown to trigger the differentiation of mESCs by promoting the T58 dependent phosphorylation and degradation of c-myc. We show that PI3K/Akt signaling, in addition to its suppression of Gsk3 activity, prevents Gsk3 from accessing nuclear substrates like c-myc by promoting its efficient nuclear export. This novel mode of Gsk3 regulation was shown to occur via the PI3K/Akt-dependent binding of the Gsk3 binding protein Frat, which we define as responsible for mediating the nuclear export of Frat. These observations define a novel mechanism explaining how PI3K/Akt signaling and GSK3 control mESC self-renewal through the regulation of c-myc.