Therefore, our findings suggest that the RictorCPrkchCKlf4 pathway has an important role in the regulation of ES cell differentiation. Results Rictor promotes ES cell differentiation The mammalian target of rapamycin (mTOR) controls cell growth and proliferation in response to nutrients and growth factors through two distinct multiprotein complexes mTORC1 and mTORC2. little is known regarding the function of mTOR signaling in the Alas2 regulation of ES cell differentiation. Here we report that Rictor, a key component of mTORC2, functions as a novel ES cell differentiation promoting factor. Mechanistically, Rictor is able to interact with Prkch and facilitate Prkch phosphorylation at Ser-642. Upon phosphorylation, Prkch promotes Klf4 phosphorylation and inhibits Klf4-dependent E-cadherin expression, thereafter leading to the ES cell differentiation. These findings reveal a novel RictorCPrkchCKlf4 pathway that plays an important role in the regulation of ES cell differentiation. Subject terms: Stem-cell research, Epigenetics Introduction Embryonic stem cells (ESCs) are distinguished by their abilities to self-renew and to differentiate into various types of cells, and have great potential for regenerative medicine [1C3]. The fate of ESCs is delicately regulated by a complex network formed by transcriptional factors, epigenetic regulators, signal receptors, and protein kinases. In mouse ESCs, the maintenance of self-renewal is primarily regulated by LIF-STAT3 [4, 5] and BMP pathway [6], while the differentiation of ESCs is promoted by MEK-ERK and Gsk3 signaling [7]. The downstream transcription factors Oct4, Sox2 and Nanog work as a central complex to activate genes that sustain the undifferentiated state and suppress genes that promote differentiation [8, 9]. The mammalian target of rapamycin (mTOR) signaling regulates cell proliferation, cell survival, and protein synthesis Sitaxsentan [10, 11]. There are two distinct types of mTOR complexes, mTORC1 and mTORC2. mTORC1 is composed of mTOR, Raptor, Sitaxsentan MLST8, PRAS40, and DEPTOR [12]. The complex is activated by insulin, growth factors, and oxidative stress, and regulates protein translation through its substrates 4E-BP1 and S6K1. mTORC2 mainly consists of mTOR, rapamycin-insensitive companion of mTOR (Rictor), MLST8, and mSIN1 [13], and responds to Rapamycin only in certain cell types under prolonged exposure. The function of mTORC2 is well understood compared to mTORC1, yet increasing evidence suggests that mTORC2 plays an important role in cancer pathogenesis, promoting tumor growth and chemotherapy resistance in cancer cells [14, 15]. As a key component of mTORC2, Rictor was originally identified as an mTOR binding partner [16]. Rictor is also shown to interact with mSIN1 and Protor1, both of which exist in mTORC2 complex [17]. It has been reported that the RictorCmTOR complex is able to phosphorylate Akt on Ser 473 and facilitate the phosphorylation of AKT on Thr 308 by PDK1 [18]. The RictorCmTOR complex also regulates the phosphorylation of PKC, thereby modulating the actin cytoskeleton [16]. In breast cancer cells, Rictor binds to PKC and regulates cancer metastasis [19]. Loss of Rictor gene in mice leads to embryo growth arrest at embryonic day 11.5 Sitaxsentan (E11.5), indicating an important role of Rictor in the regulation of embryonic development [20]. However, the function of Rictor in embryonic stem cells has not been characterized. Here we provide evidence that Rictor functions as a novel mouse ES cell differentiation promoting factor. Rictor exerts its function via interacting with Prkch and enhancing Prkch phosphorylation. Phosphorylated Prkch in turn increases phosphorylation of Klf4 and decreases its transcriptional activity toward E-cadherin. Therefore, our findings suggest that the RictorCPrkchCKlf4 pathway has an important role in the regulation of ES cell differentiation. Results Rictor promotes ES cell differentiation The mammalian target of rapamycin (mTOR) controls cell growth and proliferation in response to nutrients and growth factors through two distinct multiprotein complexes mTORC1 and mTORC2. As a key component of mTORC2, Rictor is essential for embryonic development, as evidenced by the finding that Rictor knockout leads to embryonic lethality in mice. However, it remains unknown if Rictor is involved in the regulation of maintenance or differentiation of mouse ES cells. To address this, we first compared expression of Rictor in MEF and R1 ES cells. Rictor was expressed at significantly lower levels in R1 cells compared to MEF cells (Fig.?1a, b). However, in response to retinoic acid (RA)-induced R1 ES cell differentiation, both mRNA and protein levels of Rictor were strongly upregulated (Fig.?1c, d). These data indicate that expression levels of Rictor are correlated with the differentiation state of ES cells. Open in a separate window Fig. 1 Rictor promotes ES cell differentiation. a Real-time RT-PCR analysis of Rictor mRNA expression in MEF and R1.
