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    • The ubiquitin proteasome degradation mechanism is

    2018-11-06

    The ubiquitin–proteasome degradation mechanism is associated with most intracellular proteins, including membrane-surface receptors (Gesbert et al., 2005; Martinez-Moczygemba et al., 2007; Rocca et al., 2001; Wauman et al., 2011). Over 60 Fbox proteins have been identified, but only a few, such as β-TrCP and FBXW7, are well characterized. Fbox proteins bind in the substrate-binding motif used for recognition and interaction with phosphorylated substrates (Kim et al., 2012; Skowyra et al., 1997). Recent studies indicate that post-translational modifications of iPS factors regulate their activity. For example, phosphorylation of human Sox2 at Ser249, Ser250 and Ser 251 (Van Hoof et al., 2009) inhibits Sox2 DNA binding activity (Tsuruzoe et al., 2006). Acetylation of mouse Sox2 at Lys75 by p300/CBP enhances nuclear export and degradation of Sox2 through an ubiquitin-mediated degradation pathway (Baltus et al., 2009). Furthermore, phosphorylation of human Oct4 at Ser229 by PKA might partially regulate Oct4 transactivation (Baltus et al., 2009). Phosphorylation of Nanog is associated with Pin1 (Moretto-Zita et al., 2010). These findings indicate that post-translational modifications of iPS factors are likely involved in the regulation of their activity, which could result in modulation of ES cell self-renewal activity. Herein, we identified mechanisms for the self-renewal of ES Octreotide acetate Supplier showing that regulation of Nanog protein stability occurs through ERK1-mediated phosphorylation and ubiquitination.
    Materials and methods
    Results
    Discussion Although the methylation status of gene promoters of stem cell factors is believed to be a major regulatory mechanism to govern stem cell self-renewal activity, recent studies have demonstrated that post-translational modifications, including phosphorylation, sumoylation and ubiquitination, of stem cell factors, such as Sox2 and Oct4, might play an important role in the regulation of transcriptional activity (Baltus et al., 2009; Saxe et al., 2009; Tsuruzoe et al., 2006; Van Hoof et al., 2009). Recent studies also indicate that Nanog, an ES cell-specific homeodomain protein, is required for the self-renewal of ES cells (Chambers et al., 2003; Mitsui et al., 2003). Self-renewal and maintenance of pluripotency of mouse ES cells require leukemia inhibitory factor (LIF; a member of the IL-6 cytokine family), which is used for maintenance of ES cells in an undifferentiated state (Smith et al., 1988; Williams et al., 1988). At the same time, LIF also stimulates and activates the MAP kinase pathway, the activation of which induces differentiation of ES cells. However, suppression of ERKs signaling promotes the self-renewal activity of mES cells (Burdon et al., 1999). Therefore, the ERKs signaling pathway is believed to play an important role in the regulation of ES cell self-renewal activity. In this study, we found that ERK1, but not ERK2, strongly binds with and phosphorylates Nanog in vitro (Fig. 1A, B, E). This finding is important because ERKs signaling induces ES cell differentiation (Burdon et al., 1999) and the MEK inhibitor, PD0325901, enhances ES cell self-renewal activity (Ying et al., 2008). Furthermore, our results demonstrated that ERK1 signaling negatively modulates Nanog transactivation activity (Fig. 1F, G) and enhances ubiquitination of Nanog (Fig. 3B, C). The down-regulation of the protein level of Nanog during ES cell differentiation corresponded with increased levels of phosphorylated ERKs (Fig. 2A). Therefore, we concluded that the phosphorylation of Nanog mediated by ERK1 is critical for the down-regulation of Nanog through ubiquitination-mediated protein stability. In order to regulate the protein level through the proteosomal protein degradation pathway, the binding and interaction of the ubiquitin conjugation machinery with the target protein are necessary (Hochstrasser, 1996). However, the molecular mechanism explaining how the phosphorylation of Nanog affects stability is not yet clearly understood.