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    • Introduction The phospho proteome in

    2018-11-05

    Introduction The phospho-proteome in pluripotent stem rivastigmine tartrate has been extensively and systematically studied (Van Hoof et al., 2012), and has uncovered phosphorylated residues on pluripotency factors that play important roles in establishing and maintaining pluripotency. Despite the fact that NANOG was speculated to be a phospho-protein over a decade ago (Yates and Chambers, 2005), very little information is available regarding the status and functional implications of NANOG phosphorylation. Studies coupling immunoprecipitation with mass spectrometry (IP-MS) have found that human NANOG is phosphorylated at 11 different sites by ERK2 and CDK1 in human embryonic stem cells (ESCs) (Brumbaugh et al., 2014), and that mouse NANOG is phosphorylated at four different sites (Li et al., 2011; Moretto-Zita et al., 2010) by ERK1 as well as unidentified kinases (Kim et al., 2014). The specific role of phosphorylation in regulating NANOG function, however, remains elusive. One study suggested that phosphorylation is important for maintaining NANOG stability in ESCs (Moretto-Zita et al., 2010). This study relied on ectopic expression of NANOG in HEK293 cells for identification of phosphorylation sites by IP-MS, and tested the functions of these phosphorylation sites with phospho-dead or phospho-mimic mutants in the presence of endogenous NANOG in wild-type (WT) mouse ESCs (mESCs) (Moretto-Zita et al., 2010). In contrast, another study reported that phosphorylation of NANOG by ERK1 during differentiation of ESCs decreases NANOG stability through ubiquitination-mediated degradation (Kim et al., 2014). Here we systematically investigated the function of NANOG phosphorylation in two biological settings within a physiological context where NANOG function is critical and endogenous NANOG interference with phospho-dead and phospho-mimic mutants is minimized. Our findings therefore contribute important functional data to the phospho-proteome in pluripotent stem cells, and improve our understanding of the key pluripotency regulator NANOG in controlling ESC pluripotency and somatic cell reprogramming.
    Results
    Discussion What might be the underlying cause of such context-dependent functions of NANOG phosphorylation in pluripotency and reprogramming? We did not observe any differences in protein stability or subcellular localization between NANOG WT and S65A in Nanog pre-iPSCs (Figure 4). The intrinsic transcriptional activity also does not seem to be affected in S65A mutant relative to WT NANOG, as we observed no noticeable difference in the abilities of WT and S65A NANOG to activate a Nanog enhancer-driven luciferase reporter (Figure S2C). Importantly, our highly sensitive SILAC IP-MS studies indicated that the NANOG S65A interactome in pre-iPSCs is preferentially enriched for the pluripotency factors ESRRB, OCT4, SALL4, DAX1, and TET1, compared with the WT NANOG interactome, in pre-iPSCs (Figure 4G). These factors often co-occupy ESC super-enhancers with NANOG, and have all been implicated in the reprogramming process (Costa et al., 2013; Huang and Wang, 2014). Therefore, it is highly likely that, despite minimal expression levels of DAX1, ESRRB, and SALL4, or equal abundance of TET1 and OCT4, in pre-iPSCs relative to ESCs (Figure 4E), these pluripotency factors may have a higher affinity with S65A than WT NANOG in forming active transcriptional regulatory complexes to mediate enhanced reprogramming. Is there a structural implication for such preferential association of NANOG S65A with pluripotency regulators? By applying automated protein structure prediction and modeling for full-length NANOG WT and NANOG S65A using the I-TASSER platform (Roy et al., 2010), we observed an apparent unfolding of the N-terminal domain of S65A compared with WT NANOG (Figure S2D). The N-terminal domain has been shown to be dispensable for NANOG nuclear localization, and has been proposed to serve as an interface for interaction with co-factors important for transcriptional activities of NANOG in maintaining ESC self-renewal (Chang et al., 2009; Guo et al., 2009). Therefore, loss of phosphorylation may have endowed NANOG S65A with an altered structure more conducive to association with those nuclear pluripotency regulators, leading to functional activation of the pluripotency program in reprogramming. Future studies applying X-ray crystallography to solve the full-length WT and S65A NANOG protein structures are warranted to confirm this hypothesis, which is currently a challenge in the field (Hayashi et al., 2015; Jauch et al., 2008). Alternatively, the preferential associations of S65A NANOG with these pluripotency regulators (Figure 4G) may also be due to their subtle increased protein levels that can only be detected by quantitative SILAC IP-MS we have employed. Of note, we found that the total OCT4 protein level in S65A pre-iPSCs was appreciably higher than that in WT pre-iPSCs (Figure 4E), likely resulting from endogenous Oct4 reactivation.