Serines and threonines were substituted with alanines (nonphosphorylatable) or aspartic acid (phospho-mimetic), whereas tyrosine residues were changed to either phenylalanine (nonphosphorylatable) or glutamic acid (phospho-mimetic). Open in a separate window FIGURE 1. Localization of Ago2 phosphorylation mutants. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798. subfamily, have been shown to increase stability and consequently gene-silencing function (Kirino et al. 2009). Conversely, during virus infection or cellular stress, inhibition of small RNA-dependent gene silencing is correlated with an increase in poly(ADP)-ribosylation of Argonautes (Leung et al. 2011; Seo et al. 2013). Similarly, the expression profiles of miRNAs can be significantly affected by ubiquitylation of Argonaute proteins, which results in their destabilization (Rybak et al. 2009). Finally, similar to many other proteins in eukaryotic cells, Argonaute proteins are substrates for multiple protein kinases (Zeng et al. 2008; Rudel et al. 2011; Horman et al. 2013; Shen et Dll4 al. 2013). There Chloramphenicol are four members of the subfamily in mammalian cells, but Ago2 is the only member that can cleave targeted mRNAs (Liu et al. 2004). Although Ago2 is the most widely expressed and predominant Argonaute isoform in mammalian somatic cells, the majority of endogenous miRNA-mediated gene Chloramphenicol silencing does not involve endonucleolytic cleavage of targeted mRNAs. Results from mass spectrometry analyses indicate the presence of at least seven phosphoamino acid residues in Ago2 (Rudel et al. 2011). To date, only three of these phosphoamino acid residues in Ago2 have been studied in a functional manner; however, it is evident that Argonaute phosphorylation affects small RNA-based gene silencing through multiple mechanisms. First, phosphorylation of tyrosine-393 negatively impacts the interaction between Ago2 and Dicer (Shen et al. 2013), the RNase responsible for processing duplex precursors into mature microRNAs (miRNAs). Disrupting this interaction affects miRNA maturation, particularly with respect to precursor miRNAs containing a long-loop structure. Once pre-miRNAs are processed into mature duplexes, the MID domains of Argonaute proteins anchor the 5 phosphates of guide RNA strands in RISC. However, phosphorylation of tyrosine-529 in this domain of Ago2 prevents loading of small RNAs onto RISC (Rudel et al. 2011). As such, regulated phosphorylation of tyrosine-529 may be a critical step in RISC activation. Finally, the phosphorylation status of serine-387 has been reported to suppress the endonucleolytic cleavage activity of Ago2, while enhancing the silencing of the targeted mRNAs by translational repression (Horman et al. 2013). Phosphorylation of serine-387 by AKT reportedly stimulates Ago2 interaction with GW182, a P-body resident protein and critical component of the ribonucleoprotein complex that represses translation of miRNA-targeted mRNAs. Increased interaction between phosphorylated Chloramphenicol Ago2 and GW182 may underlie the change of silencing mechanism for this protein. Consistent with this Chloramphenicol scenario is the observation that activation of the PI3K-AKT-mTOR kinase pathway in hematopoietic cells increases both expression of GW182 and association of Argonaute proteins with mRNAs in high molecular weight complexes (La Rocca et al. 2015). The association of Argonautes with high molecular weight complexes correlated with improved efficiency of slicer-independent silencing by RISC loaded with miRNAs and provides strong evidence of an intricate kinase-mediated regulatory mechanism for RNAi involving both direct and indirect modification of Argonautes. Given the large number of genes targeted by small RNA-based gene silencing, these pathways must be regulated at multiple levels. Evidence to date indicates that phosphorylation of serine-387, tyrosine-393, and tyrosine-529 regulate Argonaute activity by three different mechanisms. However, the role of phosphorylation at other sites in Ago2 has not been reported. Here, we used site-directed mutagenesis to change every known phosphoamino acid residue in Ago2 to residues that either prevent or mimic constitutive phosphorylation. The subcellular localizations as well as endonuclease-dependent and -independent gene-silencing activities of these mutants were then analyzed. Whereas most Ago2 mutants did not exhibit significant changes in localization or RNAi activity, substitution of aspartic acid for serine at position 798.