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  • The minimal segment of the SIM appears to be

    2022-01-13

    The minimal segment of the SIM appears to be a core of Topotecan HCl residues, which frequently matches the consensus sequences ΨΨΨΨ, ΨxΨΨ, or ΨΨxΨ (where Ψ is V, I, or L and x is typically D, E, S, or T) [29,31,32]. In a subset of SIMs, this hydrophobic core is flanked by serine residues and a stretch of negatively charged acidic residues. These motifs non-covalently bind SUMO either in a parallel or anti-parallel orientation, depending on the distribution of the charged residues in the SIM [29,33]. Importantly, recent studies demonstrated that serine residues adjacent to the hydrophobic core of the SIM are phosphorylated by casein kinase 2 (CK2) and that the two adjacent segments work together as a phosphoSIM. Based on a combination of biochemical, structural, and functional studies, CK2-regulated phosphoSIM modules were clearly dissected in the SUMO ligase, PIAS1 [51], the transcriptional coregulator, Daxx [52] and the tumor suppressor, PML [53], indicating that these modules not only contribute to the specificity of distinct SUMO paralogues, but also serve as critical platforms that integrate CK2- and SUMO-mediated signaling networks. Herein, we demonstrated that FAF1 contains two motifs (SIM1 and -2) (Fig. 2), which bind SUMO-1, but not SUMO-2 or SUMO-3. The hydrophobic core (112V-D-V-V115) of FAF1 SIM1 matches the V/I-V/I-X-V/I/L motif derived from PIASx [33], while the stretch of acidic residues of SIM2 closely resemble the S-D-S-D portion found in the Daxx SIM [39]. Interestingly, previous research found significant associations between CK2 and FAF1 [54]. Results showed that CK2 can phosphorylate FAF1 at serines 289 and 291 and influence its intracellular location [14,55]. However, CK2-mediated FAF1 phosphorylation does not affect the ability of FAF1 to potentiate Fas-mediated apoptosis [14]. Our data showed that only the FAF1 hydrophobic residues (SIM1; 112V-D-V-V115 or SIM2; 285V-H-M-V288) were involved in SUMO-1 interactions (Fig. 2C). In addition, mutation of the SIMs (DM) in FAF1-containing consensus sites for CK2 at positions S289 and S291 did not affect its potentiation of Fas-induced apoptosis, inhibition of NF-κB or Wnt/β-catenin signaling pathways, or subcellular localization (Fig. 3). Of note, SIM mutations abolished the ability of FAF1 to modulate MR-mediated transactivation (Fig. 4). So far, it is yet unclear how the phosphorylation of FAF1 by CK2 is regulated and whether the phosphoSIM module of FAF1 controls other physiological functions. A very recent study by Grossmann's group reported that CK2 phosphorylates the MR and influences transcriptional MR activity by affecting MR-DNA interactions with subsequent MR degradation [56]. Their study also demonstrated that CK2-dependent MR activation elevates the expression of NF-κB-regulated genes involved in inflammatory responses. Given that FAF1 was reported to regulate the NF-κB pathway, the link among FAF1/CK2/MR complex formation, phosphorylation, and aberrant NF-κB activity in inflammatory immune responses requires further investigation. We demonstrated that FAF1 physically interacts with the NTD of the MR in a SUMO-dependent manner and may function as a corepressor of MR-mediated transcription (Fig. 5). Topotecan HCl Our analyses also showed that FAF1 affects the MR N/C-interaction and stability (Fig. 6). The N/C-interaction of steroid receptors has been best characterized in the AR [57]. N/C-interactions were also reported for the ERα, PR, and MR [[42], [43], [44], [45]]. Physiologically, the N/C-interaction stabilizes the AR by reducing ligand dissociation rates and preventing receptor degradation [58]. The AR N/C-interaction is mediated by binding of the AF-2 region to specific FxxLF and WxxLF sequences in the NTD [59]. However, the mechanism of the MR N/C-interaction differs from that of the AR in that the MR lacks both of these motifs in its N-terminus. The human MR NTD contains four sumoylation sites or “synergy control (SC) motifs” at positions K89, K399, K428, and K494 [23,27]. Of note, mutations at all four sumoylation sites do not compromise the N/C-interaction of the MR, suggesting that sumoylation of the NTD is not the key point of interaction with the LBD [45]. In light of our results that FAF1/SIMs directly bind to the NTD, and the sumoylated MR, it is possible that this spatial interference and/or competition effect abrogates the ability of the MR NTD to interact with the C-terminus. Determining whether FAF1 modulates the activity of other transcription factors through sumoylation requires further investigation. On the other hand, FAF1 is involved in valosin-containing protein (VCP)-related protein ubiquitination and proteasomal degradation [7,10]. Currently the role of MR ubiquitination in its transcriptional activity has not been fully elucidated. It is perhaps of relevance that FAF1 plays a role as a scaffolding protein or a bridging factor and regulates MR protein degradation. The spatiotemporal regulation of these functions remains to be established. Taken together, our data establish a significant role of FAF1 in modulating MR transcriptional activity.