Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-. (Figure 2C), suggesting that Daxx directly activates MST1. It is also noteworthy that MST1 did not phosphorylate Daxx (Supplementary Figure S3). To examine the potential role of Daxx in IFN–induced MST1 activation in microglia, we transfected primary rat microglial cells with GFP (control) or Rabbit Polyclonal to OR5K1 Daxx siRNA oligonucleotides. Immunoblot analysis revealed that IFN- treatment increased the abundance of 173997-05-2 Daxx in the cells transfected with control siRNA, and this effect was abolished in Daxx siRNA-transfected cells (Figure 2D). Concomitantly, IFN- increased MST1 activity in control siRNA-transfected cells, and depletion of Daxx by RNAi prevented this effect of IFN-. Similar results were observed in separate experiments using BV-2 cells expressing Daxx siRNA (Supplementary Figure S4). The RNAi-mediated depletion of Daxx also abolished the phosphorylation of histone H2B on Ser14 in primary rat microglia (Figure 2E). Together, these results suggested that IFN- upregulates the expression of Daxx and that Daxx in turn mediates the activation of MST1 in microglial cells. Daxx physically associates with MST1, thereby promoting the homodimerization of MST1 We next examined which domains of Daxx and MST1 are responsible for the interaction between the two proteins. Ectopically expressed Daxx interacted with MST1 and MST1 (421C487), but not with MST1 (1C420), in 293T cells (Figure 3A). Consistent with these data, binding analysis revealed that Daxx bound to 173997-05-2 MST1 and MST1 (421C487), but not to MST1 (1C420) (Figure 3B). Furthermore, Daxx induced the activation of MST1 but not that of MST1 (1C420) (Figure 3C). Reciprocal co-immunoprecipitation analysis showed that MST1 bound to Daxx (1C500) and Daxx (1C267), but not to Daxx (268C570) (Figure 3D) or Daxx (498C740) (Supplementary 173997-05-2 Figure S5). Consistent with these binding data, ectopically expressed Daxx or Daxx (1C500) increased MST1 activity whereas Daxx (498C740) did not (Figure 3E). Figure 3 Characterization of the binding regions of MST1 and Daxx. (A) Schematic representation of the domain organization of human MST1 is shown in the upper panel. 293T cells were transfected for 48 h with a vector encoding Flag-Daxx together with vectors for … Homodimerization of MST1 contributes to the mechanism of MST1 activation (Creasy et al, 1996; Glantschnig et al, 2002). Given that Daxx binds to MST1 (421C487) that includes the SARAH domain, which mediates MST1 homodimerization (Creasy et al, 1996; Scheel and Hofmann, 2003), we examined whether Daxx might regulate the homodimerization of MST1. We thus transfected Daxx?/? mouse embryonic fibroblasts (MEFs) with plasmids encoding Daxx, Flag-MST1, and HA-MST1, and subjected the cells to co-immunoprecipitation analysis. Flag-MST1 was associated with HA-MST1 only in the Daxx?/? MEF cells reconstituted with Daxx (Figure 4A). In comparison, Flag-MST1 did not form a complex with HA-MST1 173997-05-2 (1C420) even in the cells reconstituted with Daxx (Figure 4B). Together, these results suggested that Daxx is indispensable for MST1 homodimerization, and that the SARAH domain of MST1 is required for the Daxx-dependent homodimerization of MST1. Figure 4 Daxx promotes the homodimerization of MST1. (A, B) Effect of ectopic Daxx on MST1 homodimerization in Daxx?/? MEFs. MEFs from Daxx-null mice were transfected for 48 h with the indicated combinations of plasmids encoding Flag-MST1, HA-MST1, … We next examined the effects of Daxx and its various fragments on the homodimerization and kinase activity of MST1 in Daxx?/? MEFs by transfection experiments. MST1 homodimerization was detected in Daxx?/? MEFs reconstituted with Daxx, Daxx (1C500), or Daxx (1C267), but not in those expressing Daxx (498C740) (Figure 4C). The extent of MST1 homodimerization was smaller in Daxx?/? MEFs expressing Daxx (1C267) than in those expressing Daxx or Daxx (1C500). The kinase activity of Flag-MST1 was 173997-05-2 markedly increased in the Daxx?/? cells reconstituted.