Rasd1 Modulates the Coactivator Function of NonO Figure 1. NonO is identified as a novel interacting partner of Rasd1 via affinity pull-down assay. (A) Coomassie blue stained SDS-PAGE gel of affinity pulldown assay. Ni-NTA magnetic beads were incubated either with lysate from pHis-Rasd1 transfected COS-7 cells or with lysate of empty vector transfected cells. Next, washes were conducted to remove non-specific binding proteins. His-Rasd1 bound to the magnetic beads was then incubated with PC-12 lysate. The beads were boiled to separate the protein complexes for fractionation on SDS-PAGE (12%). The 55 kDa, 50 kDa and 30 kDa bands were observed in the elute lane of His-Rasd1 (Lane 5) but not in the elute lane of the negative control (Lane 1). Protein bands were excised for further analysis using mass spectrometry. The proteins identified were NonO, Tubulin beta 5 and Rasd1, respectively. E, elute; W, wash; and I, input. (B) Co-precipitation assay was performed to study in vivo interaction between Rasd1 and NonO. COS-7 cells were co-transfected with plasmids expressing HA-Rasd1 and either GST-NonO or GST. The lysates were then incubated with GSH-linked magnetic beads to precipitate GST- tagged proteins. HA-Rasd1 was observed to co-precipitate specifically with GST-NonO but not GST (compare Lanes 1 with 2). (C) A similar interaction assay was performed for NonO-V5 and GST-Rasd1 proteins. In this experiment, COS-7 cells were co-transfected with pNonO-V5 and either pGST- Rasd1 or pXJGST. GSH-linked magnetic beads were added to the cell lysates to pull-down GST-tagged proteins. NonO-V5 was observed to be co- precipitated with GST-Rasd1 but not with GST (compare Lanes 1 with 2). (D) To study the in vivo interaction of endogenous Rasd1 and NonO, co-IP was performed on HEK293T cell lysates incubated with either rabbit anti-NonO or rabbit control IgG. Detection of the blot with anti-Rasd1 showed that RASD1 was co-IP specifically by NONO (Lane 2). (E) A similar Co-IP was conducted using mouse brain lysate incubated with either anti-Rasd1 or goat control IgG. NonO was only co-precipitated by lysate incubated with anti-Rasd1 (Lane 2). NonO-V5 is detected with anti-V5 (Invitrogen, USA, CA); GST-tagged proteins are detected with anti-GST (Santa Cruz, USA, CA); HA-Rasd1 is detected with anti-Xpress (Invitrogen, USA, CA); endogenous NonO is detected with goat anti-NONO; and endogenous Rasd1 is detected with goat anti-Rasd1. doi:10.1371/journal.pone.0024401.g001 GTP-bound state. Hence, the mutants bind a higher proportion of GTP to GDP in vivo and behave functionally as constitutively active signal transducers [20,35]. However, the mutant has an overall lower activity than the wild type. G81A, another con- stitutively active mutant of Rasd1, carries a point mutation in the G3 box guanine residue. This mutation in H-Ras has been shown to interfere with its interaction with GTPase-activating proteins (GAPs), thus leading to a protein that is consistently bound to GTP [21]. In the T38N mutant, the key residue in the G2 box is switched from threonine to asparagine. This mutation is known to severely reduce the binding affinity of Rab11 to GTP, leaving the GDP-binding properties unchanged [22]. Therefore, the mutation results in an inactive Rasd1 that is constantly bound to GDP. In a previous study [19], it was suggested that a putative nuclear translocation signal (NLS) on the C-terminal of Rasd1 permitted its nuclear translocation. Hence, we constructed a Rasd1 mutant, Del-NLS, in which the putative nuclear localisation signal was deleted, to further understand how increased nuclear presence of Rasd1 occurs in the company of NonO. We observed from our immunofluorescence study that, although all mutants (Figure 3A, 12, 19, 26 and 33) displayed similar sub-cellular distribution as wild-type Rasd1 (Figure 3A, 5), none of them displayed an increase in nuclear distribution upon co-transfection with pGST-NonO (Figures 3A: 15, 22, 29, and 36). Interestingly, NonO was observed in the cytoplasm upon co- transfection with T38N (Figure 3A, 30), unlike all other co- transfections where the sub-cellular location of NonO was observed primarily in the nucleus (Figures 3A, 9, 16, 23, and 37). Next, we compared the ability of wild-type and mutant Rasd1 to suppress CREB-mediated transcriptional activity in HEK293T cells overexpressing NonO. We observed that none of the Rasd1 mutants were able to effectively suppress CREB-mediated tran- scriptional activity when compared to the wild-type Rasd1 (compare Figure 3B, bars III with IV–VII). Interaction studies were subsequently performed, and the results show that mutants G81A and Del-NLS were unable to interact with NonO (Figure 3C, lanes 4 and 6); however, A178V and T38N were still able to interact with NonO (Figure 3C, lanes 3 and 5), and a substantial amount of NonO was present in the cytoplasm (Figure 3C, 38) in the presence of T38N. Taken together our findings suggest that interaction between NonO and wild-type Rasd1 is required for the suppression of CREB-mediated transcription. Rasd1 cooperates with full-length NonO to repress CREB activity To map the interaction domain between Rasd1 and NonO, a series of truncated NonO were constructed to determine the site at which Rasd1 binds (Figure 4A). HEK293T cells were co- transfected with pGST-Rasd1 and various plasmids containing truncated NonO. GST-pulldown was performed using cell lysate prepared from HEK293T cells transfected with the respective plasmids. All truncated constructs except NonOD2 were able to interact with Rasd1 (Figure 4B). Unlike other constructs which possessed at least one RRM domain, there was no RRM domain present in NonOD2. The results suggest that one RRM domain is sufficient for interaction with Rasd1. Next, pGST-Rasd1 was co- transfected with either NonOD1- or NonOD2-expressing plas- mids to determine if the RRM domains (NonOD1) are adequate for cooperation with Rasd1 to repress CREB’s activity. We observed that, unlike wild-type NonO, neither truncated clones were able to repress CREB’s activity in the presence of Rasd1 (compare Figure 4C, bars II with III and IV). This implies that both the RRMs and the DNA-binding region of NonO were required for functional interaction between Rasd1 and NonO. Next, we investigated if the RRM domains of NonO are responsible for the increased nuclear distribution of Rasd1 in the presence of NonO via immunofluorescence studies. The NonOD1 construct lacking the nuclear localisation motif was unable to translocate into the nucleus (Figure 4D2). In the event of co- transfection of NonOD1 with Rasd1, Rasd1 was present only in the cytoplasm, which is different in comparison to co-transfection of NonO with Rasd1 (compare Figure 3C2 with Figure 4D5). The results suggest that NonO may play a role in retaining Rasd1 in the nucleus and that the nuclear presence of both NonO and Rasd1 is required for down-regulating CREB-mediated transcrip- tional activity (Figure 4C, bar III). NR4A1 and NR4A2 are target genes regulated by both Rasd1 and NonO in the cAMP pathway Expression of both Rasd1 and NonO leads to the repression of CREB-mediated transcriptional activity (Figure 2B, bar VIII). To identify endogenous genes regulated by the combined actions of both Rasd1 and NonO, plasmids expressing Rasd1 and NonO were co-transfected into HEK293T cells. This was followed by quantitative Real time PCR to study the transcriptional activity of endogenous genes regulated by the cAMP pathway. Several endogenous genes known to have a functional CRE site were studied. This includes PER1 (Period1), CYP17, NR4A1, NR4A2, NR4A3, FOS, and Prolyl-4-hydroxylase a 1 (Prolyl a). However, out of all the genes studied in HEK293 cells, only the transcript levels of NR4A1, NR4A2 and FOS were consistently induced by forskolin (Figure S1). It has previously been shown that not all genes that contain CRE elements (such as PEPCK, BDNF, and insulin) are regulated by CREB in PC-12 cells [36]. Hence, our PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24401