Results NonO was identified as a novel interacting partner of Rasd1 via affinity pull-down assay followed by mass spectrometry analysis To facilitate our understanding of Rasd1’s physiological functions, an in vitro affinity assay was performed to identify novel interacting partners of Rasd1. COS-7 cells were used to over- express His-Rasd1 for subsequent interaction studies. The His- tagged proteins were then purified by Ni-NTA magnetic beads. This was followed by incubation with cell lysate extracted from PC-12 cells, which are known to express endogenous Rasd1 [26]. The complexes bound to Rasd1 were eluted and fractionated by SDS-PAGE. Three distinct bands were observed on the elute lane of beads bound with His-Rasd1 but not in the elute lane of negative control (compare Figure 1A, Lanes 1 with 5). These bands were excised, and mass spectrometry was conducted to determine the identity of the proteins. The band that was approx- imately 30 kDa on the gel (Figure 1A, Lane 5) was identified to be Rasd1. It was logical that the band was present in the elute lane for cells transfected with pHis-Rasd1 but not in the negative control. The next two bands at approximately 50 kDa and 55 kDa on Lane 5 were analysed with mass spectrometry as well. The bands were identified as Tubb5 and NonO, respectively (Figure 1A, Lane 5). In the present study, we focused exclusively on investigating the interaction between NonO and Rasd1. In vivo interaction study confirms NonO as a novel binding partner of Rasd1 To validate the interaction between Rasd1 and NonO, an in vivo interaction study was conducted by co-transfecting COS-7 cells with plasmids expressing either HA-Rasd1 and GST-NonO or HA-Rasd1 and GST. GST and GST-tagged proteins were purified with MagneGSTTM particles. Bound complexes were eluted and then fractionated on SDS-PAGE, followed by western blot. GST-NonO was observed to co-precipitate HA-Rasd1 specifically (Figure 1B, Lane 2). A similar observation was observed when cells were co-transfected with pGST-Rasd1 and pNonO-V5. In this case, GST-Rasd1 was purified using GSH- linked beads, and NonO-V5 was co-precipitated along with GST- Rasd1 (Figure 1C, Lane 2). Previous studies have shown that Rasd1 and NonO are expressed in HEK293T cells [13,26]; hence co-IP was carried out using HEK293T cell lysates. An antibody against NONO was used to precipitate endogenous NONO, and RASD1 was observed to be co-precipitated with NONO (Figure 1D, Lane 2). In addition, co-IP using mouse brain lysate was also performed. Rasd1 was purified by anti-Rasd1 and NonO was observed to co-purify specifically with Rasd1 (Figure 1E, Lane 2). The results obtained from co-precipitation and co-IP assays show that interaction of Rasd1 and NonO is specific and conserved across species (mouse and human). Rasd1 interacts with NonO to suppress CREB-mediated transcription Rasd1 protein has been shown to play multiple roles in the regulation of the cAMP pathway, including heterologous sensitisa- tion of adenylyl cyclase 1 via Gbc, attenuation of cAMP-stimulated hGH secretion, and inhibition of adenylyl cyclase through Gia in the HEK293T cell line [20,28,29]. Similar involvement was shown for NonO in the cAMP pathway. NonO interacts with TORC2 (transducer of regulated CREB-binding proteins 2) and functions as a co-activator to upregulate transcription of NR4A2 and FOS upon activation of the cAMP-dependent pathway; it has also been shown Rasd1 Modulates the Coactivator Function of NonO to be involved in regulation of CYP179s transcription via this pathway [13,14]. In this paper, we employed the PathDetect CREB trans-Reporting System to investigate the effects of Rasd1 and NonO on CREB-mediated gene transcription in HEK293T cells. Cells were transfected with pHis-Rasd1 along with luciferase reporter gene driven by CREB-responsive promoter and CREB- expressing vector. Prior to harvest, cells were induced with forskolin for 4 hours to study the effect of Rasd1 on the cAMP pathway. We observed that Rasd1 repressed the CREB-mediated transcription in a dose-dependent manner (compare Figure 2A, bars I with II–IV), supporting the findings reported in the previous study [19]. We transfected plasmid-expressing NonO in HEK293T cells to observe the effect of NonO on CREB’s activity via reporter gene assay. Transfection of NonO in the cells only led to a mild activation of the CREB-luciferase reported activity (compare Figure 2B, bars I with V). When plasmids expressing NonO and Rasd1 were co- transfected in HEK293T cells to study the effect of these proteins on the pathway, the CREB-luciferase reporter activity was reduced by 80% (compare Figure 2B, bars I and VIII). The up- regulation effect of NonO on the pathway was also abolished in the presence of Rasd1 (compare Figure 2B, bars V with VIII). In addition, the repressive effect of Rasd1 on the pathway was enhanced in the presence of NonO (compare Figure 2A, bar IV with Figure 2B, bar VIII). These results suggest that Rasd1 acts as a regulator of NonO to modulate its function in transcription. Rasd1 and NonO co-localise in the nucleus Co-localisation studies were performed by co-transfection of Rasd1- and NonO- expressing plasmids to study if Rasd1 and NonO influence each other’s sub-cellular localisation. Consistent with previous reports, we observed that in cells transfected with pNonO-V5, NonO mainly resides in the nucleus (Figure 3, A2) [12,13,14,15,31,32,33]. In addition, the localization of NonO was not affected by the presence of Rasd1 (Figures 3, A2 & A9). On the other hand, Rasd1 was distributed throughout the cells transfected with pHis-Rasd1 (Figure 3, A5), which is consistent with previous reports [19,34]. In the event of co-transfection of pGST-NonO and pHis-Rasd1, a substantial increase in the nuclear localisation of Rasd1 was observed when compared with transfection with Rasd1 expression vector alone (compare Figures 3, A5 and A8). The finding suggests that nuclear presence of Rasd1 is enhanced by NonO. GTP hydrolysis activity of Rasd1 is required for repression of CREB-mediated transcription As a member of the monomeric G protein family, Rasd1 possesses GTP binding and hydrolysis activity. RAS proteins are activated when GTP-bound and inactivated when GDP-bound. In order to obtain a better understanding of the mechanism by which the cooperation of Rasd1 with NonO mediates a suppressive effect, three Rasd1 mutants with point mutations of the conserved residues at the G boxes of RAS proteins were constructed. The mutants consist of two constitutively active mutants of Rasd1 – A178V and G81A – and an inactive mutant, T38N. Simi- lar Rasd1 mutants have been constructed, including H-Ras (H-Ras[A146V] and H-Ras[G60A]), and Rab11 (Rab11[S25N] and Rab11[Q70L]) [21,22,34,35]. The A178V mutation is expected to interrupt the guanyl nucleotide-binding pocket, resulting in an enhanced exchange rate of guanine nucleotides [20,35]. Since, guanyl nucleotide exchange is the rate limiting step in the activation of G proteins, and the intracellular levels of GTP is higher than GDP, an increase in the nucleotide exchange rate is supposed to lead to an increased occupancy in the active PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24401