14-3-3h Interacts with rEag1 Figure 9. Lack of effect of 14-3-3h over-expression on the total and surface expression of rEag1 protein. (A) Representative result of surface biotinylation experiments. Intact HEK293T cells were biontylinated on ice and thereafter solubilized. (Surface) Cell lysates were pulled down with streptavidin agarose beads, followed by immunoblotting with the anti-rEag1 antibody. (Input) Cell lysates were directly employed for immunoblotting analyses. Input represents 5% of the total protein used for streptavidin pull-down. Also shown at the bottom are the corresponding b-actin expression levels for each lane. The specificity of the biotinylation procedure was verified by the absence of b-actin bands in the surface fraction. (B) Quantification of total and surface expression of rEag1 in the absence or presence of 14-3-3h over-expression. The total protein density (top panel) was determined as the ratio of input signal to the cognate b-actin signal. The surface expression efficiency (bottom panel) was expressed as the ratio of surface signal to the corresponding total protein density. The mean values were subsequently normalized with respect to that of vector control. Densitometric scans of immunoblots were obtained from three independent experiments. doi:10.1371/journal.pone.0041203.g009 that the GST-N163 fusion protein displayed a notably enhanced binding efficiency than that of GST-N207, suggesting that the PAS domain per se may be sufficient to mediate the N-terminal interaction of rEag1 with 14-3-3h. Next we switched the focus to the C-terminal interaction of rEag1 with 14-3-3h. Since the rEag1 protein contains a long cytoplasmic C-terminus spanning over 470 amino acids, we generated three more GST fusion proteins that divided the C- terminus into three regions: GST-C1 (amino acids 492–724), GST-C2 (amino acids 723–848), and GST-C3 (amino acids 835– 962). GST pull-down assay indicated that GST-C1 displayed the highest 14-3-3h-binding efficiency, virtually identical with that of GST-C0 (Fig. 3B). This observation suggests that the proximal region of the rEag1 C-terminus may harbor a major 14-3-3h- interacting domain. Two structural domains have been defined for this region of the rEag1 protein: the C-linker and the cyclic nucleotide-binding homology domain (CNBHD) [1,26]. We therefore made two additional GST fusion proteins that encoded either the C-linker (GST-C1-1: amino acid 493–560) or the CNBHD (GST-C1-2: amino acids 561–672) region. As depicted in Figure 3C, 14-3-3h preferentially bound to the GST-C1-2 fusion protein, implying that the CNBHD may contribute to the C- terminal interaction of rEag1 with 14-3-3h. Interaction of 14-3-3h with rEag1 in heterolgous and native cells To further confirm the foregoing GST pull-down results, we tested the association of 14-3-3h with full-length rEag1 by transiently co-expressing myc-14-3-3h and rEag1 proteins in HEK293T cells. As shown in the left panel of Figure 4A, upon immunoprecipitating myc-14-3-3h with the anti-myc antibody, a significant rEag1 protein signal was detected on the immunoblot. Conversely, myc-14-3-3h was also immunoprecipitated with the anti-rEag1 antibody (Fig. 4A, right panel), indicating that rEag1 indeed co-existed in the same protein complex with 14-3-3h. By contrast, no significant co-immunoprecipitation pattern was found for 14-3-3h and rEag2, an isoform of rEag1 (Fig. 4A, left panel). In most cases, 14-3-3 proteins bind to targets containing specific phosphoserine or phosphothreonine motifs [27,28], although some non-consensus 14-3-3-binding phosphorylation motifs have been reported as well [29]. Moreover, 14-3-3 may also bind to unphosphorylated ligands [30,31]. Upon analyzing the amino acid sequence of rEag1, we did not find any consensus or putative 14-3-3-binding phosphorylation motif. To further address this issue, we compared the effects between a phosphatase inhibitor (okadaic acid) and a protein kinase inhibitor (staurosporine). The modulatory effects of the two agents on the status of cellular phosphorylation were confirmed by the dramatic change in the phosphorylation state of the protein kinase Akt (Fig. 4B and 4C, upper panels). As illustrated in the lower panels of Figures 4B and PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41203