on the cell surface and are often disassembled in cancer cells [48]. Caveolin 1 deleted cells become resistant to detachment-induced death [49] and caveolin 1 deletion is sufficient to drive cell transformation in NIH3T3 cells [50]. Interestingly, in NIH3T3- C2b-DN cells we observed strongly reduced matrix attachment to collagen and fibronectin and loss of contact inhibition, thus NIH3T3-C2b-DN cells were able to proliferate in multi-layers after reaching confluence without undergoing apoptosis (data not shown). Surprisingly, we found that p53 was strongly elevated in these cells (Fig. 6D). However the anti-apoptotic Bcl-2 protein showed also strongly increased expression (Fig. 6D). These protein deregulations revealed a pattern reported for cells protected from anoikis [51]. We analysed cell death induction by plating the cells on ultra-low attachment matrix. After 16 h incubation NIH3T3- C2b-DN cells showed very robust apoptosis protection measured by Caspase 3/7 activation, nevertheless the NIH3T3-C2b-WT cells had also lower apoptosis induction compared to control cells (Fig. 7A). Since inhibition of RhoA results in loss of substrate adhesion and severe cell retraction [24], we hypothesised that due to the long lasting clonal selection, cells with high kinase-dead PI3KC2b expression and hence attenuated RhoA function will gain survival advantage only under conditions when are able to upregulate signalling pathways protecting them from the RhoA- dependent lost of cell anchorage and the following apoptosis induction. We reported that PI3KC2b protects A-431 epithelial carcinoma cells from anoikis, however the mechanism remain elusive [17]. To test the hypothesis that wild-type PI3KC2b protects cells from anoikis in a RhoA dependent manner, we performed transiently expression of wild-type PI3KC2b, constitutively active RhoA and myristoylated Akt as a positive control in NIH3T3 cells. All three proteins were able to decrease significantly Caspase 3/7 activation in cells plated on ultra-low attachment matrix for 16 h, compared to NIH3T3 cells transfected with an empty vector (Fig. 7B). Moreover, transient transfection of NIH3T3-C2b-WT cells with a dominant- negative RhoA construct led to an increase in apoptosis induction compared to parental cells (Fig. 7C). These findings reveal an important role of PI3KC2b in protecting cells from detachment- induced apoptosis in a RhoA-dependent manner. PI3KC2b Expression Increases the Activation of EGFR and PDGFR Downstream Signalling The recruitment and activation of PI3KC2b downstream of the EGFR and the PDGFR has been reported [9,12]. In agreement with these reports, the results discussed above pointed to a PDGFR related PI3KC2b function in cell migratory responses. Therefore, we aimed to investigate Akt and Erk signalling pathway activation downstream of these two receptors. In line to our previous findings [10], elevated expression of wild-type PI3KC2b increased Akt pathway activation upon EGF and PDGF treatment, illustrated by increase in serine and threonine Akt phosphorylation, FOXO and S6 protein phosphorylation (Fig. 6A and B). Moreover, the extracellular regulated kinase (Erk1) showed also increased phosphorylation, compared to control cells. Surprisingly, we found very high basal Akt and FOXO phosphorylation levels in NIH3T3-C2b-DN cells. Moreover, EGF or PDGF stimulation did not further increase the Akt and FOXO phosphorylation (Fig. 6A and B). On the contrary, Erk activation was strongly abolished in these cells and the response to PDGF was clearly weaker than to EGF (Fig. 6A and B). In order to understand how these signalling differences contribute to the observed cytoskeletal rearrangements, we further studied signalling in cells grown in serum-supplemented condi- tions. Very high basal Akt and FOXO phosphorylation was Role of PI3KC2b in RhoA Activation detected in the NIH3T3-C2b-DN cells, however we did not find any striking differences in Akt and FOXO phosphorylation between NIH3T3-C2b-WT and control cells (Fig. 6C). Also the expression of the negative regulator of PI3K signalling PTEN was not consistently decreased in the NIH3T3-C2b-DN cells under study (Fig. 6D). However, we observed an induction of p53 expression in the NIH3T3-C2b-DN cells (Fig. 6D). In addition these cells displayed increased Bcl-2 expression and reduced expression of caveolin-1 and caveolin-2 (Fig. 6D). Thus it appears that NIH3T3-C2b-DN cells display prominent changes in the expression of major regulators of apoptosis and tumour suppres- sors. It is conceivable that enforced expression of dominant negative PI3KC2b in NIH3T3 cells leads to p53 induction and anoikis and that the surviving NIH3T3-C2b-DN cells have managed to escape apoptosis during the selection process by up- regulating Bcl-2 expression and activating the Akt pathway. The down-regulation of caveolin expression in NIH3T3-C2b-DN cells may contribute to the constitutive activation of the Akt pathway, by relieving an inhibitory effect on RTK signalling. Collectively, our data reveal the implication of PI3KC2b in the activation of the EGFR- and PDGFR-dependent Akt and Erk signalling cascades. PI3KC2b Mediates Migratory Response Downstream of the PDGFR Next, we attempted to analyse the migratory properties in NIH3T3 cells with deregulated PI3KC2b expression. We performed a wound healing assay under serum-supplemented conditions and in cells upon PDGF stimulation. Compared to control cells neither PI3KC2b-WT nor PI3KC2b-DN had a significant impact on the migratory cell speed in 10% serum (Fig. S2A). However, when cells were starved for 24 hours and then PDGF was added, we observed very strong migratory delay down to 80% in NIH3T3-C2b-DN cells (Fig. S2A). Interestingly, NIH3T3-C2b-WT cells were also not able to fully respond and migrated at 60% speed of control cells. These effects were not due to a different proliferation status, since NIH3T3-C2b-WT cells showed even slightly increased proliferation in serum. Surprisingly, NIH3T3-C2b-DN cells showed nearly doubled cell proliferation in low serum (Fig. S2B). These experiments reveal a PI3KC2b- dependent migratory response downstream of the activated PDGF receptor in NIH3T3 cells. Discussion Our analysis of the interacting molecules of PI3KC2b in NIH3T3 fibroblasts revealed a novel signalling complex implicat- ed in RhoA and Rac1 activation. We identified the RhoGEF Dbl as a novel interaction partner of PI3KC2b and we hypothesise that, in collaboration with Dbl, PI3KC2b can modulate RhoA and Rac1 activity and thus control cytoskeletal rearrangements, cell motility and protection against anoikis. Dbl is a crucial RhoGEF with activity towards Cdc42, RhoA and Rac1 [52]. It was also the first mammalian RhoGEF to be identified [26], but the mechanism of its activation is still not completely understood. In order to relieve its intrinsic auto-inhibitory activity, a physical interaction between the GEF’s N-terminal spectrin- and C- terminal PH-domain has to be released. This change in the inactive conformation of the protein can be triggered by diverse regulatory mechanisms including phosphorylation, or interactions with some other proteins. In our studies, the prominent binding of PI3KC2b to the spectrin- and PH- domain of Dbl was similar to the results reported for Dbl binding to the chaperone Hsc70 [31]. Hsc70 together with Hsp90 and the ubiquitin ligase CHIP keep Dbl activity in check by stabilising its inactive conformation. An in PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44945