vitro examination of the molecular mechanism of PI3KC2b/Dbl complex formation, however, revealed that neither the N-terminal sequence of the PI3K, nor Grb2, directly bound to Dbl, although both molecules were present in the PI3KC2b complex isolated from living cells. Formation of the Dbl/PI3KC2b complex was observed in serum-starved cells and the association was not further promoted by EGF or PDGF stimulation. In addition, we did not find evidence for a role of PI3KC2b in the activation of Dbl GEF activity, at least when measuring its activity in vitro. Therefore, other molecules need to be present in the cells, which mediate the interaction of the class II PI3K with Dbl and Dbl activation, resulting in the observed phenotypes in the NIH3T3 cells. The identity of these putative additional binding partners involved in the Dbl/PI3KC2b complex is briefly discussed below. Grb2 has been reported to be crucial for the EGF-dependent Dbl phosphorylation and subsequent GEF activation towards RhoA and Cdc42 [53,54]. These findings support our observation that Grb2 is present in the Dbl/PI3KC2b complex in NIH3T3 cells. Dbl phosphorylation is catalysed by the non-receptor tyrosine kinase Ack1 [54], a widely expressed protein which is recruited and activated downstream of the EGFR and PDGFR via Grb2 [55]. Furthermore, Dbl binds to newly generated phospho- inositides (mainly PI(3,4,5)P3 and PI(4,5)P2), via its PH domain thereby localizing to the plasma membrane [56]. Considering the potential of Class II PI3K to generate the same spectrum of phosphoinositides as Class I PI3K [7,9,14], this model provides a possibility for PI3KC2b to build up a complex consisting of Grb2, ACK1 and Dbl, which may be important for RhoA and Rac activation in serum-supplemented conditions and Rac activation upon EGF or PDGF stimulation. After EGF or PDGF stimulation, the Dbl/PI3KC2b complex may be recruited to the plasma membrane via Grb2 and generates PI(3,4,5)P3, which binds to the PH domain of Dbl. Grb2, on the other hand, may bind to ACK11 complexed with Cdc42. Being in close proximity, ACK1 could therefore bind to Dbl. Ack1 could phosphorylate Dbl only when in complex with Grb2 and Cdc42, which then become activated and in turn activates RhoA and Rac leading to stress fibres and membrane ruffles formation. Further investigations of the phos- phorylation status of Dbl in the NIH3T3 cells over-expressing PI3KC2b are needed to confirm this model. Surprisingly, the increased basal and PDGF-induced Rac1 activity in NIH3T3 cells expressing wild-type PI3KC2b did not correspond to increased wound healing properties, a response which can be explained by the fact that active RhoA and the generation of focal adhesions and stress fibres significantly slow down cell migration [57]. NIH3T3 cells transfected with dominant negative PI3KC2b cells failed to activate Rac1 after PDGF stimulation. This became more evident when looking at the completely abolished wound healing properties of the cells after PDGF treatment, once again confirming the importance of PI3KC2b-dependent PDGFR signalling. The RhoA-activating function of PI3KC2b also had inhibitory effects on detachment- induced apoptosis. We observed this outcome in both NIH3T3 cells stably expressing wild type PI3KC2b and in NIH3T3 cells transiently transfected with the wild type PI3KC2b. Moreover, in both systems, caspase activation was reduced down to ,65% of its original activity after plating the cells under ultra-low attachment conditions. The same effect was observed after transient overex- pression of constitutively active RhoA in NIH3T3 cells. These results are in agreement with a recent report showing that active RhoA was able to rescue NIH3T3 cells from detachment-induced apoptosis [58]. This anti-apoptotic effect under stress conditions Role of PI3KC2b in RhoA Activation could play also a role under normal conditions as we observed slightly, but significant, increase in cell proliferation in NIH3T3 cells expressing wild type PI3KC2b, compared to control cells. Even ROCK, a downstream target of RhoA has been widely accepted as mediator of apoptosis by inducing characteristic morphological changes, including cell contraction and dynamic membrane blebbing, RhoA seems to be excluded from this signalling cascade [59]. Here we provide further evidence that sustained RhoA activation provides NIH3T3 cells with pro- survival signals and enables anchorage-independent cell survival, one of the first phenomenon described for oncogenic Rho proteins [60]. Together, our data show for the first time that overexpression of PI3KC2b in NIH3T3 cells is sufficient to induce RhoA and Rac activation and thus to promote cell transforming properties, reported for both RhoA and Rac [61,62]. We have further identified the Dbl RhoGEF as a new interaction partner for PI3KC2b. Previous reports [10,17] and unpublished observations have revealed that PI3KC2b is overexpressed in different human cancer cell lines and primary tumour samples, and modulates cell survival and chemotherapy resistance [63]. Together with the present report we propose a model in which PI3KC2b can contribute to the protection of cells from anoikis and increase Rho/Rac, Akt and Erk signalling downstream of the EGFR and PDGFR. This signalling pathway may therefore contribute to the malignant progression of human cancers. Supporting Information Figure S1 PI3KC2b and Grb2 do not associate directly with Dbl. (A) GST-tagged and gluthatione-sepharose beads conjugated spectrin- and PH- Dbl domains, and GST as control, were incubated in vitro with GST-truncated soluble PI3KC2b N- terminal domain (NT-C2b) (,35 kDa) and Grb2 (26 kDa). To detect an interaction wih Dbl domains samples were subjected to immunoblotting with indicated antibodies. Experiment was repeated four times with similar results, independently of buffer used for the reaction (Brij96 or Triton 1%). One representative blot is shown (Brij96 1% used for the incubation). (B) Control in vitro reactions, which utilized immobilized on gluthatione-sephar- ose beads GST-tagged NT-C2b domain and Grb2, and GST as control, were incubated in vitro with GST-truncated soluble NT- C2b domain (,35 kDa) and Grb2 (26 kDa). Samples were subjected to SDS-PAGE analysis and the interaction between NT-C2b and Grb2 was detected with anti-GST antibody by western blot analysis. (C) Representative immunoblot of Dbl domains separated on SDS-PAGE and analysed by wester blot with anti-GST antibody. (TIF) Figure S2 PI3KC2b regulates wound healing properties. (A) Wound healing assay in NIH3T3-V, -C2b-DN and -C2b-WT cells, which were grown in 10% FCS or were serum-starved over night and stimulated with 10 nM PDGF. Migration rate was monitored for 16 h by phase contrast microscopy. Graph presents quantitative analysis of time-lapse microscopy pictures. Data are mean 6 SD of two independent experiments. (B) Cell proliferation of NIH3T3-V, -C2b-DN and -C2b-WT cells, which were growing for 48 h either in 10% or 0.5% FCS was performed. The number of viable cells was determined based on Trypan Blue exclusion and cell counting. Data are mean 6 SD of three independent experiments. *, p,0.05. (TIF) PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e44945