used recombinant GST-tagged spectrin- and PH- Dbl domains immobilized on gluthatione-sepharose beads together with the soluble N-terminal region of PI3KC2bor/and Grb2– expressed and purified in E.coli (Fig. S1). In the case of NT-PI3KC2b, we did not observe an interaction with Dbl. On the other hand, Grb2 showed an interaction, but due to detectable binding with GST control, we considered it as un-specific. The lack of association of the NT-PI3KC2b and Grb2 with Dbl domains suggests that other factors are required for the interaction in living cells (Fig. S1). Overexpression of PI3KC2b is Sufficient to Induce Cell Morphology Changes in a Kinase-dependent Manner We and others have reported that PI3KC2b interacts with different receptor tyrosine kinases such as EGFR, PDGFR and c- Kit [9,10,12]. We aimed to investigate the biological importance of PI3KC2b in NIH3T3 mouse fibroblast cells. Since these cells have very low levels of endogenous PI3KC2b expression, we established NIH3T3 clones stably expressing an epitope wild-type (WT) and a mutant kinase-dead (DN) PI3KC2b form. NIH3T3 cells stably transfected with an empty pBabeNeo vector were used as control. We utilised a mutant that we previously reported to eliminate PI3KC2b-associated lipid kinase activity by mutation of the highly conserved aspartate (DFG) to an alanine residue in the activation domain (D1213A, PI3KC2b-DN) [17]. Western blot analysis confirmed the expression of each of the constructs (Fig. 3A). For further experiments clones with high recombinant protein expression were selected. Interestingly, we observed striking morphological differences between the cell clones differentially expressing either the WT or DN PI3KC2b. In comparison to control cells, NIH3T3-C2b-WT cells displayed increased cell spreading with strong stress fibres assembly and increased membrane ruffling revealed by F-actin staining (Fig. 3B), whereas the clones expressing the kinase-dead form of PI3KC2b did not display F-actin polymerisation and presented rounded phenotype with a strongly reduced cell spreading (Fig. 3B). Staining for PI3KC2b showed cytosolic distribution with addi- tional localisation of the protein to cell extensions and cell-cell contacts (Fig. 3B). Stimulation with PDGF led to more pronounced membrane localisation of PI3KC2b protein in NIH3T3-C2b-WT cells (data not shown), revealing recruitment to the activated PDGF receptor in the cell membrane as reported [9]. In line with our previous report [17], these data demonstrate that the lipid kinase activity of PI3KC2b plays a role in modulating the actin cytoskeleton in NIH3T3 cells. PI3KC2b Regulates Rac and RhoA Activity Due to the observation, that NIH3T3-C2b-WT cells show increased spreading and membrane ruffling, two phenotypes related to the small GTPases RhoA and Rac1 [24], we analysed RhoA activity in serum-supplemented conditions and found highly elevated basal levels of GTP-loaded RhoA in NIH3T3-C2b-WT cells in comparison to control cells (Fig. 4A). Based on recent observation that PI3KC2b modulates cell migration in response to lysophosphatidic acid (LPA) [16], we treated serum-starved cells with LPA, a commonly used RhoA activator [42]. NIH3T3-C2b- WT cells showed increased RhoA activation, but also higher basal activity in low serum, compared to control cells (Fig. 4A). NIH3T3-C2b-DN cells displayed only a minimal response to LPA treatment (Fig. 4A). To confirm these observations we used a different approach, making use of the fact that RhoA activates a MADS-Box transcription factor termed serum response factor (SRF) [43]. SRF controls growth factor regulated immediate-early genes such as c-fos and cytoskeletal actin [44]. RhoA downstream targets such as mDia and LIM kinase 1 effectively regulate SRF by Role of PI3KC2b in RhoA Activation modulating the activity of a SRF co-factor termed MAL [45]. MAL is constitutively bound to monomeric actin and thus sequestered in the cytosol. Activation of RhoA in response to serum leads to actin polymerisation, MAL release from G-actin and its translocation to the nucleus, where it binds and activates SRF [45]. In a transactivation luciferase assay we determined up to 5-fold MAL-dependent SRF response in NIH3T3-C2b-WT cells after serum stimulation as compared to control cells (Fig. 4B). In contrast, NIH3T3-C2b-DN cells showed only minimal SRF activation (Fig. 4B). We next analysed Rac activity in serum-supplemented condi- tions. NIH3T3-C2b-WT cells showed significantly elevated Rac GTP loading, whereas the three DN clones tested had reduced Rac activity compared to control cells (Fig. 4C). Next we investigated Rac activation downstream of the PDGFR. We found strongly increased Rac GTP levels upon PDGF stimulation in NIH3T3-C2b-WT cells compared to control cells (Fig. 4D). Phosphorylation of the downstream Rac target SAPK/JNK was strongly elevated as well (Fig. 4D). Interestingly, in NIH3T3-C2b- DN we observed down to 50% reduced Rac activation and accordingly very weak SAPK/JNK phosphorylation (Fig. 4D). Taken together, these results indicate that PI3KC2b is implicated in the LPA and serum dependent activation of RhoA and in the Rac activation upon PDGF treatment. The Assembly of the Dbl - PI3KC2b Complex is not Modulated by PI3K Activity or Cell Stimulation with EGF or PDGF The data presented above suggested that the catalytic activity of PI3KC2b may modulate activation of Rho/Rac proteins via Dbl. In order to further investigate the importance of PI3KC2b kinase activity for its interaction with Dbl, we assessed the ability of PI3KC2b WT and DN to interact with Dbl in transiently transfected HEK293 cells and stably transfected NIH3T3 cells. In HEK293 cells, both PI3KC2b WT and DN interacted with endogenous or ectopically expressed (HA-tag) Dbl in a similar manner (Fig. 5A). This observation was confirmed in NIH3T3- C2b-WT and NIH3T3-C2b-DN cells (Fig. 5B). We next assessed whether the Dbl - PI3KC2b complex was constitutively assembled, or induced by cell stimulation with polypeptide growth factors. The Dbl - PI3KC2b complex could be detected in serum-starved NIH3T3-C2b-WT cells, and the interaction was apparently not increased by cell stimulation with EGF or PDGF from 59 up to 309 (Fig. 5C). Finally, we investigated whether the catalytic activity of PI3KC2b contributes to the activation of Dbl GEF activity. In transiently transfected HEK293 cells, ectopic expression of PI3KC2b WT or DN did not alter the GEF activity of endogenous Dbl towards RhoA (Fig. 5D). Taken together, these results indicate that PI3KC2b lipid kinase activity is not required for its association with Dbl, and does not modulate Dbl activity measured in vitro. PI3KC2b Protects NIH3T3 Cells from Detachment- induced Death (Anoikis) Forkhead fox O (FOXO) family of transcription factors have been reported to control the expression of caveolin-1 [46]. Phosphorylated FOXO remains in the cytosol [47] and is not able to activate Caveolin 1 transcription. Based on these reports we investigated Caveolin 1 and 2 expression and found strong protein downregulation of both proteins (Fig. 6D). These two isoforms are crucial for the proper formation and function of caveolae: membrane invaginations serving as signalling platforms PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44945