endosomal myotubularin PI3K phosphatase Mtm-1 coregulates the PI(3)P pool, which in turn promotes F-actin reorganization and modulates protrusion formation [21]. D. melanogaster Pi3K68D is a homologue of human PI3KC2b and human Mtm genes present high functional conservation with the D. melanogaster MTM family members, which suggests that the MTM/Class II PI3K pathway might be important for similar biological responses in mammalian cells [21]. However, the main players in the regulation of actin dynamics are GTP-binding proteins known as RhoGTPases. A recent report in vascular smooth muscle cells of spontaneously hypertensive rats showed that Class II PI3KC2a is involved in Ca2+-induced Rho signaling pathway activation, which stimulates the interaction of myosin II (MII) with actin filaments, which in turn leads to increased cell contractility [22]. RhoGT- Pases proteins cycle between inactive GDP- and active GTP- bound states and hydrolyse GTP to GDP. This process is tightly regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). The best studied Rho GTPases under the control of PI3K activity are RhoA, Rac1 and Cdc42 [23]. Rac1 is involved in the formation of membrane extensions (lamellipodia or ruffles) along the leading edge of the cell. Cdc42 controls the formation of spike-like membrane extensions (filopo- dia), involved in the recognition of the extracellular environment. RhoA stabilises the cell body and promotes cell attachment to the extracellular matrix by inducing the formation of stress fibres and focal adhesions [24]. Ligand binding to RTK, integrins and GPCR results in PI3K activation and accumulation of PI(3,4)P2 and PI(3,4,5)P3 in the cell membrane. This process is spatially defined by the site of ligand concentration and results in the local membrane recruitment of GEFs [25]. The dbl oncogene was the first GEF identified by cloning from human diffuse B-cell lymphoma and subsequently from nodular poorly differentiated lymphoma (NPDL), which upon transfection into NIH3T3 cells induced cellular transformation [26,27]. The oncogenic form of dbl arises from full length dbl as a result of amino acid truncation of its N-terminus, which is crucial for the increased transforming activity of dbl oncogene [28,29]. The mechanism of proto-Dbl regulation is strongly dependent on its multi-domain structure. As many other GEFs family members, it contains Dbl homology- Pleckstrin homology (DH-PH) tandem domains. These domains are respectively responsible for the intrinsic GEF catalytic activity toward RhoGTPases and the intracellular distribution to the plasma membrane or to the cytoskeletal matrix [30–32]. The spectrin domain of proto-Dbl located at the N-terminus regulates Dbl activity by indirect binding to its PH-domain, thus blocking the access to the catalytic DH domain and masking the intracellular targeting function of the PH domain [31–33]. Many GEFs exist in an inactive or partially active state maintained by complex intra- and intermolecular interactions. These inhibitory conformations can be altered by upstream regulatory signals, which involve phosphorylation, oligomerization, interactions with heterotrimeric G-protein subunits, protein kinases, adaptor or scaffolding proteins, or phosphoinositol lipids, and result in intracellular translocation and stimulation of the GEF catalytic activity [34–36]. Once GEFs are at the membrane, they assemble into multi-protein complexes with PI3K, RhoGTPases and actin- binding proteins [37]. These multi-protein complexes are crucial for the proper localisation to dynamic actin structures and the efficient activation of RhoGTPases [37,38]. In our previous study, we reported the implication of PI3KC2b in a multi-protein complex regulating Rac activity in A-431 epithelial carcinoma cells [17]. In order to investigate the function of PI3KC2b in non-tumor cells, we established clones of NIH3T3 Role of PI3KC2b in RhoA Activation fibroblast stably expressing either the wild-type or dominant- negative form of the enzyme. According to our previous data, we found elevated Rac/JNK activation upon PDGF stimulation in PI3KC2b wild-type expressing cells. We further observed increased cell spreading and strong stress fibres formation due to elevated RhoA activity. We show that serum stimulation leads to strong serum response factor (SRF) induction downstream of RhoA and polymerised actin. This finding led us to the identification of a RhoGEF Dbl as interaction partner of PI3KC2b. Hence, we hypothesise that in NIH3T3 cells PI3KC2b is involved in a complex comprising Dbl and regulates RhoA and Rac1 activity. This signalling cascade further protected the cells from detachment-induced apoptosis. Materials and Methods Reagents and Antibodies The following antibodies were used: PI3KC2b was described in [7], 9E10 myc epitope, p115RhoGEF, Dbl, Caveolin-2, Grb2, Src, RhoA, GFP, p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Caveolin-1, Akt (pan), phospho-Akt (Ser473), phospho- Akt (Thr308), phospho-SAPK/JNK (Thr183/Tyr185), phospho- S6 ribosomal protein (Ser240/244), phospho-GSK-3a/b (Ser21/ 9), phospho-FOXO1 (Thr24)/FOXO3a (Thr32), phospho-Erk1/ 2 (Thr202/Tyr204), p44/42 MAPK, Bcl-2, Bcl-XL, PTEN, phospho-Bad (Ser136) (Cell Signalling Technology, Danvers, MA, USA); FITC-conjugated anti-mouse and anti-rabbit anti- bodies, b-actin, anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA); anti-rabbit-IgG (R&d Systems, Minneapolis, MN,USA); Rac1 (BD Transduction Laboratories, USA). Anti-EE antibody was kindly provided by Julian Downward (CRUK). Alexa Fluor 555 was obtained from Invitrogen. Recombinant EGF, PDGF BB were purchased from Calbiochem, La Jolla,CA,USA. Oleoyl-L-a- lysophosphatidic acid (LPA) was purchased from Sigmla-Aldrich, St Louis, MO, USA. The SRF luciferase reporter plasmid p3DA-Luc has been described previously [39]. Cell Culture Mouse NIH3T3 fibroblast, HEK293 and COS-1 cells were purchased from the American Type Culture Collection. Mouse NIH3T3 fibroblast, HEK293 and COS-1 cells were grown in DMEM (Life Technologies/Invitrogen) supplemented with 10% (v/v) FCS and penicillin/streptomycin/L-glutamine, and passaged every 3 to 5 days by trypsinization. Cultures were incubated in a humidified atmosphere of 5% CO2 at 37uC. Stably transfected NIH3T3 clones were grown in the presence of 0.8 mg/ml G418. Retroviral Infection NIH3T3 cell lines stably expressing Glu-tagged PI3KC2b wild- type (WT) were described [9]. The cDNA construct encoding myc-tagged dominant-negative kinase-dead PI3K-C2b (DN) [17] was subcloned into the pBabe Neo Vector. Retroviral packaging BOSC 23 cells were transfected using a Ca2+ phosphate protocol. NIH3T3 cells were grown up to 40–60% confluence and infected with the recombinant retrovirus for 48 h at 37uC. The cultures were split 1:10 in DMEM- 10% FCS and 0.8 mg/ml G418 containing medium. Colonies of resistant cells appeared after 1 week. Single colonies were picked and expanded in selective medium. Protein expression was confirmed by western blot analysis. All experiments were done with at least two clones from each transfection. Cells transduced with pBabe Neo vector without insert were used as controls. PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44945