Phosphoinositide 3-Kinase C2b Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl Karolina Błajecka1., Marin Marinov2., Laura Leitner3, Kristin Uth1, Guido Posern3, Alexandre Arcaro1* 1 Department of Clinical Research, University of Bern, Bern, Switzerland, 2 Department of Oncology, University Children’s Hospital Zurich, Zurich, Switzerland, 3 Department of Molecular Biology, AG Regulation of Gene Expression, Max-Planck-Institute of Biochemistry, Martinsried, Germany Abstract The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2b. Dbl was co-immunoprecipitated with PI3KC2b in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2b in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2b strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2b over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant- negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal- regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2b expression. Furthermore, increased expression of PI3KC2b protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2b modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA. Citation: Błajecka K, Marinov M, Leitner L, Uth K, Posern G, et al. (2012) Phosphoinositide 3-Kinase C2b Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl. PLoS ONE 7(9): e44945. doi:10.1371/journal.pone.0044945 Editor: Pontus Aspenstrom, Karolinska Institutet, Sweden Received December 20, 2011; Accepted August 14, 2012; Published September 12, 2012 Copyright: 2012 Błajecka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Swiss National Science Foundation (grants 3100A0-105321 and 31003A-120294). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexandre.arcaro@dkf.unibe.ch . These authors contributed equally to this work. Introduction The phosphoinositide-3 kinase (PI3K) family comprises evolu- tionary conserved lipid kinases characterised by their ability to phosphorylate phosphoinositides at the D-3 position of the inositol ring. This reaction leads to the production of essential for the cell second messengers as PI(3)P, PI(3,4)P2 and PI(3,4,5)P3 [1]. The subsequent membrane recruitment of enzymes recognizing and binding D-3 phosphorylated phosphoinositides leads to their activation, which results in the initiation of multiple signalling cascades providing growth, survival and motility signals to the cell. The biological functions of PI3K have been widely investigated by the use of transgenic mouse models and in vitro cell systems. It became clear that PI3K are extremely important for cell proliferation, differentiation, chemotaxis and motility. Further- more, they are implicated in the embryonic development, immunity and the development of diseases such as cancer, inflammation and diabetes [1–3]. The family of PI3K comprises three classes, based on structural similarity and in vitro substrate specificity. Class I PI3K is divided into Class IA, comprising three isoforms activated by growth factor receptor tyrosine kinases (RTKs) and Class IB with a single member activated by the Gbcsubunit downstream of activated G-protein coupled receptors (GPCR) [1]. The main product of Class I PI3K activity, the lipid second messenger PI(3,4,5)P3, activates a plethora of downstream molecules by binding to their pleckstrin-homology (PH) domains [4]. To one of the best studied effectors belong the serine/ threonine kinase Akt [5] and RhoGEFs [6]. Class II PI3K consists of three members termed PI3KC2a, PI3KC2b and PI3KC2c. In vitro, the main substrates of these enzymes are PI and PI(4)P [7,8]. Class II PI3K are activated downstream of RTK (EGFR, PDGFR and Kit), cytokine receptors, insulin and integrins [9–13]. Localisation of PI3KC2a in the trans-Golgi apparatus and in clathrin vesicles indicates function in clathrin assembly and clathrin mediated trafficking [14,15]. Further studies have revealed that PI3KC2b is implicated in the cytoskeletal regulation and migration of cancer cells [16– 18]. However, the specific functions of Class II PI3K in response to their activators remain to be fully understood. The control of actin filaments dynamic is crucial for cell morphology, adhesion and movement [19]. PI3Ks regulate cell polarity and motility by controlling multiple proteins and protein complexes implicated in cytoskeletal rearrangement and actin dynamics [20]. It was recently demonstrated in Drosophila melanogaster hemocytes that Pi3K68D in collaboration with PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44945