Figure 7. PI3KC2b increases MAPK and Akt signalling down- stream of EGFR and PDGFR. (A) and (B) NIH3T3-V, -C2b-DN and - C2b-WT cells were serum-deprived overnight and stimulated for 10 min. with EGF (A) or PDGF (B) as indicated. Cell lysates were analysed by immunobloting and MAPK and Akt pathway activation was assessed with indicated phospho-specific antibodies. (C) and (D) Lysates of NIH3T3-V, -C2b-DN and -C2b-WT cells grown in 10% FCS were analysed for activated signalling molecules implicated in the cell cycle control and caveolae formation such as p53, Bcl2, PTEN and caveolin 1, 22. WT1, 22 and DN1, 22 indicate individual clones. doi:10.1371/journal.pone.0044945.g007 Role of PI3KC2b in RhoA Activation with lysates of NIH3T3 cells stably expressing Glu-tagged wild- type (WT) PI3KC2b form. As shown in Fig. 1A, we detected a binding of the recombinant PI3KC2b with endogenously expressed RhoGEF Dbl, which belong to one of the best studied regulators of RhoGTPases [36]. Our further analysis showed that PI3KC2b co-immunoprecipitated Grb2 and Src (Fig. 1A). Grb2 has been reported to serve as an adaptor protein for PI3KC2b downstream of the EGFR in HEK293 cells [12], whereas Src is involved in EGFR and PDGFR downstream signalling in NIH3T3 fibroblasts [41]. The results revealed an endogenous PI3KC2b/Dbl complex in the NIH3T3 parental cell line (Fig. 1B). To confirm our findings, we selected IMR5, a neuroblastoma cancer cell line, which expresses relatively high levels of PI3KC2b and Dbl, and performed co-immunoprecipitation with anti-Dbl and anti-PI3KC2b antibodies. As depicted in Figure 1B, PI3KC2b associated with Dbl in the IMR5 cells confirming the formation of an endogenous protein complex. We were further interested in studying the molecular mechanism of PI3KC2b/Dbl complex assembly. Pull down experiments with GST-fused PI3KC2b N-terminal and C2 C-terminal domain and lysates of HEK293 cells transfected with HA-tagged proto-Dbl revealed that the N-terminal sequence of the PI3K is involved in the interaction with Dbl (Fig. 1C). The N terminus of PI3KC2b (in particular proline-rich regions) was previously shown to mediate PI3KC2b association with the activated EGF receptor through the Grb2 adaptor protein [12]. Interestingly, our pull down experiments with purified GST-fused Grb2 and lysates of HEK293 cells transfected with HA-tagged proto-Dbl showed binding of Grb2 to Dbl, and confirmed the interaction with PI3KC2b (Fig. 1D). Additionaly, Grb2 complex formation with endogenous Dbl was detected in untransfected HEK293 cells, which confirms the relevance of this interaction. These findings demonstrate the formation of a multi-protein complex comprising PI3KC2b, Grb2 and Dbl, which may be functional in normal cells (NIH3T3), as well as in cancer cells (IMR5). Whether the protein-protein interactions in this complex are direct or mediated by other possible interaction partners was further elucidated in an in vitro binding assay. Dbl RhoGEF Domains Bind to PI3KC2b with Different Affinities Considering the multi-domain structure of Dbl, we were interested in uncovering which part of the protein sequence is involved in the interaction with PI3KC2b. Pull down experiments with glutathione-sepharose beads from COS-1 cells co-transfected with GST-fused Dbl N-terminal-, spectrin-, onco-, PH- mutants and wild-type PI3KC2b showed a prominent interaction of the kinase with the Dbl spectrin- and PH- domain (Fig. 2). On the other hand, none of the Dbl domains bound to Grb2, whereas Grb2 associated with full-length HA-proto-Dbl immunoprecipi- tated with anti-Dbl antibody and immobilized on beads. The N- terminal spectrin- and C-terminal PH- domain of Dbl were previously shown to associate with the chaperone Hsc70. This interaction results in Dbl adopting an inactive conformation, which in turn blocks an access of GTPases to its DH- catalytic domain [31,33]. Furthermore, it was suggested that the spectrin domain regulates the level, localization, and activity of proto-Dbl through the interaction with Hsc70-Hsp90 and the ubiquitin ligase CHIP [32]. However, further investigations are required to exactly assess the contribution of PI3KC2b to the regulation of Dbl activity. The binding of Dbl spectrin and PH domains to PI3KC2b in co-transfected COS-1 cells led us to test whether this interaction is direct. Therefore, an in vitro binding assay was performed, which PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44945