Phosphoinositide 3-Kinase C2 Regulates Cytoskeletal Organization and Cell Migration via Rac-dependent Mechanisms

Supplemental Material

This article contains the following supporting material:

• Movie 1 - Control and PI3KC2β-transfected A-431 cells were plated on 100-mm plates in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). For live imaging, cells were analyzed with an Olympus IX-70, collecting images every 30 sec for up to 30 min. Images were compiled into movies by using the program metamorph.
• Movie 2 -

• Figure 1 - Evaluation of lipid kinase activity of PI3KC2βƒnproteins and morphological effects of ectopic expression of wild type and kinase dead PI3KC2β. (A) Wild type and kinase dead PI3KC2β D1213A were immunoprecipitated from the respective A-431 cell lines with the 9E10 Myc monoclonal antibody and lipid kinase activity was assessed in the presence of calcium ions as previously reported (Arcaro et al., 1998). (B-C) Phase contrast images of growing control A-431 cells and 3 A-431 cell lines stably expressing wild type PI3KC2β (Myc-4, Myc-6) and cell lines expressing kinase-dead PI3KC2β (PI3KC2β DN-32).

• Figure 2 - Mechanism of resistant to anoikis in PI3KC2β-transfected A-431 cells. A-431 and A-431-C2β WT cells were transfected with RNAi oligonucleotide targeting Rac1 (A and C), AKT1 and/or 2 (G and C), MKK7 (H and C), or scrambled (Sc) control oligonucleotides (A, C, G and H) as indicated. 48h later cells were either analysed by Western-blotting for downregulation of target-proteins in RNAi treated cells (C) or placed for 12h in anchorage-free conditions in the presence or absence of PD098059 (F), SB202190 (F), Rapamycin (G) or E-cadherin neutralising antibodies (D). (C) “A” indicates A-431 while “M” indicates A-431-C2β WT cells. Actin immunodetection was used as a loading control. Cells were then stained using propidium iodide (A, B, F, G and H) and the proportion of cells in Sub-G1 determined by flow cytometry. (D) A-431-C2β-WT cells display an E-cadherin-dependent increase in cell-to-cell contacts as compared to A-431 cells. Confocal microscopy of A-431 and A-431-C2β WT cells treated with and without E-Cadherin neutralising antibodies for 12 h. Actin staining was achieved using Alexa 488-Phallodin. (A to H) are representative results from a minimum of three independent experiments.

• Figure 3 - Proposed model of complex assembly and recruitment to activated EGFR-1. Under basal conditions, Grb2 is complexed with PI3KC2β through its C-terminal SH3 domains. Upon EGF stimulation, the macromolecular complex of PI3KC2β-Grb2-Abi1-Eps8-Sos is recruited to activated EGFR-1 either directly on pY1068 through Grb2 recruitment or indirectly on pY992 through complex formation with Shc. EGF activates class II PI3K lipid kinase activity and initiates signal propagation, which mediates Rac activation, cell-cell adherens junction assembly and F-actin polymerisation.