Fig. 6. Binding of Crk and p85/PI 3-kinase to phosphorylated Cbl mediates lamellipodia formation in PC12 cells. (A) PC12 cells stably expressing the PDGFR ß-receptor and overexpressing Pyk2 and Cbl-CT (proline-rich regions, acidic box and leucine zipper domain) were analyzed for the formation of lamellipodia following stimulation with PDGF-BB for 48 hours. Cbl was detected with anti-Cbl (C-15) and Pyk2 with anti-Pyk2 (NT) antibodies. Cbl was visualized with swine anti-rabbit TRITC secondary antibodies, Pyk2 with donkey anti-goat Alexa 488 and F-actin with AMCA-labeled phalloidin. The boxed regions are enlarged in the panels below to show lamellipodia. (B) PC12-PDGFR cells were transiently transfected with Pyk2 and wild-type Cbl or Cbl mutants (Cbl-Y700F, Cbl-Y700/774F or Cbl-Y700,731,774F=Cbl-3YF) and stimulated with 100 ng/ml PDGF-BB for 48 hours. Cells were then subjected to immunofluorescence with goat polyclonal anti-Pyk2 (N-19) and rabbit polyclonal anti-Cbl (C-15) antibodies followed by incubation with Alexa-488-conjugated donkey anti-goat and TRITC-labeled swine anti-rabbit antibodies. Cells overexpressing Pyk2 and Cbl or Cbl mutants were quantified for the presence of neurite lamellipodia. Percentages for each construct relative to the wild-type control were determined as described in Materials and Methods. The error bars represent the standard error of the mean. (C) PC12-PDGFR cells overexpressing Pyk2 and Cbl were stimulated with 100 ng/ml PDGF-BB for 48 hours and treated with 100 mM LY294002 (a PI 3-kinase inhibitor) for 30 minutes, 4 hours or 6 hours. Cells overexpressing Pyk2 and Cbl were visualized by immunofluorescence as in B. Transfected cells showing neurite lamellipodia were quantified for each time period and compared to untreated cells. Percentages were determined as described in Materials and Methods. The error bars represent the standard error of the mean. (D) PC12-PDGFR cells overexpressing Pyk2, Cbl and wild-type CrkII, CrkII-SH2M or CrkII-SH3M were stimulated for 48 hours with PDGF-BB and quantified for the presence of neurite lamellipodia. Percentages for each construct were determined as in Materials and Methods. The error bars represent the standard error of the mean. (E) Model of interactions between Cbl, ArgBP2 and Pyk2 in lipid rafts and of how growth factor signals might regulate lamellipodia formation via this complex. ArgBP2 associates via its SH3 domains with proline-rich motifs in Pyk2 (via SH3A) and Cbl (via SH3B and SH3C) and is able to recruit Pyk2 and Cbl to lipid rafts via its SoHo domain. Growth factor stimulation promotes Pyk2 activation and its autophosphorylation on Y402. This leads to activation of Src family kinases, which are able to phosphorylate tyrosines in the carboxyl termini of Pyk2 and Cbl (Y700, Y731 and Y774). This in turn allows Pyk2 and Cbl to recruit several effectors involved in regulating the cytoskeleton. In particular, Cbl recruits Crk and the p85 subunit of PI 3-kinase, which are involved in Rac-dependent lamellipodia formation in the neurite growth cone.