Overactivation of phospholipase C-γ1 renders platelet-derived growth factor β-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis

We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) beta-receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF beta-receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engstrom, U., Wernstedt, C., Heldin, C.-H., and Ronnstrand, L. (1996) EMBO J. 15, 5299-5913). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma 1 (PLC-gamma 1), measured as inositol-1,4,5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma 1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma 1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-gamma 1. To assess whether increased activation of PLC-gamma 1 is the cause of the hyperchemotaetic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wildtype or a catalytically compromised version of PLC-gamma 1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-gamma 1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-gamma 1 mutant had no effect on PDGF-BB-induced chemotaxis. Furthermore, in cells expressing normal levels of PLC-gamma 1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-gamma 1 was not inhibited by the PI3-kinase inhibitor LY294002. These observations suggest the existence of two different pathways which mediate PDGF-induced chemotaxis; depending on the cellular context, the PI3-kinase pathway or the PLC-gamma 1 pathway may dominate.