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Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis

Rönnstrand, Lars LU ; Siegbahn, Agneta; Rorsman, Charlotte; Johnell, Matilda; Hansen, Klaus and Heldin, Carl-Henrik (1999) In Journal of Biological Chemistry 274(31). p.22089-22094
Abstract
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, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general... (More)
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, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-gamma1. To assess whether increased activation of PLC-gamma1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wild-type or a catalytically compromised version of PLC-gamma1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-gamma1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-gamma1 mutant had no effect on PDGF-BB-induced chemotaxis. Furthermore, in cells expressing normal levels of PLC-gamma1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-gamma1 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-gamma1 pathway may dominate. (Less)
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@article{7d6ce19f-c098-43e9-a9e8-640e2b4595b5,
  abstract     = {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, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-gamma1. To assess whether increased activation of PLC-gamma1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wild-type or a catalytically compromised version of PLC-gamma1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-gamma1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-gamma1 mutant had no effect on PDGF-BB-induced chemotaxis. Furthermore, in cells expressing normal levels of PLC-gamma1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-gamma1 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-gamma1 pathway may dominate.},
  author       = {Rönnstrand, Lars and Siegbahn, Agneta and Rorsman, Charlotte and Johnell, Matilda and Hansen, Klaus and Heldin, Carl-Henrik},
  issn         = {1083-351X},
  keyword      = {Platelet-Derived Growth Factor beta
Receptors,Platelet-Derived Growth Factor/genetics/*physiology
Recombinant Proteins/chemistry/metabolism
Transfection
Type C Phospholipases/chemistry/*metabolism,4,5-Trisphosphate/metabolism
Inositol Phosphates/metabolism
Isoenzymes/chemistry/*metabolism
Kinetics
Maleimides/pharmacology
Morpholines/pharmacology
Peptide Mapping
Phosphatidylinositol 3-Kinases/*metabolism
Phospholipase C gamma
Phosphorylation
Platelet-Derived Growth Factor/pharmacology
Point Mutation
Receptor,Enzymologic/drug effects
Indoles/pharmacology
Inositol 1,Amino Acid Substitution
Animals
Cell Line
Chemotaxis/*physiology
Chromones/pharmacology
Doxycycline/pharmacology
Enzyme Activation
Enzyme Inhibitors/pharmacology
Gene Expression Regulation},
  language     = {eng},
  number       = {31},
  pages        = {22089--22094},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis},
  volume       = {274},
  year         = {1999},
}