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

Research output: Contribution to journalArticle

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.

Details

Authors
  • Lars Rönnstrand
  • Agneta Siegbahn
  • Charlotte Rorsman
  • Matilda Johnell
  • Klaus Hansen
  • Carl-Henrik Heldin
External organisations
  • External Organization - Unknown
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medicinal Chemistry

Keywords

  • 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
Original languageEnglish
Pages (from-to)22089-22094
JournalJournal of Biological Chemistry
Volume274
Issue number31
Publication statusPublished - 1999
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes

Bibliographic note

The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Experimental Clinical Chemistry (013016010)