Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-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., Engström, U., Wernstedt, C., Heldin, C.-H., and Rönnstrand, 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.
Rönnstrand
L
L
Ludwig Institute for Cancer Research, Biomedical Centre, Box 595, S-751 24 Uppsala, Sweden. Lars.Ronnstrand@LICR.uu.se
Siegbahn
A
A
Rorsman
C
C
Johnell
M
M
Hansen
K
K
Heldin
C H
CH
D019943:Amino Acid Substitution; D000818:Animals; D000077214:Becaplermin; D002460:Cell Line; D002633:Chemotaxis / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017354:Point Mutation; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D002867:Chromones; D004791:Enzyme Inhibitors; D007211:Indoles; D007295:Inositol Phosphates; D007527:Isoenzymes; D008301:Maleimides; D009025:Morpholines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D015544:Inositol 1,4,5-Trisphosphate; D019869:Phosphatidylinositol 3-Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; C070515:bisindolylmaleimide I; D004318:Doxycycline