Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis
(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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https://lup.lub.lu.se/record/1782650
- author
- Rönnstrand, Lars LU ; Siegbahn, Agneta ; Rorsman, Charlotte ; Johnell, Matilda ; Hansen, Klaus and Heldin, Carl-Henrik
- publishing date
- 1999
- type
- Contribution to journal
- publication status
- published
- subject
- 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
- in
- Journal of Biological Chemistry
- volume
- 274
- issue
- 31
- pages
- 22089 - 22094
- publisher
- American Society for Biochemistry and Molecular Biology
- ISSN
- 1083-351X
- language
- English
- LU publication?
- no
- additional info
- 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)
- id
- 7d6ce19f-c098-43e9-a9e8-640e2b4595b5 (old id 1782650)
- date added to LUP
- 2016-04-04 07:10:24
- date last changed
- 2019-05-22 02:18:06
@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}}, 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}}, language = {{eng}}, number = {{31}}, pages = {{22089--22094}}, publisher = {{American Society for Biochemistry and Molecular Biology}}, 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}}, }