Overexpression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis

Jiankang Chen, Jorge Capdevila, Raymond C. Harris

Research output: Contribution to journalArticle

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Abstract

We have previously reported that 14,15-epoxyeicosatrienoic acid (14,15- EET) is a potent mitogen for the renal epithelial cell line, LLCPKcl4. This mitogenic effect is dependent upon activation of a protein-tyrosine kinase cascade that results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET also activated Src in these cells, we stably transfected LLCPKcl4 with an expression construct of the C-terminal Src kinase (CSK), which inhibits Src family kinase activity. In vitro Src kinase activity assays confirmed that in empty vector-transfected cells (Vector cells), 14,15-EET increased Src kinase activity, while in clones overexpressing CSK mRNA and immunoreactive protein (CSK cells), 14,15-EET-induced activation of Src was almost completely blocked (94% inhibition). Of interest, epidermal growth factor (EGF) and fetal bovine serum (FBS) also increased Src activity in Vector cells, but not in CSK cells, further confirming the ability of CSK overexpression to prevent Src activation. CSK cells failed to increase [3H]thymidine incorporation in response to exogenous 14,15-EET. In contrast, both EGF and FBS significantly increased [3H]thymidine incorporation in CSK cells. Immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting with an antibody against extracellular signal-regulated kinase (ERK) indicated that in CSK cells, 14,15-EET failed to activate ERK1 and ERK2; however, EGF- and FBS-induced activation of ERKs was not different from that seen in Vector cells. In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 μM PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14,15-EET exerts its mitogenic effects predominantly through a Src kinase-mediated pathway, which is the most upstream signaling step determined to date in the 14,15-EET-activated tyrosine kinase cascade in renal epithelial cells.

Original languageEnglish (US)
Pages (from-to)13789-13792
Number of pages4
JournalJournal of Biological Chemistry
Volume275
Issue number18
DOIs
StatePublished - May 5 2000
Externally publishedYes

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Phosphorylation
Tyrosine
src-Family Kinases
Chemical activation
Epidermal Growth Factor
Protein-Tyrosine Kinases
Thymidine
Phosphatidylinositol 3-Kinase
Epithelial Cells
Serum
CSK tyrosine-protein kinase
14,15-epoxy-5,8,11-eicosatrienoic acid
Phosphotyrosine
Kidney
Antibodies
Extracellular Signal-Regulated MAP Kinases
Protein C
Mitogen-Activated Protein Kinases
Mitogens
Immunoprecipitation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Overexpression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis. / Chen, Jiankang; Capdevila, Jorge; Harris, Raymond C.

In: Journal of Biological Chemistry, Vol. 275, No. 18, 05.05.2000, p. 13789-13792.

Research output: Contribution to journalArticle

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