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

We have previously reported that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a potent mitogen for the renal epithelial cell line, LLCPKc14, This mit ogenic effect is dependent upon activation of a protein-tyrosine kinase cas cade that results in activation of mitogen-activated protein kinase and pho sphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET a lso activated Src in these cells, we stably transfected LLCPKc14 with an ex pression construct of the C-terminal Src kinase (CSK), which inhibits Src f amily kinase activity. In vitro Src kinase activity assays confirmed that i n empty vector-transfected cells (Vector cells), 14,15-EET increased Src ki nase activity, while in clones overexpressing CSK mRNA and immunoreactive p rotein (CSK cells), 14,15-EET-induced activation of Src was almost complete ly 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 p revent Src activation. CSK cells failed to increase [H-3]thymidine incorpor ation in response to exogenous 14,15-EET. In contrast, both EGF and FBS sig nificantly increased [H-3]thymidine incorporation in CSK cells, Immunopreci pitation with anti-phosphotyrosine antibodies and immunoblotting with an an tibody against extracellular signal-regulated kinase (ERK) indicated that i n 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 c ells, In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 mu M PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14,15-EET exerts its mito genic effects predominantly through a Src kinase-mediated pathway, which is the most upstream signaling step determined to date in the 14,15-EET-activ ated tyrosine kinase cascade in renal epithelial cells.