Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on Ras in human hepatic stellate cells

Focal adhesion kinase (FAK) is a widely expressed nonreceptor tyrosine kina se found in focal adhesions. FAK has been indicated as a point of convergen ce of other signaling pathways including platelet-derived growth factor (PD GF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of pas as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosp horylation in human hepatic stellate cells (HSCs), liver-specific pericytes primarily involved in the pathogenesis of liver fibrosis. For this purpose , cells were first subjected to retroviral-mediated gene transfer with a do minant-negative mutant of pas (N17Ras), This resulted in a marked inhibitio n of PDGF-induced FAK tyrosine phosphorylation together with the expected r eduction of PDGF-induced extracellular signal-regulated kinase activity (ER K), Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylatio n, ERK activity and intracellular calcium increase, as well as the biologic al effects of this growth factor, (i.e., mitogenesis and cell migration) we re effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransfera se I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of f arnesyltransferase, resulted in detectable effects only at high doses. Take n together, these results establish that pas operates as a protein-linking PDGF-beta receptor to FAK in human HSCs, and that signaling molecules requi ring geranylgeranylation may also be involved in this process.