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 kinase found in focal adhesions. FAK has been indicated as a point of convergence of other signaling pathways including platelet-derived growth factor (PDGF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of Ras as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosphorylation 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 dominant-negative mutant of Ras (N17Ras). This resulted in a marked inhibition of PDGF-induced FAK tyrosine phosphorylation together with the expected reduction of PDGF-induced extracellular signal-regulated kinase activity (ERK). Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylation, ERK activity and intracellular calcium increase, as well as the biological effects of this growth factor, (i.e., mitogenesis and cell migration) were effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransferase I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of farnesyltransferase, resulted in detectable effects only at high doses. Taken together, these results establish that Ras operates as a protein-linking PDGF-beta receptor to FAK in human HSCs, and that signaling molecules requiring geranylgeranylation may also be involved in this process.
Carloni
V
V
Dipartimento di Medicina Interna, Università di Firenze, Florence, Italy. v.carloni@dfc.unifi.it
Pinzani
M
M
Giusti
S
S
Romanelli
R G
RG
Parola
M
M
Bellomo
G
G
Failli
P
P
Hamilton
A D
AD
Sebti
S M
SM
Laffi
G
G
Gentilini
P
P
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D000077214:Becaplermin; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051231:Farnesyltranstransferase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D008099:Liver / Q000201:enzymology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017368:Protein Prenylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D019000:Phosphotyrosine; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D010738:Type C Phospholipases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium