Regulation of endothelin-1- and lysophosphatidic acid-stimulated tyrosine phosphorylation of focal adhesion kinase (pp125fak) in Rat-1 fibroblasts.
The characteristics of protein tyrosine phosphorylation were examined in Rat-1 fibroblasts in response to endothelin-1 (ET-1) and 1-oleoyl-lysophosphatidic acid (LPA). Both agonists stimulated the biphasic tyrosine phosphorylation of at least three major proteins of approx. 120 kDa (pp116, pp120 and pp130) and two of 80 kDa (pp80 and pp70). Immunoprecipitation experiments indicated that the pp120 protein corresponded to the recently described focal adhesion protein kinase pp125fak. Phorbol 12-myristate 13-acetate, alone or in combination with the calcium ionophore A23187, also stimulated the phosphorylation of pp125fak but to a smaller extent than LPA or ET-1. Removal of both extracellular and intracellular Ca2+ did not significantly reduce LPA- and ET-1-stimulated tyrosine phosphorylation of pp125fak. In cells where protein kinase C activity was down-regulated or inhibited, ET-1-stimulated tyrosine phosphorylation of pp125fak was reduced to a greater extent than phosphorylation in response to LPA. In addition, ET-1-stimulated tyrosine phosphorylation of pp80 was decreased by 50-70% in response to protein kinase C inhibition at both 2 and 60 min whereas LPA-stimulated tyrosine phosphorylation of this protein was only reduced at 2 min. Pretreatment with pertussis toxin reduced the tyrosine phosphorylation of pp42 and pp44 forms of mitogen-activated protein kinase in response to both ET-1 and LPA but reduced the tyrosine phosphorylation of pp125fak only in response to LPA. These results indicate agonist-specific differences in the regulation of pathways mediating the tyrosine phosphorylation of pp125fak and other target proteins.
Saville
M K
MK
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, U.K.
Graham
A
A
Malarkey
K
K
Paterson
A
A
Gould
G W
GW
Plevin
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006706:Homeostasis; D007163:Immunosorbent Techniques; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D016232:Endothelins; D008246:Lysophospholipids; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D000001:Calcimycin; D014443:Tyrosine; D004533:Egtazic Acid; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium