Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway.
Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21ras through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21ras and to stimulate DNA synthesis in these cells. LPA and, more effectively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p125FAK) but not the 120-kDa p21ras GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the p42/p44 mitogen-activated protein (MAP) kinases, paralleling p21ras activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 microM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21ras and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21ras activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21ras, and tyrosine phosphorylation of the other substrates, including p125FAK, is associated with phospholipase C activation.
Hordijk
P L
PL
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
Verlaan
I
I
van Corven
E J
EJ
Moolenaar
W H
WH
D037361:Adenylate Cyclase Toxin; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D037342:Pertussis Toxin; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine; D014443:Tyrosine / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000470:Alkaloids; D015815:Cell Adhesion Molecules; D016232:Endothelins; D008246:Lysophospholipids; D010566:Virulence Factors, Bordetella; D014443:Tyrosine; D004815:Epidermal Growth Factor; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D019311:Staurosporine