Angiotensin II-induced protein tyrosine phosphorylation in neonatal rat cardiac fibroblasts.
Angiotensin II has been demonstrated to act as a growth factor in rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to angiotensin II remain to be elucidated. This study was designed to determine whether angiotensin II stimulated tyrosine phosphorylation of proteins in cardiac fibroblasts. Immunoblot analysis demonstrated rapid tyrosine phosphorylation of distinct substrates of 125, 95, 46-60, and 44 kDa in response to 10 nM angiotensin II. Tyrosine phosphorylation was maximal at 5 min and persisted for at least 180 min. Additional tyrosine-phosphorylated proteins of 185, 145, and 85 kDa were detected in response to 10 ng/ml platelet-derived growth factor BB. A cluster of 75-80-kDa proteins were phosphorylated in response to angiotensin II, phorbol ester, and platelet-derived growth factor. Angiotensin II-induced tyrosine phosphorylation was unaffected by phorbol ester-sensitive protein kinase C down-regulation and could be partially blocked by pertussis toxin pretreatment. Angiotensin II stimulation resulted in increased cytosolic tyrosine kinase activity which was recovered by immunoprecipitation. Immunoblot analysis demonstrated tyrosine phosphorylation of p44MAPK, and, in addition, we demonstrated for the first time tyrosine phosphorylation of p125FAK, p46SHC, and p56SHC in response to angiotensin II. The finding that angiotensin II and platelet-derived growth factor stimulated tyrosine phosphorylation of p46SHC and p56SHC suggested that this protein may serve as a common tyrosine kinase substrate in the mitogenic signaling cascade induced by G-protein-coupled receptors and growth factors and is consistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts.
Schorb
W
W
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Peeler
T C
TC
Madigan
N N
NN
Conrad
K M
KM
Baker
K M
KM
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D015815:Cell Adhesion Molecules; D010703:Phorbol Esters; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000804:Angiotensin II; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins