ABSTRACT
-PYK2, a recently identified Ca2+-sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca2+-dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT1 receptor antagonist and an intracellular Ca2+ chelator. A Ca2+ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT1 receptor uses Ca2+-dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.
Subject(s)
Angiotensin II/physiology , Aorta, Thoracic/physiology , ErbB Receptors/physiology , Muscle, Smooth, Vascular/physiology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Angiotensin II/pharmacology , Animals , Aorta, Thoracic/drug effects , Calcium/metabolism , Cells, Cultured , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , Focal Adhesion Kinase 2 , Humans , Models, Biological , Muscle, Smooth, Vascular/drug effects , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptors, Angiotensin/drug effects , Receptors, Angiotensin/physiology , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ ionophore and completely inhibited by an intracellular Ca2+ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.
Subject(s)
Adaptor Proteins, Signal Transducing , Angiotensin II/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium/metabolism , ErbB Receptors/metabolism , Muscle, Smooth, Vascular/enzymology , Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Tyrphostins , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Aorta, Thoracic/enzymology , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , GRB2 Adaptor Protein , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Nitriles/pharmacology , Phosphorylation , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , src Homology DomainsABSTRACT
For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr76gag, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used. One hybridoma culture produced antibodies that were directed against the p19 portion of the gag precursor. However, no antibodies directed against pp60src could be detected in any of the hybridoma supernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies belonged to the immunoglobulin G subclass 2b. The antibodies precipitated Pr76gag and the processed virion-associated p19, as well as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone marrow cells. Also, viral ribonucleoprotein complexes were specifically precipitable, indicating that they contain p19 molecules.
