ABSTRACT
The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gßγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.
Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein-Tyrosine Kinases/physiology , Receptor, IGF Type 1/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Cell Culture Techniques , Coronary Vessels/cytology , Coronary Vessels/metabolism , Enzyme Activation , ErbB Receptors/antagonists & inhibitors , GTP-Binding Protein beta Subunits/antagonists & inhibitors , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/antagonists & inhibitors , GTP-Binding Protein gamma Subunits/metabolism , Humans , Insulin-Like Growth Factor I/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Myocytes, Smooth Muscle/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/genetics , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Swine , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Release of angiotensin II (Ang II) after vascular injury promotes tissue repair by stimulating phenotypic modulation of smooth muscle cells, which enables cell proliferation and migration. This process requires cytoskeleton remodeling, which involves cortactin, a scaffold protein that is phosphorylated by Src kinase in response to Ang II. Since insulin-like growth factor (IGF)-1 receptor transactivation mediates intracellular signals originating from the Ang II type 1 (AT1) receptor in a Src kinase-dependent manner, we examined whether IGF-1 receptor transactivation was also required for cortactin phosphorylation. Treatment of quiescent smooth muscle cells with Ang II resulted in both cortactin phosphorylation and its translocation to the plasma membrane. Both events were prevented by 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo(3,4-d)pyrimidin-4-amine (PP1), a Src kinase inhibitor, and by AG1024, an inhibitor of the IGF-1 receptor tyrosine kinase. Additionally, PP1 and AG1024 blocked the association of cortactin with actin-related protein (Arp) 3, an actin nucleation factor. These results indicate that Src kinase and the IGF-1 receptor kinase are necessary for activating cortactin. Phosphorylation of Src kinase in Ang II-treated cells was subsequently examined and was shown to be prevented by AG1024. Furthermore, Src kinase phosphorylation was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphatidylinositol (PI) 3-kinase. These data establish that IGF-1 receptor transactivation is required for Src kinase-mediated cortactin phosphorylation and cytoskeletal reorganization in response to Ang II.
Subject(s)
Angiotensin II/pharmacology , Cortactin/metabolism , Receptor, IGF Type 1/metabolism , src-Family Kinases/metabolism , Actin-Related Protein 3/genetics , Actin-Related Protein 3/metabolism , Animals , Blotting, Western , Fluorescent Antibody Technique , Immunoprecipitation , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Kinase C/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/metabolism , Swine , Transcriptional Activation , Tyrphostins/pharmacology , src-Family Kinases/antagonists & inhibitorsABSTRACT
ADP-ribosylation has been coupled to intracellular events associated with smooth muscle cell vasoreactivity, cytoskeletal integrity and free radical damage. Additionally, there is evidence that ADP-ribosylation is required for smooth muscle cell proliferation. Our investigation employed selective inhibitors to establish that mono-ADP-ribosylation and not poly(ADP-ribosyl)ation was necessary for the stimulation of DNA synthesis by mitogens. Mitogen treatment increased concomitantly the activity of both soluble and particulate mono-ADP-ribosyltransferase, as well as the number of modified proteins. Inclusion of meta-iodobenzylguanidine (MIBG), a selective decoy substrate of arginine-dependent mono-ADP-ribosylation, prevented the modification of these proteins. MIBG also blocked the stimulation of DNA and RNA synthesis, prevented smooth muscle cell migration and suppressed the induction of c-fos and c-myc gene expression. An examination of relevant signal transduction pathways showed that MIBG did not interfere with MAP kinase and phosphatidylinositol 3-kinase stimulation; however, it did inhibit phosphorylation of the Rho effector, PRK1/2. This novel observation suggests that mono-ADP-ribosylation participates in a Rho- dependent signalling pathway that is required for immediate early gene expression.
