The angiotensin II type-1 (AT1) receptor, a G protein-coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-gamma 1 and to be activated by G protein-coupled receptors. We hypothesized that pp60c-src associates with the AT1 receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II-stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II-stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1 receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1 receptor, the AT1 receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1 receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1 fusion protein to bind proteins from cell lysates stimulated by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)
"Several earlier studies on AT1aR signalling have described angiotensin II mediated tyrosine phosphorylation –. Proteins harbouring these are often low abundant  and in addition current enrichment methods favour peptides phosphorylated on serines or threonines . This leads to tyrosine phosphorylation being significantly underreported; in the two studies the fraction of tyrosine sites was 0.7% and 3.3% for the studies by Christensen and co-workers and Xiao and co-workers respectively (Figure 2B). "
[Show abstract][Hide abstract] ABSTRACT: Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.
PLoS ONE 04/2014; 9(4):e94672. DOI:10.1371/journal.pone.0094672 · 3.23 Impact Factor
"Studies in a variety of Ang II target cell types have shown that the Ang II activation of different pathways is time dependent. For example, activation of the G-protein-dependent pathway and generation of IP 3 occurs in seconds, while MAPK and JAK/STAT activation occurs in minutes to hours after initial AT1R activation (Ishida et al., 1995; Schmitz et al., 1998). While many of the Ang II signaling cascades have been defined, an understanding of the downstream changes in gene expression has moved ahead very slowly, often through the definition of one gene-target at a time. "
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (Ang II) is the key peptide hormone in the renin–angiotensin–aldosterone system (RAAS). Its ability to regulate levels of circulating aldosterone relies on actions on adrenal glomerulosa cells. Many of the Ang II effects on glomerulosa cells involve a precisely coordinated regulation of signaling cascades and gene expression. The development of genome-wide gene arrays has allowed the definition of transcriptome-wide effects of Ang II in adrenocortical cells. Analysis of the Ang II gene targets reveals broad effects on cellular gene expression, particularly the rapid induction of numerous transcription factors that may regulate long-term steroid metabolism and cell growth/proliferation. Herein we discuss the Ang II-induced genes in adrenocortical cells and review the progress in defining the role of these genes in zona glomerulosa function.
"There is also an increasing recognition for a critical role of reactive oxygen species (ROS) generation in triggering the responses of Ang II (Zafari et al. 1998; Zhang et al. 2005) and ET-1 (Cheng et al. 2003; Daou and Srivastava 2004). Both receptor and nonreceptor protein tyrosine kinases (PTK) have been shown to be activated by Ang II (Du et al. 1996; Ishida et al. 1995; Eguchi et al. 1999; Rocic et al. 2001) and ET-1 (Iwasaki et al. 1999; Kodama et al. 2002; Robin et al. 2002; Daou and Received 17 August 2006. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 7 March 2007. "
[Show abstract][Hide abstract] ABSTRACT: Transactivation of epidermal growth factor receptor (EGFR) is a well-documented mechanism by which vasoactive peptides and H2O2 elicit their cellular responses. However, a role for the insulin-like growth factor type-1 receptor (IGF-1R) transactivation in mediating the effects of angiotensin II (Ang II) and H2O2 in vascular smooth muscle cells from different artery types have also been recently recognized. By using a series of pharmacological inhibitors of various growth factor receptor tyrosine kinases and a direct analysis of the phosphorylation status of the beta-subunit of IGF-1R, a requirement of this growth factor receptor in Ang II and H2O2 response has been demonstrated. This review discusses some of the studies that highlight the importance of IGF-1R transactivation in mediating Ang II- and H2O2-induced mitogen-activated protein kinase and protein kinase B signaling pathways.
Canadian Journal of Physiology and Pharmacology 02/2007; 85(1):105-11. DOI:10.1139/Y06-101 · 1.77 Impact Factor
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