M. Showkat Ali

Angiotensin II signal transduction pathways

It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin-angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.

Phosphorylation of p130Cas by Angiotensin II Is Dependent on c-Src, Intracellular Ca2+, and Protein Kinase C

p130Cas is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130Cas tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca2+ chelator BAPTA-AM but not the Ca2+ channel blocker verapamil. The Ang II-induced tyrosine phosphorylation of p130Cas was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Calpha and the cell adhesion signaling molecule pp120 formed temporal associations with p130Cas in response to Ang II. c-Src was found to associate with p130Cas in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II-induced tyrosine phosphorylation of p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway.

The critical role of c-Src and the Shc/Grb2/ERK2 signaling pathway in angiotensin II-dependent VSMC proliferation

Angiotensin II promotes vascular smooth muscle cell proliferation through the actions of the G protein-coupled AT(1) receptor. Recent evidence suggest that the tyrosine kinase c-Src may mediate this proliferative response. c-Src can signal through multiple intracellular signaling pathways including (1) the Shc/Grb2/ERK2 pathway, (2) the signal transducers and activators of transcription (STATs), (3) the focal adhesion kinase (FAK) signaling pathway, and (4) the phosphatidylinositol 3-kinase (PI3K) signaling pathway. In this study, we sought to determine the extent to which c-Src mediates vascular smooth muscle cell proliferation through the Shc/Grb2/ERK2 signaling pathway. Here we demonstrate that treatment of vascular smooth muscle cells with angiotensin II results in activation of the Shc/Grb2/ERK2 signaling pathway as measured by (1) increased Shc tyrosine phosphorylation, (2) increased c-Src/Shc cellular co-localization, (3) increased Shc/Grb2 co-association, and (4) ERK2 activation. Furthermore, these events are critically dependent on c-Src as pharmacological inhibition of c-Src activity blocked all these cellular occurrences. Most importantly, angiotensin II-dependent cellular proliferation was measured in the presence and absence of c-Src and MEK pharmacological inhibitors. We found that pharmacological inhibition of either c-Src or ERK2 completely eliminated angiotensin II-dependent cellular proliferation. Thus, the data suggest that c-Src and the Shc/Grb2/ERK2 signaling pathway play a critical role in angiotensin II-mediated VSMC proliferation.

The Angiotensin II–Dependent Association of Jak2 and c-Src Requires the N-Terminus of Jak2 and the SH2 Domain of c-Src

The binding of angiotensin II (Ang II) to AT1 is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2.

Cytoskeletal remodeling in vascular smooth muscle cells in response to angiotensin II-induced activation of the SHP-2 tyrosine phosphatase.

Angiotensin II is an octapeptide that regulates diverse cellular responses including the actin cytoskeletal organization. In this study, stable cell lines overexpressing wild‐type or catalytically inactive SHP‐2 were employed to elucidate the signaling pathway utilized by the SHP‐2 tyrosine phosphatase that mediates an angiotensin II‐induced reorganization of the actin cytoskeleton in vascular smooth muscle cells (VSMC). The expression of wild‐type SHP‐2 prevented an angiotensin II dependent increase in stress fiber formation. In contrast, the catalytically inactive mutant SHP‐2 increased stress fiber formation. Additional observations further established that SHP‐2 regulates the reorganization of the actin cytoskeleton through RhoA‐ and Vav2‐dependent signaling pathways. The expression of wild‐type SHP‐2 caused a dephosphorylation of several focal adhesion associated proteins including paxillin, p130Cas, and tensin in VSMC. This dephosphorylation of focal adhesion associated proteins was accompanied by significantly decreased numbers of focal adhesions within cells. These results demonstrate a unique role for SHP‐2 in the regulation of the cellular architecture of VSMC, suggesting the possibility that this phosphatase might be instrumental in vascular remodeling.

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells

This work investigated the role of Ca2+ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca2+ chelator BAPTA-AM, but not by the Ca2+ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca2+. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca2+ mobilization and extracellular Ca2+ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca2+ mobilization and heterotrimeric G protein activation.