Thrombin Receptor Signaling to Cytoskeleton Requires Hsp90

Abstract

Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione Stransferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90⅐PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombinmediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombininduced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.Thrombin is a serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes, and tumor metastasis (1-4). In brain, thrombin regulates the viability of neurons and astrocytes by increasing survival under conditions of hypoglycemia and oxidative stress and inducing apoptosis under other conditions (5-8). Thrombin is also chemotactic for macrophages and mitogenic for smooth muscle cells, fibroblasts, and astrocytes and induces secretion of growth factors and cytokines from fibroblasts and smooth muscle cells (9). Most of the thrombinmediated effects are preceded by morphological changes in cells that follow activation of a seven-transmembrane G proteincoupled receptor called protease-activated receptor-1 (PAR-1)

Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38

“…In this context, we previously reported that the proper assembly of focal adhesions, through a process that requires HSP90-dependent phosphorylation of focal adhesion kinase (FAK), was required to allow the SAPK2/p38 remodeling of F-actin into stress fibers in response to VEGF (Rousseau et al, 2000). In accordance, FAK is tyrosine phosphorylated downstream of RhoA in Swiss 3T3 cells activated with serum and HSP90 is required for the activation of RhoA and cytoskeleton remodeling in response to thrombin (Barry and Critchley, 1994;Pai et al, 2001;Pai and Cunningham, 2002). A complementarity between the RhoA and Cdc42/SAPK2/p38 pathways in regulating actin remodeling has also been reported in response to TGFb in pancreatic carcinoma cells (Edlund et al, 2002).…”

Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting RhoA Signaling

“…Furthermore, inactivation of RhoA has been shown to improve barrier function (33,34). Other studies have shown that vascular endothelial growth factorand thrombin-induced RhoA activity was inhibited by the Hsp90 inhibitor geldanamycin (35,36). S.M.B.…”

Heat shock protein inhibitors increase the efficacy of measles virotherapy

“…25 GA has been previously shown to inhibit RhoA activation. 26 Western immunoblotting was performed following immunoprecipitation to assess active RhoA expression. The results showed gradual decrease of RhoA activity starting at 6 h following GA treatment with RhoA activity becoming undetectable at 12 h after GA treatment (36 h post-MV-CEA infection).…”

“…25 RhoA is regulated by heat shock proteins: HSP90 is required for RhoA activation in thrombin-induced signaling to cytoskeleton, and the activation of RhoA could be specifically inhibited by GA in this reaction. 26 Furthermore, RhoA co-precipitates with HSP70. 46 Although there is no information regarding the role of RhoA in measlesinduced fusion, because of its regulation by heat shock proteins and its role in fusion induced by other paramyxoviruses we hypothesized that changes in RhoA activity could explain the observed augmentation of fusion and cytopathic effect.…”