The Tumor Suppressor Hamartin Enhances Dbl Protein Transforming Activity through Interaction with Ezrin

Abstract

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between ␤1 and ␤2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.ERMs (ezrin, radixin, moesin) 2 are ubiquitously expressed proteins that serve as membrane-cytoskeleton linkers and control diverse actin-based cellular functions including cell adhesion, cell motility, and protein localization, and participate in signaling pathways (1). ERM proteins share a conserved structure containing two domains: the C-terminal domain, that binds to actin, and the N-terminal FERM domain, which binds to protein targets at the plasma membrane and with several signaling proteins (2-14). In the inactive conformation the C terminus binds to the N terminus, thus preventing both interaction with membrane targets and actin. Activation of ERM proteins causes the release of this intramolecular binding and subsequent association of ERMs with the cytoskeleton and membrane proteins.Two distinct signals have been proposed as effectors of ERM protein activation: the phosphorylation, by Rho-dependent kinase ROCK, of a conserved C-terminal threonine residue, localized in a consensus C-terminal region of ERM proteins (15)(16)(17)(18)(19)(20), and the binding of phosphatidylinositol-4,5-biphosphate (21-23), produced by the activity of phopshatidylinositol 4-phosphate 5-kinase (24, 25), which in turn is stimulated by activated ROCK (26). Thus Rho, upon activation by guanine nucleotide exchange factors, may act as an upstream activator of ERMs.On the other hand, in vitro and in vivo studies have indicated that ERM proteins can act as upstream regulators of Rho GTPases by binding to the Rho GDP dissociation inhibitor (RhoGDI). This association is thought to displace RhoGDI from Rho GTPases, allowing them to be activated by their specific guanine nucleotide exchange factors (2). In this regard, a functional dependence of Rho GEFs on ezrin has been shown (27) and the association of ERM proteins with Rho GEF Dbl has been demonstrated (28 -30). Moreover, association of ezrin with a novel GEF that activates the small GTPase RhoG has been reported (8). Therefore, ERM proteins may act as upstream activators of Rho GTPases not only through their association with Rho GDI but also through their interaction with R...

Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

“…As a follow-up, we looked further into the downstream signaling of EZR, which we hoped might explain our pro-self-renewal TIC phenotype. EZR has been associated with a number of different downstream signaling pathways, including the AKT, MAPK/ERK, and MAPK14/p38 pathways [ 28 – 30 ]. While AKT and MAPK/ERK pathways remained unchanged under hypoxia and after EZR inhibition (Fig.…”

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

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Macropinocytosis activated by oncogenic Dbl enables specific targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells

“…Proto-Dbl, onco-Dbl, and their limited domain gene fragments ( Figure 1A) were previously constructed and the GST-Nterminal cloned into pEBG plasmids for overexpression. [22][23][24] Recombinant Dbl plasmids were transient transfected by Lipofectamine ® 3000 according to the manufacturer's instructions. Two kinds of Dbl knockdown approaches were used: the siRNA oligo pool (GenePharma) was used for short-term gene silencing in most uptake-checking assays.…”

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