Identification and functional implication of a Rho kinase-dependent moesin-EBP50 interaction in noradrenaline-stimulated artery

Abstract

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.

Binding of EBP50 to Nox organizing subunit p47 phox is pivotal to cellular reactive species generation and altered vascular phenotype

“…Rho kinase 1 (ROCK1) has been indirectly linked to EBP50 because it has been shown that ERM family proteins, which associate with EBP50, are substrates of ROCK1 (11,36). Because p47 phox possesses phosphorylation sites that regulate its activity, and given our present findings demonstrating p47 phox association with EBP50, we tested whether ROCK1 kinase associates with p47 phox in vascular tissues.…”

“…Prior reports drew our attention to a possible molecular interaction between EBP50 and p47 phox . First, EBP50's interaction with actin cytoskeletal elements (11,48,49) piqued our interest in that p47 phox is known to bind actin as it translocates to and activates the Nox catalytic core (2,(50)(51)(52)(53). Moreover, the modulatory role of protein kinase C (PKC) at key sites on both molecules suggested a possible common pathway (2,9,54).…”

“…ROCK1 has been implicated to mediate association of ERM family proteins to EBP50 by directly phosphorylating them (11). This raised the possibility that ROCK1 may interact with p47 phox to play a role in facilitating the latter's association with EBP50 and, henceforth, cytoskeletal elements.…”

“…Because EBP50 reportedly associates with its binding partners via hallmark PDZ domains (PDZ1 and PDZ2) (10,11,17), we searched for potential PDZ-binding motifs in p47 phox . Fig.…”

“…Ezrin-radixin-moesin (ERM) binding phosphoprotein 50 (EBP50; aka NHERF1) is a widely expressed PDZ domain-containing scaffolding protein that associates with the actin cytoskeleton and plasma membrane by virtue of its binding to the ERM family of proteins (10)(11)(12)(13)(14). Recently, Bisello and coworkers (15)(16)(17) showed that EBP50 plays a role in neointimal hyperplasia and…”