Abstract
The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY477 present in ezrin’s C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.
Highlights
The activation of the Met receptor by its ligand, the Hepatocyte Growth Factor (HGF), elicits complex biological responses in epithelial cells
We identified an interaction of ezrin with WWP1/AIP5/Tiul1 a type E3 ubiquitin ligase that belongs to the HECT family (Homologous to the E6-associated protein C terminus) [11]
Ezrin Interacts with WWP1 through its PPVY477 Motif To confirm the interaction between WWP1 and ezrin we performed experiments to pull down Flag-WWP1 expressed in 293T cells with various GST-ezrin domains
Summary
The activation of the Met receptor by its ligand, the Hepatocyte Growth Factor (HGF), elicits complex biological responses in epithelial cells. It triggers cell scattering and motility and promotes morphogenic programs when cells are cultured in three-dimensional matrix [1]. Ezrin belongs to the ERM (Ezrin, Radixin, Moesin) protein family. The activity of these proteins is negatively regulated by an intramolecular interaction between their N-terminal and C-terminal domains that masks their membrane and actin cytoskeleton binding sites [2]. Following HGF stimulation of epithelial cells, ezrin is phosphorylated at specific tyrosine residues involved in various signalling pathways [4,5]. The association of ERM (Ezrin, Radixin, Moesin) proteins with CD44v6, a coreceptor of Met, is necessary for HGF-mediated activation of Ras by the guanine nucleotide exchange factor Sos [7]. Ezrin phosphorylation at tyrosine 477 is required for HGF-induced cell scattering through the recruitment and activation of the Fes kinase [9]
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