Dataset Information

ß1 integrin binding phosphorylates ezrin at T567 to activate a lipid raft signalsome driving invadopodia activity and invasion.

ABSTRACT: Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na(+)/H(+) exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.

SUBMITTER: Antelmi E

PROVIDER: S-EPMC3782503 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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ß1 integrin binding phosphorylates ezrin at T567 to activate a lipid raft signalsome driving invadopodia activity and invasion.

Antelmi Ester E Cardone Rosa A RA Greco Maria R MR Rubino Rosa R Di Sole Francesca F Martino Nicola A NA Casavola Valeria V Carcangiu Marialuisa M Moro Loredana L Reshkin Stephan J SJ

PloS one 20130924 9

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na(+)/H(+) exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in ...[more]

PMID: 24086451

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Project description:Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (?(v)?(3) and ?(5)?(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for ?(v)?(3) and ?(5)?(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both ?(v)?(3) and ?(5)?(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, ?(v)?(3) sequesters strongly into raft-like domains and ?(5)?(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

Dataset Information

ß1 integrin binding phosphorylates ezrin at T567 to activate a lipid raft signalsome driving invadopodia activity and invasion.

Publications

ß1 integrin binding phosphorylates ezrin at T567 to activate a lipid raft signalsome driving invadopodia activity and invasion.

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