The importance of active ERM proteins stabilization by NHERF1/EBP50 in tumor progression

MORALES FC; TAKAHASHI Y; KREIMANN EL; GEORGESCU MM

Anaheim Convention Center. Anaheim, Orange County, CA, USA

Congreso; 96th Annual Meeting, American Association for Cancer Research, AACR; 2005

American Association for Cancer Research

NHERF1/EBP50 was identified as a regulator factor of the Na+/H+ exchanger isoform 3 (NHE3). EBP50 is a 358-residue adaptor molecule that has two PDZ domains and a C-terminal ezrin-radixin-moesin (ERM)-binding sequence that interacts to the N-terminal domain of the ERM proteins, including merlin, the product of the neurofibromatosis-2 (NF2) tumor suppressor gene. While the loss of expression of merlin was involved in cancer progression, less is known about EBP50’s role on cell growth. In order to understand the regulation of ERM proteins and EBP50 in progression of cancer we used glioblastoma cell lines to identify the pattern of expression of these proteins. Out of 10 glioblastoma cell lines tested, three showed a high expression level of EBP50 and ezrin compared to normal tissue while merlin is downregulated. This finding suggests a correlation in between the expression levels of EBP50 and ERM proteins. To investigate the in vivo role of EBP50 in tumor formation and metastasis we generated mice with targeted disruption of the EBP50 gene. Embryonic stem cells were electroporated with the targeting construct that replaced the first 4 exons of the EBP50 gene with a neomycin cassette flanked by loxP sites. We confirmed that EBP50 was efficiently disrupted in mice by Southern and Western blot analysis. EBP50 is mainly expressed in kidney and intestine of wild type (WT) mice and ezrin is expressed in parallel with EBP50. Purification of kidney and intestine brush border membranes (BBM) from both WT and knockout (KO) mice showed that ezrin and moesin are downregulated in the BBM fraction of KO mice, suggesting a regulation of ERM proteins by EBP50. We also found that EBP50 is necessary for the maintenance of active phosphorylated ERM at the BBM. Immunofluorescense experiments showed that indeed ezrin is downregulated from the apical membrane of polarized epithelial cells of KO mice. Kidney and intestine BBM were fractionated in a Sephadex S-300 gel filtration column in order to identify protein complexes with ERM proteins. Our results showed that EBP50 co-fractionates with ezrin in WT BBM. Electron microscopy analysis of KO intestinal epithelium cells revealed disorganized microvilli and an altered actin-rich terminal web region suggesting a loss of organization of the apical cytoskeleton. In this work we propose a mechanism in which EBP50 is necessary for the recruitment of active ERM proteins to the plasma membrane and we discuss the implications of cell polarity disruption for tumorigenesis.