The loss of EBP50 induces changes in cell migration and enhances cell transformation

KREIMANN EL; TAKAHASHI Y; MORALES FC; GEORGESCU MM

New Braunfels, Texas, USA

Workshop; Spring Genes & Development Retreat. MD Anderson Cancer Center; 2005

MD Anderson Cancer Center

NHERF1/EBP50 is and adaptor protein containing two PDZ domains that was initially identified as a cofactor essential for cAMP-mediated inhibition of the activity of Na+/H+ exchanger 3 (NHE3), a subtype of the Na+/H+ exchanger of kidney proximal tubule brush border membranes.  It was independently identified as a 50 kDA Ezrin binding phosphoprotein.  EPB50 protein interacts with the Ezrin-radixin-moesin (ERM) family of proteins which directly binds to the actin cytoskeleton through their C-terminal domain. The ERM proteins are involved in epithelium cell membrane polarization, cell shape determination and are important components of transduction pathways like EGF and PDGF. It has been shown that PDGF stimulation induces ruffling in the cell membrane and these cellular processes or “ruffles” (actin-rich protrusions) are the major driving force for cell movements. Because EBP50 also binds to PDGF receptor and enhances its effect, we believe that EPB50 could be involved in the ruffles formation and cell migration. We are using a genetic approach, disrupting the EBP50 gene in mice, in order to define the importance of the interaction between EBP50 and ERM proteins in cell growth and motility, as well as the effect of cell transformation.              Mouse embryonic fibroblasts (MEFs) from the EBP50(-/-) and EBP50(+/+) littermates were isolated from E14 embryos. After the individual isolation, the cells with identical genotype were pooled and both populations were immortalized with the SV40 virus. We performed migration and proliferation assays to characterized the EBP50(-/-) and EBP50(+/+) MEFs in the parental and SV40 transformed populations.  We also studied the anchorage-independent growth in soft agar of the SV40 MEFs.  The cell migration was studied using the wound-healing assay.  For the soft agar assay, 5 104 cells/100 µl of 10 % FBS DMEM were mixed with 0.4 % agar and plated on a 60 mm plate previously covered by 0.5 % bottom agar, and cultured for 5 weeks.  The experiment was done in triplicates.             We found alterations in the migration pattern of the parental and immortalized EBP50(-/-) and (+/+) cells, but we did not see a difference in the proliferation of the parental EBP50(-/-) MEFs when compared with EBP50(+/+).  Only the EBP50(-/-) MEFs are capable of forming colonies in soft agar, but no colonies formed in the EBP50(+/+) plates. This finding clearly shows that the lack of the EBP50 function potentiates cell transformation, suggesting that the EBP50 gene could act as a tumor suppressor gene in the cell.  The molecular mechanisms are under investigation.