A FERM domain governs apical confinement of PTP-BL in epithelial cells

PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins, Here w e show by transient expression of modular protein domains of PTP-BL in epit helial MDCK cells, that the presence of a FERM domain in the protein is bot h necessary and sufficient for its targeting to the apical side of epitheli al cells. Furthermore, immune-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revea led that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FE RM domain moiety with the membrane-cytoskeleton linker ezrin, However, at t he electron microscopy level this colocalization cannot be confirmed nor ca n we detect a direct interaction by immunoprecipitation assays. Fluorescenc e recovery after photobleaching (FRAP) experiments show that PTP-BL confine ment is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that rel ocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly r estricted subcellular compartmentalization and its specific associative pro perties may provide the appropriate conditions for regulating substrate spe cificity and catalytic activity of this member of the PTP family.