Mutagenesis of the phosphatidylinositol 4,5-bisphosphate (PIP2) binding site in the NH2-terminal domain of ezrin correlates with its altered cellulardistribution

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing liposomes via its NH2-terminal domain. Using internal deletions and COOH-terminal truncat ions, determinants of PIP, binding were located to amino acids 12-115 and 2 33-310, Both regions contain a KK(X)(n)K/RK motif conserved in the ezrin/ra dixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 d id not affect cosedimentation of ezrin 1-333 with PIP2-containing liposomes , but their combination almost completely abolished the capacity for intera ction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduc ed lipid interaction, but combined with the double mutation K253N, K254N, t he interaction of PIP2 with ezrin 1-333 was strongly inhibited. Similar dat a were obtained with full-length ezrin. When residues 253, 254, 262, and 26 3 were mutated in full-length ezrin, the in vitro interaction with the cyto plasmic tail of CD44 was not impaired but was no longer PIP2 dependent. Thi s construct was also expressed in COS1 and A431 cells. Unlike wild-type ezr in, it was not any more localized to dorsal actin-rich structures, but redi stributed to the cytoplasm without strongly affecting the actin-rich struct ures. We have thus identified determinants of the PIP, binding site in ezri n whose mutagenesis correlates with an altered cellular localization.