A HIGHLY-EXPRESSED 81-KDA PROTEIN IN IMMORTALIZED MOUSE FIBROBLASTS -ITS PROLIFERATIVE FUNCTION AND IDENTITY WITH EZRIN

An 81 kDa protein was found to be highly expressed in spontaneously im mortalized CD1-ICR mouse fibroblasts, RS-4, as compared to normal fibr oblasts. RS-4 cells have a reduced serum requirement and exhibit multi layered growth in vitro but are not tumorigenic, The protein was purif ied from RS-4 cell extracts and used to obtain a polyclonal antibody t hat specifically immunoprecipitated an 81 kDa protein from cell lysate s. Immunocloning of its cDNA and sequence analysis revealed its identi ty with ezrin, an F-actin binding protein that is a component of the c ortical cytoskeleton, Microinjection of the purified IgG fraction of t he anti-p81 antiserum into the cytoplasm of RS-4 cells blocked their e ntry into S phase suggesting that the protein has a proliferative func tion, Immunostaining of normal mouse tissues showed that the expressio n of p81/ezrin was highest in proliferating cell populations. Unlike R S-4, NIH3T3 cells exhibit contact inhibition and express levels of p81 /ezrin similar to those of normal fibroblasts, When NIH3T3 cells were transfected with p81/ezrin cDNA they lost contact inhibition and thus resembled RS-4 cells, The study demonstrates a proliferative function of p81/ezrin and suggests its involvement in pathways that negatively regulate contact inhibition.