BETA-CAP73 - A NOVEL BETA-ACTIN-SPECIFIC BINDING-PROTEIN

Whereas actin-binding proteins (ABPs) regulate network formation durin g the cell cycle, it is not known whether ABPs also function to seques ter or target isoactins to specific subcellular compartments. Recently , we have shown that ezrin indirectly associates with beta, but not al pha actin filaments in a calcium- and cytochalasin-sensitive manner [S huster and Herman, 1995: J. Cell Biol. 128:837-848]. To identify the b eta actin-specific binding protein that fosters ezrin-beta actin inter actions, we developed an isoactin affinity fractionation and F-isoacti n overlay/Western blotting technique. Results reveal that a 73 kd poly peptide that co-precipitates with ezrin and beta actin [Shuster and He rman, 1995: J. Cell Biol. 128:837-848] can also binds directly to fila ments of beta, but not or actin by isoactin overlay. In an effort to e stablish whether p73 plays a role in regulating beta actin dynamics in cells, we produced monoclonal antibodies by immunizing BALB/c mice wi th p73-containing lamellar lysates or high salt elutions from beta act in affinity columns. Two monoclonal antibodies were cloned that react with p73 present in fractions released from beta actin Sepharose-4B or purified to homogeneity by DEAE chromatography. Anti-p73 Western blot s reveal that there is a 16-fold difference in p73 binding to beta act in vs. alpha actin affinity columns when experiments are performed in physiological salts. ?To characterize p73-beta actin binding in vitro and establish whether p73 binds along the lengths or at the barbed end of the beta actin filament, we asked whether cytochalasin D (CD) coul d displace p73 pre-bound to beta actin-Sepharose 4B. Anti-p73 Western blotting reveals that nanomolar concentrations of CD are capable of se lectively eluting p73 and ezrin from beta actin Sepharose 4B, indicati ng that p73 binds beta actin via the barbed end. Simultaneous double a ntibody localization studies using anti-beta actin IgG and anti-p73 Ig M reveal that p73 and beta actin are co-localized in the forward aspec ts of motile cytoplasmic domains, in close proximity to the plasma mem brane. Because of its isoform-specific interactions with the barbed en d of beta actin filaments, we have named this molecule beta cap73. The se results indicate that isoform-specific actin-binding proteins can b e identified from cortical cytoplasm, and suggest that beta cap73 may not only act to spatially regulate the intracellular distribution of i soactins, but may also facilitate forward protrusion formation through the regulated release of free filament ends during cell motility. (C) 1996 Wiley-Liss, Inc.