PHOSPHORYLATION OF HUMAN FASCIN INHIBITS ITS ACTIN-BINDING AND BUNDLING ACTIVITIES

Human fascin is an actin-bundling protein that is thought to be involv ed in the assembly of actin filament bundles present in microspikes as well as in membrane ruffles and stress fibers. We have found that hum an fascin is phosphorylated in vivo upon treatment with 12-O-tetradeca noylphorbol-13-acetate, a tumor promoter. The in vivo phosphorylation is gradually increased from 0.13 to 0.30 mol/mol during 2 h of treatme nt, concomitant with the disappearance of human fascin from stress fib ers, membrane ruffles, and microspikes. Human fascin can also be phosp horylated in vitro by protein kinase C at the same sites as observed i n vivo as judged by phosphopeptide mapping. The extent of phosphorylat ion depends on pH: the stoichiometries are 0.05, 0.38, and 0.6 mol of phosphate/mol of protein at pH 7.0, 6.0, and 5.0, respectively. Phosph orylation greatly reduces actin binding of human fascin, while lowerin g the pH to 6.0 alone does not affect fascin-actin binding. With the i ncorporation of 0.25 mol of phosphate/mol of protein, the actin bindin g affinity is reduced from 6.7 x 10(6) to 1.5 x 10(6) m(-1). The actin bundling activity is also decreased. These results suggest that phosp horylation of fascin plays a role in actin reorganization after treatm ent with 12-O-tetradecanoylphorbol-13-acetate.