IDENTIFICATION OF AN ACTIN-BINDING REGION AND A PROTEIN-KINASE-C PHOSPKORYLATION SITE ON HUMAN FASCIN

Fascin is a 5538-kDa actin-bundling protein, the actin binding of whic h is regulated by phosphorylation (Yamakita, Y., One, S., Matsumura, F ., add Yamashiro, S, (1996) J. Biol. Chem. 271, 12632-12638). To under stand the mechanism of fascin-actin interactions, we dissected the act in binding region and its regulatory site by phosphorylation of human fascin. First, we found that the C-terminal half constitutes an actin binding domain, Partial digestion of human recombinant fascin with try psin yielded the C-terminal fragment with molecular masses of 32, 30, and 27 kDa. The 32- and 27-kDa fragments purified as a mixture formed a dimer and bound to F-actin at a saturation ratio of 1 dimer:11 actin molecules with an affinity of 1.4 x 10(6) M(-1). Second, we identifie d the phosphorylation site of fascin as Ser-39 by sequencing a tryptic phosphopeptide purified by chelating column chromatography followed b y C-18 reverse phase high performance liquid chromatography. Pep tide map analyses revealed that the purified peptide represented the major phosphorylation site of in vivo as well as in vitro phosphorylated fas cin. The mutation replacing Ser-39 with Ala eliminated the phosphoryla tion-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fa scin.