Identification of an actin binding region and a protein kinase C phosphorylation site on human fascin.
Fascin is a 55-58-kDa actin-bundling protein, the actin binding of which is regulated by phosphorylation (Yamakita, Y., Ono, S., Matsumura, F., and Yamashiro, S. (1996) J. Biol. Chem. 271, 12632-12638). To understand the mechanism of fascin-actin interactions, we dissected the actin 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 trypsin 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 identified the phosphorylation site of fascin as Ser-39 by sequencing a tryptic phosphopeptide purified by chelating column chromatography followed by C-18 reverse phase high performance liquid chromatography. Peptide map analyses revealed that the purified peptide represented the major phosphorylation site of in vivo as well as in vitro phosphorylated fascin. The mutation replacing Ser-39 with Ala eliminated the phosphorylation-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fascin.
Ono
S
S
Department of Molecular Biology and Biochemistry, Nelson Laboratory, Busch Campus, Rutgers University, Piscataway New Jersey 08855-1059, USA.
Yamakita
Y
Y
Yamashiro
S
S
Matsudaira
P T
PT
Gnarra
J R
JR
Obinata
T
T
Matsumura
F
F
D000199:Actins / Q000378:metabolism*; D000409:Alanine; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C020324:fascin; D011493:Protein Kinase C; D000409:Alanine