Mutational analysis on the function of the SWAP-70 PH domain.
SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3))-binding protein, which is suggested to be involved in membrane ruffling, cooperating with activated Rac. Various point mutations were introduced in the PH domain. Substitutions of alanines for the positively charged amino acids within the first loop abolished the binding activity of the PH domains to PtdIns(3,4,5)P(3). The PtdIns(3,4,5)P(3) binding activity was required for translocation of SWAP-70 to the membrane, enhancement of membrane ruffling by the overexpressed protein, or the dominant-negative effect of a mutant lacking the carboxyl terminal region in membrane ruffling. When Rac was overexpressed, the above mutants were translocated to the membrane and exhibited a dominant-negative effect on membrane ruffling without PtdIns(3,4,5)P(3)-binding activity. These results suggest that the PtdIns(3,4,5)P(3)-binding activity is dispensable for these events when SWAP-70 and Rac interacts efficiently. These results implicate that binding of SWAP-70 to PtdIns(3,4,5)P(3) may facilitate the recruitment of SWAP-70 to activated Rac.
Wakamatsu
Isamu
I
Division of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Ihara
Sayoko
S
Fukui
Yasuhisa
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D020744:rac GTP-Binding Proteins