Direct binding of SWAP-70 to non-muscle actin is required for membrane ruffling.
Membrane ruffling induced by growth factor stimulation is caused by actin remodeling, which is mediated by various signaling molecules including Rac. We have shown that SWAP-70, which binds phosphatidylinositol trisphosphate, is one such molecule required for membrane ruffling in mouse kidney cells. Here, we show that SWAP-70 directly binds to F-actin. The bacterially expressed C-terminal region of SWAP-70 co-sedimented with non-muscle F-actin, suggesting direct binding of SWAP-70 to F-actin. The binding was much weaker in muscle F-actin. A truncated mutant of SWAP-70 containing only the C-terminal region readily colocalizes with F-actin, supporting this idea. Full-length SWAP-70 does not colocalize with F-actin unless cells are stimulated with growth factors, suggesting the presence of a stimuli-dependent regulatory mechanism for actin-binding activity in vivo. Overexpression of the mutant SWAP-70 lacking this binding domain inhibits the membrane ruffling induced by epidermal growth factor stimulation in COS7 cells. This dominant-negative effect is also observed in membrane ruffling induced by a dominant-active Rac, suggesting that SWAP-70 cooperates with Rac. These results suggest that the binding activity of SWAP-70 to non-muscle F-actin is required for membrane ruffling.
Ihara
Sayoko
S
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.
Oka
Tsutomu
T
Fukui
Yasuhisa
Y
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006133:Growth Substances / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000199:Actins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D020662:Guanine Nucleotide Exchange Factors; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; C491873:SWAP70 protein, human; D051057:Proto-Oncogene Proteins c-akt