Cooperation of DEF6 with activated Rac in regulating cell morphology.
Rho-family GTPases have been implicated in actin remodeling and subsequent morphologic changes in various cells. DEF6, a pleckstrin homology domain-containing protein, has been reported to regulate Rho-family GTPases as a guanine nucleotide exchange factor. Here, we demonstrate that DEF6 also has the property of cooperating with activated Rac1. DEF6 bound selectively to Rac1 loaded with GTP. The interaction is mediated by the effector domain of Rac1. Overexpression of GFP-DEF6 together with constitutively active Rac1 in COS-7 cells significantly changed their cell shape; this was not seen in the absence of activated Rac1. This effect of DEF6 on cellular morphology was shown to be independent of its guanine nucleotide exchange activity. Because DEF6 does not contain any sequences previously known to interact with Rac, we explored the domain necessary for the binding. The amino-terminal portion and central parts of DEF6 were required for the binding. Finally, we succeeded in creating mutants of DEF6 with point mutations in the amino-terminal portion, which abrogate the binding to activated Rac1. These mutants did not exhibit the morphologic change in COS-7 cells when they were co-expressed with activated Rac1. These results suggest that DEF6 not only activates Rho-family GTPases but also cooperates with activated Rac1 to exert its cellular function.
Oka
Tsutomu
T
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; D019556:COS Cells; D048430:Cell Shape; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
C479750:DEF6 protein, human; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins