RESUMO
SWAP-70 translocates to the plasma membrane in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner and contributes to membrane ruffling. It binds to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) through its PH domain, which is essential for the membrane translocation after EGF stimulation. We examined the behavior of the SWAP-70s which have mutations in the beta3/beta4 loop of the PH domain. The two mutants fused to green fluorescent protein (GFP) carrying the mutations failed to translocate to the plasma membrane. The sole PH domains carrying the same mutations behaved similarly. The PtdIns(3,4,5)P(3) binding activity of two mutants was comparable to that of the wild-type protein. These results suggest that translocation of SWAP-70 largely depends on the activity of the PH domain, and that not only PtdIns(3,4,5)P(3) binding activity, but also some additional activity of the PH domain is required for the translocation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína/fisiologia , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Nucleares/genética , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico/fisiologiaRESUMO
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.
