TY - JOUR
T1 - Targeting of Tiam1 to the plasma membrane requires the cooperative function of the N-terminal pleckstrin homology domain and an adjacent protein interaction domain
AU - Stam, J C
AU - Sander, E E
AU - Michiels, F
AU - van Leeuwen, F N
AU - Kain, H E
AU - van der Kammen, R A
AU - Collard, J G
PY - 1997/11/7
Y1 - 1997/11/7
N2 - The Rho-like GTPases Cdc42, Rac, and Rho play key roles in the regulation of the actin cytoskeleton and are implicated in transcriptional activation and cell transformation. We have previously identified the invasion-inducing Tiam1 gene, which encodes an activator of Rac. In fibroblasts, Tiam1 induces Rac-mediated membrane ruffling, which requires the N-terminal pleckstrin homology (PHn) domain. Here we show that this PHn domain is part of a protein interaction domain, which mediates membrane localization of Tiam1. After subcellular fractionation, up to 50% of Tiam1 is recovered in the Triton X-100-insoluble high speed pellet that contains small protein complexes. The regions in Tiam1 that are responsible for these protein interactions comprise the PHn domain, an adjacent putative coiled coil region (CC), and an additional flanking region (Ex). Deletions in each of these regions abolish membrane localization of Tiam1 and membrane ruffling, suggesting that they function cooperatively. Indeed, only polypeptides encompassing the PHn-CC-Ex region, and not the PHn-CC or the Ex region, localize at the membrane. These results indicate that the N-terminal PH domain is part of a larger functional Tiam1 domain that mediates protein complex formation and membrane localization of Tiam1.
AB - The Rho-like GTPases Cdc42, Rac, and Rho play key roles in the regulation of the actin cytoskeleton and are implicated in transcriptional activation and cell transformation. We have previously identified the invasion-inducing Tiam1 gene, which encodes an activator of Rac. In fibroblasts, Tiam1 induces Rac-mediated membrane ruffling, which requires the N-terminal pleckstrin homology (PHn) domain. Here we show that this PHn domain is part of a protein interaction domain, which mediates membrane localization of Tiam1. After subcellular fractionation, up to 50% of Tiam1 is recovered in the Triton X-100-insoluble high speed pellet that contains small protein complexes. The regions in Tiam1 that are responsible for these protein interactions comprise the PHn domain, an adjacent putative coiled coil region (CC), and an additional flanking region (Ex). Deletions in each of these regions abolish membrane localization of Tiam1 and membrane ruffling, suggesting that they function cooperatively. Indeed, only polypeptides encompassing the PHn-CC-Ex region, and not the PHn-CC or the Ex region, localize at the membrane. These results indicate that the N-terminal PH domain is part of a larger functional Tiam1 domain that mediates protein complex formation and membrane localization of Tiam1.
KW - 3T3 Cells
KW - Animals
KW - Binding Sites
KW - Blood Proteins/chemistry
KW - COS Cells
KW - Cell Membrane/metabolism
KW - Centrifugation, Density Gradient
KW - Guanine Nucleotide Exchange Factors
KW - Mice
KW - Microscopy, Immunoelectron
KW - Mutagenesis, Site-Directed
KW - Phosphoproteins
KW - Protein Binding
KW - Proteins/genetics
KW - T-Lymphoma Invasion and Metastasis-inducing Protein 1
KW - Tumor Cells, Cultured
U2 - 10.1074/jbc.272.45.28447
DO - 10.1074/jbc.272.45.28447
M3 - Article
C2 - 9353304
VL - 272
SP - 28447
EP - 28454
JO - The Journal of biological chemistry
JF - The Journal of biological chemistry
SN - 0021-9258
IS - 45
ER -