F-actin-dependent translocation of the Rap1 GDP/GTP exchange factor RasGRP2.
RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors.
Centro de Investigación del Cáncer, University of Salamanca-Consejo Superior de Investigaciones Cientiíficas, Campus Unamuno, E-37007 Salamanca, Spain.
Mariía J
MJ
Caloca
José L
JL
Zugaza
Miguel
M
Vicente-Manzanares
Francisco
F
Sánchez-Madrid
Xosé R
XR
Bustelo
D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D014162:Transfection; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D016023:Integrins; C057811:MCF2 protein, human; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; C415483:RASGRP2 protein, human; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; C496618:VAV1 protein, human; C093651:VAV2 protein, human; D004247:DNA; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins