RRC ID 42117
Author Takaya A, Ohba Y, Kurokawa K, Matsuda M.
Title RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells.
Journal Mol Biol Cell
Abstract RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
Volume 15(6)
Pages 2549-57
Published 2004-6
DOI 10.1091/mbc.e03-11-0857
PII E03-11-0857
PMID 15034142
PMC PMC420081
MeSH Animals COS Cells Cell Line Cell Membrane / metabolism Cell Movement* Chlorocebus aethiops Dogs Enzyme Activation / drug effects Epidermal Growth Factor / pharmacology* Epithelial Cells / drug effects* Epithelial Cells / enzymology Epithelial Cells / metabolism Fluorescence Resonance Energy Transfer Guanosine Triphosphate / metabolism Kidney / cytology Kidney / drug effects* Kidney / enzymology Kidney / metabolism Pseudopodia / drug effects Pseudopodia / enzymology* Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism cdc42 GTP-Binding Protein / metabolism rac1 GTP-Binding Protein / metabolism ral GTP-Binding Proteins / antagonists & inhibitors ral GTP-Binding Proteins / deficiency ral GTP-Binding Proteins / genetics ral GTP-Binding Proteins / metabolism* ral Guanine Nucleotide Exchange Factor / metabolism ras Proteins / metabolism
IF 3.905
Times Cited 46
Human and Animal Cells