The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.
n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.
Kozma
R
R
Department of Neurochemistry, Institute of Neurology, London, United Kingdom.
Ahmed
S
S
Best
A
A
Lim
L
L
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins / Q000502:physiology*; D020848:Chimerin 1; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D051379:Mice; D008845:Microinjections; D009419:Nerve Tissue Proteins / Q000494:pharmacology / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D011554:Pseudopodia / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D016596:Vinculin / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins
D000199:Actins; D018797:Cell Cycle Proteins; D020848:Chimerin 1; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate