Nerve growth factor activates persistent Rap1 signaling in endosomes.
We investigated a role for endogenous Rap1, a small monomeric GTP-binding protein of the Ras family, in nerve growth factor (NGF) signaling in PC12 cells. Although both epidermal growth factor (EGF) and NGF caused transient activation of Ras, only NGF induced the activation of Rap1. Moreover, Rap1 activation was sustained for hours, an effect that matched the sustained activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate the molecular basis for Rap1 activation, we examined complexes containing C3G, a guanine nucleotide exchange factor for Rap1, and CrkL, an adapter protein known to influence Rap1 signaling. NGF induced the formation of a long-lived complex containing C3G/CrkL/Shp2/Gab2/TrkA. Linking the complex to Rap1 activation, we coprecipitated activated TrkA and activated MAPK with activated Rap1 in NGF-treated cells. Confocal microscopy and subcellular fractionation showed that activated Rap1 and the other proteins of the signaling complex were present in endosomes. Pretreatment of PC12 cells with brefeldin A (BFA), which disrupts the Golgi and endosomal compartments, had little effect on Ras activation but strongly inhibited NGF-induced Rap1 activation and continuing MAPK activation. We propose that endosomes are a site from which NGF induces the prolonged activation of Rap1 and MAPK.
Wu
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C
Departments of Neurology and Neurological Sciences and of Pediatrics and the Program in Neuroscience, Stanford University School of Medicine, Stanford, California 94305, USA. cbwu@stanford.edu
Lai
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CF
Mobley
W C
WC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism; D015801:Helminth Proteins / Q000378:metabolism; D015151:Immunoblotting; D020935:MAP Kinase Signaling System / Q000187:drug effects; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000378:metabolism; D016716:PC12 Cells; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D020917:Receptor, trkA; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D002352:Carrier Proteins; C490893:Gab2 protein, rat; D020735:Guanine Nucleotide-Releasing Factor 2; D015801:Helminth Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016222:Fibroblast Growth Factor 2; C095048:microfilarial sheath protein, Helminth; D020126:Brefeldin A; D004815:Epidermal Growth Factor; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D020827:rap1 GTP-Binding Proteins