A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes.
Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein-Raf1 and the Raichu-KRas probe, we identified for the first time in vivo-active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14-MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.
Lu
Albert
A
Departament de Biologia Cellular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Serveis Cientificotècnics, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.
Tebar
Francesc
F
Alvarez-Moya
Blanca
B
López-Alcalá
Cristina
C
Calvo
Maria
M
Enrich
Carlos
C
Agell
Neus
N
Nakamura
Takeshi
T
Matsuda
Michiyuki
M
Bachs
Oriol
O
D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D014709:Vero Cells; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D000736:Androstenes; D002147:Calmodulin; D002966:Clathrin; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D004815:Epidermal Growth Factor; C040929:bafilomycin A1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C430804:Raichu-Ras protein; D018631:ras Proteins