title: H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes.
abstract: Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011-3260, USA.
authors.forename: Jodi
authors.initials: J
authors.lastname: McKay
authors.forename: Xing
authors.initials: X
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Ding
authors.forename: Janice E
authors.initials: JE
authors.lastname: Buss
authors.forename: Linda
authors.initials: L
authors.lastname: Ambrosio
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction; D022161:Transport Vesicles / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D019908:Proto-Oncogene Proteins c-raf; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)