CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1813 (2011) 298–307 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes Jodi McKay 1,2, Xing Wang 1,3, Jian Ding 4, Janice E. Buss 5, Linda Ambrosio ⁎ Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011-3260, USA article info abstract Article history: Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may Received 23 June 2010 play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated Received in revised form 2 November 2010 subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we Accepted 29 November 2010 show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Available online 8 December 2010 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 Keywords: fi Ras internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are suf cient to mediate Endocytosis localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6- Arf6 associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on Protein trafficking EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the Endosomes plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and Clathrin dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the Raf-1 specificity of Ras:effector interaction. Endosomal-recycling center © 2010 Elsevier B.V. All rights reserved. 1. Introduction identifiable by the presence of the small GTPase Arf6 that participates in vesicular trafficking [14,15]. Although the site of Ras signaling is widely depicted as the plasma The internalization pathways of the H-Ras and K-Ras4B proteins membrane, endocytosis of Ras pathway components is required to appear to differ. In neurons, calcium signals trigger Ca++/calmodulin achieve efficient and specific spatio-temporal activation of downstream binding to K-Ras4B and cause the release of the K-Ras/calmodulin cascades [1–3]. Ras proteins have now been shown to be distributed on, complex from the plasma membrane [16]. This transient, cytosolic and to produce significant signals from, internal membranes [4–6].The complex is subsequently targeted to Golgi or recycling endosomes means by which Ras proteins localize to these internal sites are varied [17]. Internalization of K-Ras can also be mediated through vesicular and complicated [7]. Some of these routes may depend on release of Ras endocytic pathways. Lu et al. [18] found K-Ras4B associated with from the plasma membrane, while others likely require transfer of EEA1/Rab7-bearing early endosomes, as well as membranes of late membrane tethered Ras between membranous compartments via endosomes, lysosomes and multivesicular bodies in COS-1 cells. vesicular budding or fusion mechanisms. The classic vesicular internal- For newly synthesized H-Ras, a small portion of the protein is ization route is via clathrin-mediated endocytosis. Formation of delivered to Golgi membranes, and from there, these molecules board clathrin-dependent traffic machineries requires involvement of clathrin outward-bound exocytic vesicles [19,20]. However, the majority of coat and adapter proteins, which are the key components of this newly synthesized H-Ras appears to initially traffic to the plasma pathway. Although not as well characterized as clathrin-mediated membrane via a non-conventional pathway that may be non- endocytosis, several pathways for clathrin-independent endosomal vesicular in nature [21]. Additional studies indicate that palmitoylated entry of proteins have been described [8–13]. One of these is a pathway H-Ras that has already reached the plasma membrane can be de- palmitoylated and released into the cytosol. The farnesylated but non- acylated protein can then be re-palmitoylated, and thus captured on ⁎ Corresponding author. Tel./fax: +1 515 294 0453. E-mail address: [email protected] (L. Ambrosio). internal membranes, to join with those H-Ras molecules traveling to 1 These authors contributed equally. the plasma membrane via the biosynthetic vesicular pathway [20,22]. 2 Current address: Department of Biology and Chemistry, Morningside College, Sioux City, Like K-Ras, there is evidence that H-Ras can leave the plasma IA 51104, USA. membrane and enter vesicular endocytic pathways. H-Ras has been 3 Current address: Fermentas Life Sciences, Dengliang Road, Shenzhen 518054, China. repeatedly found on internal vesicles with all the characteristics of 4 Current address: Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA. endosomes [23–25]. A previous study has shown that both Rab5 and 5 Current address: TechLab, 2001 Kraft Drive, Blacksburg, VA 24060, USA. Rab11 regulate translocation of H-Ras to the pericentriolar recycling 0167-4889/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2010.11.019 J. McKay et al. / Biochimica et Biophysica Acta 1813 (2011) 298–307 299 center [26]. However, Rab5 functions on endosomal membranes and NIH 3T3 cells were cultured at 37 °C and 10% CO2 in Dulbecco's sorting endosomes that are derived from both clathrin-mediated and modified Eagle's medium supplemented with 10% calf serum plus clathrin-independent pathways [11,26], thus, its presence does not penicillin, streptomycin, glutamine and sodium pyruvate. For immu- define a specific class of endosomes. nofluorescence and immuno-isolation, NIH 3T3 cells were transfected Several clues to the identity of H-Ras endocytic pathways have with Effectene (Qiagen) and used after 24 hours or as mentioned in been derived from the identification of components from H-Ras the results. Stable lines contained H-Ras-61L or H-Ras-WT (pZIPneo associated vesicles. One study demonstrated that H-Ras appears to be vector), or GFP-H-Ras-61L cDNA (pEGFP-C3 vector). Cells treated with present on endosomes containing epidermal growth factor receptor EGF (Gibco BRL) were cultured as described above but incubated with (EGFR) [24]. Vesicles captured by magnetic beads coated with EGF 5 or 10 ng/ml of EGF at 37 °C for the time as indicated, followed by contained EGFR along with endogenous Ras isoforms. These endo- washing with phosphate-buffered saline and fixation. some preparations also contained clathrin protein [27], suggesting cargo, including Ras, on these captured vesicles may be derived from a 2.2. Antibodies clathrin-dependent pathway. However, EGFR can be internalized via both clathrin-dependent and independent mechanisms. Thus, the Primary antibodies used for immunofluorescence included rat identity and types of H-Ras associated vesicles remained undefined. monoclonal anti-Ras sc34 (Santa Cruz Biotechnology); the mouse Recently, Porat-Shliom et al. [28] has reported that endosomes in a monoclonals anti-Ras 610010 (BD Bioscience; Fig. 4) and anti-Ras clathrin-independent pathway associated with Arf6 contained H-Ras. LA069 (Quality Biotech); and the rabbit polyclonals anti-c-Raf-1 sc227 They proposed an elegant model for sequential production of and anti-clathrin heavy chain sc9069 (Santa Cruz Biotechnology), phosphoinositides on these endosomes accompanied by signaling of anti-EEA1P1-063(Affinity BioReagents), anti-hemagluttinin H-Ras to the phosphatidyl inositol 3-kinase (PI3-K) effector pathway. PRB101P (Convance) and anti-giantin PRB114C (Convance). Primary However, this study did not, in a systematic way, rule out the possibility antibodies used for immuno-isolation included mouse monoclonal that H-Ras can also enter cells via clathrin-mediated endocytosis. anti-Ras LA069 (Quality Biotech), rat monoclonal anti-Ras sc34 (Santa Ras effector proteins can be internalized by clathrin-dependent Cruz Biotechnology), and rabbit polyclonals anti-EEA1 P1-063 and clathrin-independent means. Effector components that are (Affinity BioReagents), and anti-hemagluttinin (HA) PRB101P (Con- involved in PI3-K signaling, as well as downstream components in vance). Primary antibodies used for immunoblotting included mouse the Raf effector branch of the Ras pathway, including KSR1 and ERK, monoclonals anti-Ras 610010 and anti-c-Raf 61051 (BD Biosciences), have been found on clathrin-independent endosomes [28,29]. anti-GFP 1814460 (Roche), and rabbit polyclonals anti-hemagluttinin However, it is not clear if Raf-1 is also present on these vesicles, as PRB101P (Convance) and anti-EEA1 P1-063 (Affinity BioReagents). Raf-1 has been observed on early endosomes, co-localizing with the Secondary antibodies from Molecular Probes included goat anti- EEA1 marker [25]. It has also been demonstrated that expression of mouse IgG coupled to Alexa Fluor 350, goat anti-rat IgG coupled to the constitutively active Rab5-Q79L protein causes some H-Ras, but, Alexa Fluor 488, and the Alexa Fluor 594 coupled goat anti-mouse IgG, notably,