RAF Kinases and Endocytosis Figure 1. Differential localization of wild type and mutant forms of RAF in yeast. A. N- and C-terminal deletions or point mutations were expressed as GFP fusions in yeast. Staining patterns of mutants are shown on the right. The largest C- and N-terminal deletions showing the ‘‘wild type’’ localisation are 1–388 and 88–606. Lipid binding domains (see also Figure S2), are indicated by yellow boxes. B. Localization of RAF isoforms in yeast. Only GFP-A-RAF localizes to small dots in the cell cortex, which accumulate to tips of small buds and to necks of larger buds (upper row). Induced polarization of yeast by a-factor leads to relocation of GFP-A-RAF to the tip of mating projections called shmoos (lowermost row). C. Localization patterns of C- and N-terminal deletion mutants. The smallest N-terminal deletion (88–606) retains wild type distribution. C-terminal deletions that lost the presumptive PA binding motif, while retaining the PtdIns(4,5)P2 binding motif in CRD (see the scheme above) are homogenously distributed to plasma membrane. The same magnification was used throughout. doi:10.1371/journal.pone.0004647.g001 led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of A-RAF, termed DA-RAF1,2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation [19]. DA-RAF2 contains the first 153 aar of A- RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic AR149 function, supporting a role of A-RAF regulated ERK signaling at endosomes that is controlled by AR149/DA-RAF2 and targets ARF6. Materials and Methods If not otherwise stated reagents were of p.a. purity (Sigma, USA), restriction enzymes were from New England Biolabs (USA) and Fermentas (Lithuania). Plasmids used in this study Deletion mutants of A-RAF were generated by insertion of PCR products into the pUG36 plasmid. In the case of N-terminal mutants of A-RAF, BamHI and HindIII recognition sequences have been attached to the sequence of forward and reverse primers respectively. Primers for C-terminal mutants of A-RAF contained SmaI and HindIII respectively. Doubly truncated mutants contained BamHI and XhoI restriction sites. PCR products were generated using primers listed in Table S1. For expression of the GFP fusion proteins in mammalian cells we used pEGFP-C-1 and pDS-Red2 (Invitrogen, USA). ARF6 constructs tagged with hemagglutinin (HA) in pLNCX, FLAG- EFA6 and GST-GGA3 were generous gift from Margaret Chou, University of Pennsylvania. GFP-tagged ARF6 was kindly provided by Antoine Galmiche. GFP-ARF6(Q67L) and GFP- ARF6(T27N) were generated by site directed mutagenesis (QuikChange, Stratagene) with the primers listed in Table S1. GFP-A-RAF in pEGFP-C1 was kindly donated by Angela Baljuls. Cell culture and cell fractionation Media and reagents were purchased from Invitrogen (USA). HeLa, NIH 3T3 and COS7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. For starvation, DMEM was supplemented with 0,03% FBS. Cell transfections utilized jetPEI (Biomol). Subcellular fractionation of HeLa cell lysates employed ‘‘ProteoExtract Subcellular Proteome Extraction Kit ‘‘(Calbio- chem) according to manufacturer’s instructions. Fluorescence Microscopy Fluorescence microscopy was done with an Openlab software (Improvision, UK) controlled inverted DMIRBE microscope (Leica, Germany) with Leica oil immersion objective. All images were captured and stored as Openlab LIF files. Images were subsequently processed using Photoshop software. Yeast live cell imaging. Yeast cells were transferred into a self-made chamber slide for imaging. Fixed yeast cells imaging. Cells were fixed in 3.7% paraformaldehyde in PBS, washed and subsequently digested for 1 hour with lyticase (Sigma). After washing and mounting, samples were either stored at 4uC or processed for imaging. Mammalian cells imaging. Cells were grown on cover slips, treated with growth factors or serum as indicated and subsequently fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100. Stainings were performed with specific antibodies and fluorescently labeled secondary antibodies. Indirect immunofluorescence after cytosol depletion HeLa cells were grown on coverslips overnight. After two washes with PBS cells were treated with 0,05% digitonin in isotonic sucrose buffer for 4 min on ice [20]. After digitonin treatment, cells were fixed with 3.7% paraformaldehyde in microtubule-stabilizing buffer (MSB; 0.1 M PIPES, pH 6.9, 2 mM MgCl2, 2 mM EGTA), washed and subsequently permeabilized with 0,1% w/v Saponin in MSB with 0.5% BSA for 10 min. To stain non-cytosolic A-RAF, cells were incubated with anti-A-RAF antibodies (Santa Cruz, USA) in combination with anti b-Tubulin antibodies (Chemicon International) at concentration of 20 mg/ml in MSB buffer with 0,5% BSA and 0,1% Saponin at room temperature for 2 h. Unbound antibodies were removed by 3 washes with the same buffer. The coverslips were incubated with appropriate secondary antibody (conjugated to TRITC or CY5) diluted 1:200 for 1 h. After three washes with MSB and brief wash with deionized water the coverslips were mounted using MOWIOL (Calbiochem, USA). siRNA-mediated depletion of human A-RAF For generation of A-RAF specific siRNA we used ‘‘X-tremeGENE siRNA Dicer Kit’’ (Roche). Prepared siRNA mix containied about 15 different siRNAs. The target sequence for human A-RAF located at the 39end of A-RAF coding region was from ‘‘Human esiRNA resource’’ (German Resource Center for Genome). Scrambled siRNA was from QIAGEN. Transfection was carried out using 2 mg of siRNA mixture and 10 ml of ‘‘X-tremeGENE siRNA Transfection Reagent’’ (Roche) for 6-well culture plates, according to the instructions provided by the manufacturer. Transferrin internalization HeLa cells were grown on coverslips overnight, transfected with either GFP or GFP-fused AR149, ARF6(Q67L), ARF6(T27N) respectively for 48 h. PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4647