RAF Kinases and Endocytosis Figure 3. Localization of GFP-A-RAF, GFP-AR149 and endogenous A-RAF in mammalian cells. A. Top row: HeLa cells were transiently transfected with pEGFP bearing the indicated genes for 2 days. GFP fusion proteins were detected by fluorescence microscopy. GFP-A-RAF is present throughout the cytoplasm and accumulates around the nucleus. In contrast, GFP-AR149 labels punctate structures often aligned on strings. Strings were disassembled by treatment with Nocodazole. A C-RAF fragment orthologous to AR149 is GFP-C-RAF(C4). Representative images are shown. Scale bar = 10 mm. Middle row: HeLa cells were cotransfected with RFP-AR149 and GFP-ARF6 as described above. RFP and GFP fluorescences were recorded separately. Bottom row: HeLa cells were cotransfected with Myc-A-RAF and GFP-ARF6. After two days transfected cells were treated with digitonin to extract cytosol and processed for detection of A-RAF and ARF6 as described in Figure 3C. Boxed areas are shown at higher magnification. Arrows indicate vesicles with colocalization. Representative images are shown. Scale bar = 10 mm. B. Cells were fractionated using ‘‘ProteoExtract Subcellular Proteome Extraction Kit’’ (Calbiochem) and analyzed by immunoblotting. antibodies against following proteins were used as compartmental markers: vimentin as cytoskeletal marker, PARP as nuclear marker, 2MPK as cytosolic marker. Both A-RAF and AR149 are co- fractionating predominantly with cytoskeleton and cytosol. Small portion of AR149 was also found in plasma membrane fraction. C. HeLa cells were treated with digitonin to extract cytosol. After fixation and washing, immunofluorescence microscopy with antibodies against A-RAF and against b- tubulin was carried out. The boxed area is shown at higher magnification. Small A-RAF positive vesicles are at the periphery and line microtubules (arrows). Representative images are shown. Scale bar = 10 mm. doi:10.1371/journal.pone.0004647.g003 contrast, mutant protein with substitution of basic residues in the PA binding site led to homogeneous distribution in the plasma membrane, the typical pattern for C-terminally truncated mutants (Figure S2). We conclude that the A-RAF specific punctate pattern requires both, the PS/PIP2 and PA lipid binding sites. Expression of AR149 in yeast causes growth inhibition, defects in cell polarization, in nuclear morphology and in membrane trafficking In the course of deletion analysis, we noted that cells expressing several C-terminally truncated mutants showed homogenous membrane localization, different from that of full-length protein. In addition, a significant fraction of cells expressing the C-terminal deletion mutants was larger and apolar (round in shape instead of oval) as compared to GFP or GFP-A-RAF expressing cells (see Figure 2A, B). Polarization during cell growth by directed membrane trafficking is essential for cell division, therefore we hypothesized that GFP-AR149 expressing cells may exhibit retarded cell cycle progression and as a consequence loss of viability. To test this, we grew GFP, GFP-A-RAF and GFP- AR149 in liquid culture, under promoter de-repressing conditions. Cells expressing GFP-AR149 showed sustained growth inhibition. The difference of 28 minutes in duplication time confirms that expression of GFP-AR149, but not that of GFP or full-length A- RAF exhibits an inhibitory effect on expressing cells (Figure 2E). We stained actin and DNA in GFP-AR149 and GFP-A-RAF expressing cells. In agreement with the apolar phenotype, no polymerized actin could be detected in the large round cells expressing higher levels of GFP-AR149 (Figure 2A arrows). Intriguingly, in the same cells (large, apolar) no intact nuclei could be detected by DAPI staining (Figure 2C). Viability tests showed about 20% decrease in survival rate of GFP-A-RAF149 expressing cells, which is in good agreement with the percentage of apolar cells found in the culture. In a FM4-64 uptake assay we found that yeasts expressing the AR-149 were affected in endocytosis (Figure 2D) conversely endocytosis was unaffected when GFP-A-RAF was expressed (Figure S1D). To test the effect of the fusion partner and of high level expression of A-RAF and of AR149 on yeasts, these proteins were expressed as GST-fusions under the strongly inducible GAL10 promoter. Cells were transformed with GST-A-RAF or GST-AR149, streaked on glucose- (‘‘promoter off’’) or galactose (‘‘promoter on’’) media and grown at 30uC. Figure S1C shows that whereas all three strains grew well in the repressive medium, in the inductive medium the GST-AR149 expressing strain did not grow. Taken together, AR149, but not full-length A-RAF, localizes homogeneously to the plasma membrane, and blocks yeast cell polarization, actin polymerization, and endocytosis, resulting in cell growth inhibition. Moreover cells overexpressing AR149 from a strong GAL10 promoter were not viable (Figure S1C). Localization of A-RAF and AR149 in human cell lines The dramatic differences in localization between B- and C-RAF versus A-RAF and its N-terminal fragment, AR149, observed in yeast, prompted us to analyze the latter proteins in mammalian cell lines. AR149 expression had a strong negative effect on actin polymerization in yeast (Figure 2A). To test this effect in mammalian cells, we chose NIH 3T3 cell line known for extended fibers of polymerized actin called stress fibers. Similar to yeast, stress fibers in NIH 3T3 cells were diminished by GFP-AR149 expression (Figure S3). This effect was also described for DA- RAF1, 2 by Yokoyama et al. (2007). We next examined the subcellular localization of A-RAF and AR149 in HeLa cells. Cells were transfected with GFP-tagged A- RAF and AR149 respectively. For control, an N-terminal fragment of C-RAF (aar 1–256) described originally as a dominant inhibitory RAF mutant [29] that corresponds to AR149 (named C-RAF-C4) was included in the analysis. As observed in yeast, AR149 was unique in that it labeled the plasma membrane. Additionally in HeLa cells ‘‘beads on a string’’ structures (Figure 3A, top panel) were seen, demonstrating isotype specificity of the lipid binding site in CR1 as B- and C-RAF were reported to localize instead to ER/Golgi complex or mitochondria respec- tively [30,20]. Disruption of microtubules by Nocodazole treatment separated beads from the strings (Figure 3A, top panel). The stained vesicular tubular structures most likely represent recycling endosomes. To test this hypothesis, we studied the localization of RFP-AR149 and that of an established endosomal marker, ARF6. As documented in Figure 3A, middle panel, there is a high degree of colocalization between RFP-AR149 and GFP- ARF6. ARF6 was reported to regulate central steps of endocytosis and recycling of endocytosed material by early and recycling endosomes (see [16] for review). Like nearly all GTPases, ARF6 functions as a molecular switch alternating between GTP-bound (active) and GDP bound (inactive) states. Consequently, overex- pressed GTP-, GDP-locked or nucleotide-free mutants of ARF6 (Q67L, T27N and N121I respectively) have distinct dominant effects on endosomal morphology and endocytosis [31]. In co-transfected cells we found colocalization of RFP-AR149 (Figure 3A, middle panel) or wild type A-RAF (Figure 3A, bottom panel) with GFP-ARF6 in tubular vesicular structures. In summary, AR149 and wild type A-RAF, but not C-RAF-C4 localizes to tubular endosomes, as proven by specific colocalization with ARF6. PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4647