RAF Kinases and Endocytosis Figure 4. Expression of GFP-AR149 inhibits maturation of endosomes. GFP-AR149-transfected and control (GFP-transfected) cells were incubated with fluorescent Tfn for indicated times. Tfn containing vesicles accumulate in the pericentriolar area of control cells, but remain in the GFP-AR149 positive vesicles scattered throughout the cytosol of GFP-AR149 transfected cells. Representative images are shown. doi:10.1371/journal.pone.0004647.g004 A fraction of endogenous A-RAF localizes to vesicles located along microtubuli Considering the dominant negative effect of AR149 on endocytosis in yeast, we set out to determine whether endogenous A-RAF in HeLa cells was localized on endocytic vesicles. After cytosol depletion, fixation and immunostaining with specific antibodies the remaining endogenous full-length A-RAF was found associated with vesicular structures. A-RAF positive vesicles were localized in the periplasmic area and at nearly each plus end of microtubules (Figure 3C). A significant fraction of A- RAF-positive punctate structures is lining the microtubular network that extends to the perinuclear area. A-RAF siRNA removed the punctate labeling, but not the diffuse staining over the nucleus that was also seen with normal rabbit IgG, (Fig S4C) and is therefore considered non-specific background. Consistently in cell fractionation experiments a significant part of A-RAF fractionates with cytoskeletal, but not with the nuclear fraction (Figure 3B). Overexpressed AR149 exhibits a dominant inhibitory effect on transport and traps internalized transferrin in ARF6 and RAB11 positive endosomes We asked whether overexpression of AR149 affects endocytic trafficking in mammalian cells. First, uptake and recycling of Tfn was investigated. Tfn is internalized upon binding to its receptor via a clathrin-dependent pathway and, after pH-regulated release, recycles back from the recycling/late endosomes. These endosomes are characterized by their accumulation in the pericentriolar space. GFP or GFP-AR149 transfected HeLa cells were incubated with Alexa-546 labelled Tfn for the indicated time (Figure 4). In control cells, after 30 to 40 minutes, endocytosed Tfn accumulated in the pericentriolar compartment (Figure 4, GFP panel). In GFP-AR149 expressing cells, relocation of endocytosed Tfn to pericentriolar compartments was significantly inhibited (Figure 4, GFP-AR149 panel). The difference is clearly visible at the 40 min timepoint, where transfected and non-transfected cells are next to each other. Therefore expression of AR149 does not influence the endocytic uptake of Tfn, but strongly inhibits its relocation to recycling endosomes. To pinpoint the cellular endosome compartment, which Tfn is trapped in, EEA1, RAB11 and ARF6 were used as markers for early and recycling endosomes. The data clearly show significant overlap between AR149, internalized Tfn and ARF6- as well as between AR149, Tfn and RAB11-positive vesicles (Figure 5). Consistently, EEA1 positive vesicles did not accumulate Tfn (Figure S5). Thus we conclude that the block in endocytic trafficking is at the level of tubular recycling endosomes. siRNA silencing of A-RAF and MEK inhibition mimicks the AR149 overexpression phenotype The splice variant DA-RAF was shown to function as an effective inhibitor of RAF-ERK signaling [19]. As AR149 is expected to share this function, AR149 overexpression or A-RAF depletion should have the same effect on Tfn trafficking. For depletion we prepared a set of A-RAF specific siRNAs and transfected them into HeLa cells. Degree of depletion and specificity of A-RAF siRNAs were determined by immunoblotting. As documented in Figure S4A significant depletion of A-RAF but not of the other two RAF isoforms was achieved. Semi- quantitative RT PCR showed that siRNA down-regulates A- RAF mRNA selectively without affecting the mRNA levels of DA- RAF (Figure S4B), which was expressed at a lower level than full length A-RAF in this cell line. To test the effect of A-RAF depletion on Tfn trafficking, A-RAF siRNA-transfected, and control (scrambled siRNA-transfected) cells were subjected to a Tfn uptake assay. Similar to GFP-AR149 expressing cells, defective or significantly delayed accumulation of endocytosed Tfn in the pericentriolar space was observed (Figure 6A) indicating that A-RAF kinase activity and thus the activation of the mitogenic cascade is required for normal Tfn trafficking. Consistent with a requirement of localized ERK activation, chemical inhibition of the mitogenic cascade by U0126, a specific inhibitor of the RAF effector MEK, prevented the aggregation of Tfn positive endosomes in a similar way as A-RAF depletion (Figure 6A). As the phenotypes of A-RAF depletion and mitogenic cascade inhibition overlap, we conclude that localized ERK signalling is required for endosomal maturation and AR149/DA-RAF func- tions as a dominant negative inhibitor of A-RAF on endosomes. Expression of GDP-locked ARF6(T27N) also blocks accumulation of Tfn in the pericentriolar endosomal compartment Dominant negative ARF6 has been shown previously to interfere with internalisation of Tfn [15]. To test whether the pattern of Tfn accumulation by ARF6(T27N) and AR149 are comparable transfected HeLa cells were examined for co- localization of internalized Tfn and ARF6(T27N) (Figure 6B). Inspection of Figure 4 and Figure 6A reveals similar re-distribution of Tfn in HeLa cells in which A-RAF has been knocked down or inhibited (AR149 or U0126) and HeLa cells that express ARF6(T27N). This data suggests that ARF6 may be a target for regulation by A-RAF on endosomes. Tfn accumulates in ARF6 positive and RAB11 positive vesicles after A-RAF knock down To characterize the endosome compartment in which Tfn accumulates after A-RAF knock down ARF6 and RAB11 were used as markers (Figure 7). Additionally we included EEA1 as marker of early endosomes (Figure S6). Co-localisation experi- ments in HeLa cells identify vesicles with accumulated Tfn as RAB11- and ARF6- positive. No EEA1 Tfn double positive vesicles were found. We conclude that the block in Tfn trafficking in the absence of A-RAF lies between tubular- and TGN- assotiated recycling endosome compartments. AR149 Interacts with ARF6 independent of nucleotide status We have shown that AR149 and both (active and inactive) ARF6 mutants partially colocalize on endocytic vesicles and the plasma membrane (Figure S7). We were interested whether interaction of these two proteins could be shown at the biochemical level. HeLa cells were co-transfected with GFP, PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4647