RAF Kinases and Endocytosis Figure 6. siRNA mediated A-RAF depletion, MEK inhibition and expression of ARF6(T27N) also block accumulation of Tfn in the pericentriolar endosome compartment. A. HeLa cells transfected with A-RAF siRNA, scrambled siRNA or treated with MEK inhibitor UO126 were subjected to Tfn uptake assay. Lack of accumulation of endocytosed Tfn in the pericentriolar compartment is similar to AR149-expressing cells (compare with Figure 4). Representative images are shown. Scale bar = 10 mm. B. HeLa cells were transfected with ARF6(T27N) before assay for fluorescent Tfn uptake. ARF6(T27N) positive vesicles accumulate internalised Tfn. The pattern of distribution is similar to that in A-RAF siRNA and to U0126 treated cells. Representative images are shown. Scale bar = 10 mm. doi:10.1371/journal.pone.0004647.g006 accumulation in yeast are known to contain proteins associated with early steps of endocytosis such as AP180 as well as Pan1p, that is associated with cortical actin patches. It is tempting to speculate that in mammalian cells the localization of A-RAF not only brings A-RAF into the neighbourhood of ARF6 on tubular endosomes, but additionally involves interaction with mammalian homologs of AP180/Pan1p that function at the interface of clathrin coated vesicles and an actin cytoskeleton regulatory complex. Such complexes are essential for endocytosis and also linked with the microtubule network [42]. Consistently GFP- AR149 decorated predominantly tubulo-vesicular endosomes, as confirmed by co-localization with ARF6 (Figure 3, middle panel). These endosomes lost their beads on the strings appearance upon Nocodazole treatment (Figure 3, top panel). Role of A-RAF and AR149 in endocytosis Definitions of endocytic compartments are poorly standardized [43]. Here we used definitions given in [44], where endocytosed Tfn is first enclosed in small vesicles. These vesicles fuse to form early endosomes. Further maturation of early endosomes through tubular recycling endosomes leads to the pericentriolar TGN- associated recycling compartment. AR149 is targeted to tubular endosomes where it colocalizes with ARF6. Exogenous expression of AR149 or dominant negative ARF6(T27N) mutant causes trapping of endocytosed Tfn in tubular endosomes, which prevents transfer to pericentriolar recycling compartment and subsequent return to the plasma membrane. AR149 and ARF6 occur in the same complex as demonstrated both by fluorescence microscopy and coprecipitation experiments (Figure 8A). A-RAF kinase was previously reported to participate in regulation of caveolae/raft-mediated endocytosis by stabilizing the caveolar coat [36,45]. Our discovery of a requirement for A- RAF activity at a step subsequent to endocytosis, in the transfer to the recycling compartment, point to a broader role of A-RAF in membrane trafficking that additionally involves feedback regula- tion by the inhibitory splice variants DA-RAF 1,2 [19]. Our data suggest that DA-RAF does not necessarily work as a general inhibitor of mitogenic signalling as initially described [19]. More likely, due to its unique intracellular localization, AR149/DA- RAF primarily inhibits just a specific endosome-associated branch of mitogenic signalling, which is responsible for regulation of receptor recycling and/or restoration of signalling molecules. The classical mechanism of cytoskeleton dependent endocytosis is subdivided in two parts: first, a short-distance step in the cortical area depends on actin and second long-distance microtubule dependent vesicular transport [42]. Our micrographs do not provide time resolution, but considering our functional analyses, it is conceivable that A-RAF functionally associates with the endosomes shuttling along microtubules between plasma mem- brane and the perinuclear recycling compartment. From the functional analysis of Tfn endocytosis (Figure 6A), we conclude that not only localization to endosomes but also activity of A-RAF kinase in the mitogenic cascade on endosomes are a prerequisite for the translocation of Tfn-positive endosomes to the pericentriolar region. ARF6 regulation by A-RAF and AR149 Our data suggest that A-RAF functions upstream of ARF6. There has been an earlier report by Robertson [31], who suggested, based on epistasis experiments with ERK and ARF6 that ERK functions upstream and downstream of ARF6. Several other reports also suggested dependence of ERK activation on ARF6 [17,46], which is in direct contrast with the results of our epistasis analysis. This contradiction may be explained by a pleiotropic effect of active ARF6, which is known to block internalization of activated receptors [15], leading to sustained signalling through the mitogenic cascade. Our data additionally suggest EFA6 as a cooperation partner of A-RAF in the activation of ARF6. Of note inspection of the primary structure of EFA6 revealed multiple potential ERK/MAP kinase phosphorylation sites that suggest that EFA6 is a substrate of ERK downstream of A-RAF. There are several examples for crosstalk between endocytosis and signal transduction [47,48]. In addition, there is a growing body of evidence that at least part of mitogenic signalling takes place on endosomes: for example, the MEK partner MP1 was isolated as a component of endosomal vesicles and associates with MEK and participates in signal transduction [49]. Another scaffold of the mitogenic cascade, KSR1, was also shown to localize and mediate signalling to endosomes [31]. Model of A-RAF function in regulation of endocytosis The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of A-RAF function shown in Figure 9. Stimulation of growth factor receptors is followed by RAS activation. RASNGTP recruits RAFs and leads to assembly of the RAF-MEK-ERK module at membranes. The three RAF isozymes become activated consecutively and mediate diversification of the signal to different subcellular compartments. A-RAF activation is delayed [50] because it has a corequirement for ERK phosphorylation [35], which uncouples A-RAF from B- and C-RAF that in turn are inhibited by feedback phosphorylation [33,34]. Due to the unique localization of A-RAF to endosomes, this delay is optimal for regulation of later endocytic events such as recycling of receptors [51]. A-RAF function at endosomes also involves the mitogenic cascade and triggers ARF6 activation possibly via EFA6. A-RAF involvement in ARF6 dependent endocytic recycling provides a new perspective for explaining the phenotype of A-RAF knock out mice, which exhibit severe neurological defects such as ataxia, rigidity of the musculature and continuous tremor [8]. Intriguingly, A-RAF, ARF6 and EFA6 are strongly expressed in Purkinje cells of mouse cerebellum [52,53]. Along this line, DA- RAF expression is particularly strong in brain [19]. Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons, which may further stress the role of A-RAF in the nervous system. PLoS ONE | www.plosone.org 13 February 2009 | Volume 4 | Issue 2 | e4647