RAF Kinases and Endocytosis Figure 5. AR149 traps inernalized transferrin in ARF6 and RAB11 positive endosomes. HeLa cells were transfected with RFP-AR149 and either GFP-ARF6 or RAB11 as indicated. Uptake of fluorescent Tfn was examined after 30 and 60 minutes. Representative images show that Tfn is trapped by AR149 in GFP-ARF6 positive and in GFP-RAB11 positive vesicles. Enlarged areas are marked by boxes. Arrows indicate co-localization. doi:10.1371/journal.pone.0004647.g005 GFP-AR149 and HA-tagged ARF6 wild type, GTP-locked (Q67L) or GDP-locked (T27N) mutants. After immunoprecipitation with anti-GFP antibodies, the precipitates were separated by SDS PAGE and tested for bound ARF6 by immunoblotting against HA. In a reverse experiment ARF6 mutants were first precipitated and the presence of GFP-A-RAF in the precipitate was demonstrated by immunoblotting with anti-GFP antibodies. Figure 8A documents that similar amounts of ARF6, ARF6(Q67L) and ARF6(T27N) co-precipitated with GFP- AR149, irrespective of which protein was precipitated first. Thus interaction of A-RAF with ARF6 is independent of the ARF6 nucleotide status. A-RAF and AR149 regulate ARF6 activation Next we addressed whether overexpression of dominant negative AR149 has an effect on activation of ARF6. ARF6 interacts specifically with its effector GGA3 in a nucleotide dependent manner. Therefore binding of GGA3 can be used as readout of the activation status of ARF6 [23]. In a control experiment, cell lysates from COS7 cells transfected with wild type, dominantly activated (Q67L) and dominant negative (T27N) mutants of HA-ARF6 were incubated with immobilized GST- GGA3 and washed. As shown in Figure 8B, GGA3 pulls effectively ARF6(Q67L), and less effectively ARF6wt. In contrast, no binding of ARF6(T27N) to GGA3 could be detected. In subsequent experiments, COS7 cells were starved and induced with EGF, a strong elicitor of mitogenic signalling. Activated ARF6 was pulled-down by immobilized GGA3 as described above. Large amounts of ARF6 were pulled-down from EGF stimulated cells compared to non-stimulated cells (Figure 8C). Thus, ARF6 is activated by the mitogenic cascade. This activation is significantly diminished by the coexpression of AR149 (Figure 8E) or A-RAF knock down (Figure 8F). Of note pulled-down ARF6 contains coexpressed AR149, which precludes competition of AR149 and GGA3 for binding to activated ARF6. To pinpoint the position of AR149 action in a signalling pathway, COS7 cells were co-transfected with HA-ARF6 and either AR149, EFA6 or both. It has been previously described that overexpression of the ARF6 exchange factor, EFA6, increases the amount of ARF6 bound to GST-GGA3 beads. As depicted in Figure 8D, less ARF6 was pulled down in the presence of AR149. Similarly, A-RAF knock down reduced the amount of ARF6 that could be pulled-down with GST-GGA3. EFA6 partially rescued this effect (Figure 8F). We conclude that AR149 co-expression or A-RAF knock down negatively affects the activation of ARF6 by EFA6, i.e. A-RAF functions upstream of ARF6. Discussion A-RAF is a distinct member of the RAF kinase family In this work we unveil an unexpected link between A-RAF and regulation of ARF6 activity. A-RAF possesses several features that set it apart from the other RAF kinases. A-RAF maps to the X chromosome and is the only steroid hormone-regulated RAF isoform, [32]. A-RAF protein has substitutions in a negatively charged region immediately upstream of the kinase domain (N-region), which is at least partially responsible for its low basal activity [12]. In contrast to C-RAF [33,34], feedback phosphorylation of A-RAF by its downstream effector, ERK, has an activating effect on its kinase activity [35]. Interestingly, out of 590 kinases tested, the three RAF isoforms were among 208 kinases affecting clathrin- or caveolae-dependent endocytosis [36]. si-RNA mediated silencing of A-RAF inhibits, whereas that of C- and B-RAF activate SV40 uptake. Role of lipid binding domains, identification and properties of inhibitory A-RAF, AR149 Our initial studies on RAF distribution in a heterologous yeast system showed that of the three RAF isoforms, only A-RAF located to the cell cortex of yeast cells (Figure 1). This pattern resembles that of components of the endocytic machinery, such as Sla1p, Sla2p, clathrin light chain protein Clc1p [37]. Differences in localization of A-RAF and AR149 can be explained by the existence of two lipid-binding domains in the structure of A-RAF, each domain binding to specific sets of lipids. The unique distribution pattern of A-RAF in yeast resembles the distribution of a PtdIns(4,5)P2 sensor, AP180 N-terminal homology (ANTH) domain [37]. Consistently, A-RAF is the only RAF isoform, which binds to immobilized PtdIns(4,5)P2 [27]. Using a set of point mutations, Johnson et al. (2005) located PtdIns(4,5)P2 binding site in the RAS binding domain of A-RAF. In contrast, we mapped PtdIns(4,5)P2 binding site to the cysteine rich domain of A-RAF because a RAS binding domain deleted mutant (88–606) still shows wild type distribution. In agreement with Johnson et al (2005), replacement of arginine 52 in the RBD with leucine, which prevents binding of C-RAF RBD to RAS [38], disturbs the membrane localization of both full- length A-RAF and AR149 in yeast (Figure 1). We interpret this discrepancy by an interaction between RBD and CRD. It is known that RBD and CRD bind to RAS cooperatively [39]. The R52L mutation in the RBD may cause structural rearrangements disabling the interaction of the PtdIns(4,5)P2 binding site in the CRD with membranes. CRDs are conserved among RAF isoforms, but only the A-RAF CRD possesses the unique property of PtdIns(4,5)P2 binding [27]. However as we show here the PtdIns(4,5)P2 binding site per se is not sufficient to specify the A-RAF wild type localization pattern. The wild type pattern of membrane binding requires in addition the PA binding site in the catalytic domain. In regard to the colocalization of A-RAF with ARF6 in mammalian cells, it is noteworthy that ARF6 is known to regulate the PtdIns(4,5)P2 content of membrane microdomains to which it binds. In yeast Arf3p is the functional homolog of ARF6. Arf3p regulates PtdIns(4,5)P2 levels, endocytosis and actin polymeriza- tion [40,41]. Therefore it is likely that the dominant lethal phenotype of AR149 in yeast is mediated by interference with Arf3p. Localization of A-RAF and AR149 in mammalian cells The differences between RAF isoforms were most pronounced in yeast, where evidence for membrane attachment was restricted to A-RAF. The punctate cortical structures that are sites of A-RAF PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4647