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 α-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.
Figure 2.
Physiological effects of AR149 overexpression in yeast.
A. Cells expressing high amounts of GFP-AR149 fail to form cortical actin patches. Cells expressing GFP-A-RAF or GFP-AR149 were grown, fixed as described in “Experimental procedures”. Polymerized actin was visualized with Alexa Fluor 546-conjugated Phalloidin. Note that cells expressing higher levels of GFP-AR149 but not of full length A-RAF (bright green cells marked with arrows) lost polymerized actin. B. GFP-AR149 expression inhibits ∝-factor induced shmoo formation. Cells transformed with pUG36-AR149 (right) or A-RAF(303–606) (left) were treated with ∝-factor for 2 h. Note increased overall size and absence of shmoos in cells expressing GFP-AR149. C. Changes in nuclear morphology of GFP-AR149 expressing cells. DNA of the nucleus and mitochondria was stained with DAPI. Morphology of nuclei of AR149 expressors varied from fragmented (left) to completely dispersed (right). D. FM-4-64 uptake is affected in AR149-expressing yeast. Yeast transformed with pUG36-AR149 and untransformed control were incubated with lipophilic styryl dye FM 4–64 for indicated time, washed and observed by fluorescence microscopy. Transition of the red fluorescence from periplasmic endocytic sites to vacuoles is clearly visible at 20 minutes in control cells, but not in GFP-AR149 expressing cells. E. Growth inhibition of AR149 expressing cells. Cells transformed with pUG36, pUG36-A-RAF or pUG36-AR149 were grown under selective conditions in fresh selective medium. Cell proliferation was monitored by spectrophotometry.
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 µm. 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 µm. 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 β-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 µm.
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.
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.
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 µm. 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 µm.
Figure 7.
Tfn accumulates in ARF6 positives, RAB11 positives vesicles after A-RAF knock down.
HeLa cells were cotransfected with either GFP-ARF6 or GFP-RAB11 and A-RAF siRNA. Tfn uptake was assayed as before. Tfn was found to co-localize with ARF6 or RAB11 as was observed in AR149 expressing cells. Enlarged areas are marked by boxes. Arrows indicate co-localization. Representative images are shown. Scale bar = 10 µm.
Figure 8.
Down regulation of A-RAF by either AR149 or A-RAF siRNA interferes with activation of ARF6.
A. Interaction of AR149 and ARF6. COS7 cells were co-transfected with GFP-AR149 and either HA-tagged ARF6wt, GTP-locked [ARF6(Q67L)] or GDP-locked [ARF6(T27N)]. After immunoprecipitation with α-GFP antibodies, co-precipitated ARF proteins were detected with α-HA antibodies. In the second experiment, precipitation and detection antibodies were exchanged. Expression levels in whole cell lysates (WCL) is shown in two bottom panels. Empty vectors were used for control (bottom panel). B. GGA3 interacts with ARF6•GTP. COS7 cells were transfected with wild type, GTP-locked (Q67L) or GDP-locked (T27N) HA-ARF6. Proteins pulled-down by incubation with GST-GGA3-Sepharose were detected with α-HA antibodies. C.,E. AR149 suppresses EGF-stimulated ARF6 activation. COS7 cells, transfected with either ARF6 alone or ARF6+GFP-AR149, were treated with EGF for 10 min and subjected to GGA3 pull-down. Bound protein was analysed by immunoblotting. Note decrease in the amount of ARF•GTP by AR149 coexpression. AR149 remains in the pulled-down ARF6 complex confirming the immunoprecipitation data. D. AR149 inhibits ARF6 activation by EFA6. COS7 cells were transfected with HA-ARF6 and either AR149, EFA6, or both. ARF6•GTP was pulled-down by GST-GGA3. AR149 decreases EFA6-stimulated ARF6 activation. F. ARF6 activation by EGF or EFA6 requires A-RAF. ARF6 activation was assessed by GGA3 pull-down. The amount of A-RAF and ARF6 protein were determed by Western blotting (WB). Treatment conditions are as indicated.
Figure 9.
Model of A-RAF and AR149/DA-RAF function in regulation of endocytosis.
Activation of receptor tyrosine kinase (here EGF receptor) leads to RAS-mediated activation of RAF kinases. RAF isoforms sort into different membrane microdomains, such as A-RAF into PtdIns(4,5)P2 rich domains. Activated ERK has opposing effects on A-RAF and C-RAF. Whereas A-RAF is activated, C-RAF becomes inactivated by feedback phosphorylation. A-RAF bound to PtdIns(4,5)P2 rich membranes continues to signal on endosomes leading to ARF6 activation. AR149/DA-RAF locates to recycling endosomes and blocks ERK activation in this compartment. See the main text for details. EE - early endosome, RC – recycling compartment, PM – plasma membrane. Red arrows indicate positive regulation of the process. Pale brown color indicates PtdIns(4,5)P2 rich membrane microdomains.