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One-way membrane trafficking of SOS in receptor-triggered Ras activation

View Article: PubMed Central - PubMed

ABSTRACT

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis.

No MeSH data available.


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PR domain dependent SOS endocytosis mediates signal attenuation(a) Confocal data in 3D rendering showing SOSFL enriched vesicle-like structures appearing away from the cell-bilayer contact zone at late time points (≈10–30 min. after cell landing). Bar: 3 µm.(b) Statistics of cells displaying internal SOS puncta as shown in a. N cells;N SLB samples;N cell cultures: FL 97;4;2, HDPC 75;3;2, HDPC-SH2 78;3;2. Error bars indicate SD over the different SLB samples.(c) Localization of EGFP-tagged full length SOS1 in COS-1 cells stimulated with EGF for the indicated time points. Bar: 10 µm.(d) Colocalzation of internalized SOS1 with the Rab5 endosomal marker in COS-1 cells stimulated for 25 minutes with EGF. Images shown are representative of colocalization pattern observed in >75% of the cells in 3 independent experiments (25 cells analyzed per experiment). The enlarged inset in the merge is also plotted in Supplementary Figure 7d as separate image channels. Bar: 10 µm.(e) Kinetics of SOS localization to endocytic vesicles in EGF stimulated COS-1 cells. EGFP-tagged SOSFL is compared to a full length SOS1 molecule with a functionally impaired allosteric pocket (SOSFL-L687E, R688A). Representative images accompany the bar graph. The results represent an average of two independent experiments (25 cells counted per condition for each experiment).Bar: 10 µm.Source data for plots and graphs are available online.
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Figure 7: PR domain dependent SOS endocytosis mediates signal attenuation(a) Confocal data in 3D rendering showing SOSFL enriched vesicle-like structures appearing away from the cell-bilayer contact zone at late time points (≈10–30 min. after cell landing). Bar: 3 µm.(b) Statistics of cells displaying internal SOS puncta as shown in a. N cells;N SLB samples;N cell cultures: FL 97;4;2, HDPC 75;3;2, HDPC-SH2 78;3;2. Error bars indicate SD over the different SLB samples.(c) Localization of EGFP-tagged full length SOS1 in COS-1 cells stimulated with EGF for the indicated time points. Bar: 10 µm.(d) Colocalzation of internalized SOS1 with the Rab5 endosomal marker in COS-1 cells stimulated for 25 minutes with EGF. Images shown are representative of colocalization pattern observed in >75% of the cells in 3 independent experiments (25 cells analyzed per experiment). The enlarged inset in the merge is also plotted in Supplementary Figure 7d as separate image channels. Bar: 10 µm.(e) Kinetics of SOS localization to endocytic vesicles in EGF stimulated COS-1 cells. EGFP-tagged SOSFL is compared to a full length SOS1 molecule with a functionally impaired allosteric pocket (SOSFL-L687E, R688A). Representative images accompany the bar graph. The results represent an average of two independent experiments (25 cells counted per condition for each experiment).Bar: 10 µm.Source data for plots and graphs are available online.

Mentions: Over time, the initially scattered BCR clusters concatenated and moved toward the center of the synapses formed between the B cells and the SLB. About 15–20 minutes after cell landing, a large central cluster appeared, a phenomenon commonly referred to as ‘BCR capping’56 (Fig. 6c and Supplementary Movie 1). SOSFL initially moved with the activated BCR, but at later time points we found that it was depleted from the central BCR cluster (Fig. 6d,e). Thus, SOSFL leaves the plasma membrane at the site of the central BCR cluster, which also correlates with attenuation of SOSFL-driven Ras-ERK signaling at later time points (Fig. 4g). Confocal fluorescence microscopy revealed the appearance of punctate SOS structures located inside the cells, reminiscent of endocytic vesicles (Fig. 7a). Moreover, these vesicle-like structures appeared only for SOSFL but not for SOSHDPC or the chimeric SOSHDPC–SH2 and only on bilayers displaying the BCR activating antibody (Fig. 7a,b). These observations suggest that removal of SOS1 from the membrane in a BCR signal dependent process requires the C-terminus.


One-way membrane trafficking of SOS in receptor-triggered Ras activation
PR domain dependent SOS endocytosis mediates signal attenuation(a) Confocal data in 3D rendering showing SOSFL enriched vesicle-like structures appearing away from the cell-bilayer contact zone at late time points (≈10–30 min. after cell landing). Bar: 3 µm.(b) Statistics of cells displaying internal SOS puncta as shown in a. N cells;N SLB samples;N cell cultures: FL 97;4;2, HDPC 75;3;2, HDPC-SH2 78;3;2. Error bars indicate SD over the different SLB samples.(c) Localization of EGFP-tagged full length SOS1 in COS-1 cells stimulated with EGF for the indicated time points. Bar: 10 µm.(d) Colocalzation of internalized SOS1 with the Rab5 endosomal marker in COS-1 cells stimulated for 25 minutes with EGF. Images shown are representative of colocalization pattern observed in >75% of the cells in 3 independent experiments (25 cells analyzed per experiment). The enlarged inset in the merge is also plotted in Supplementary Figure 7d as separate image channels. Bar: 10 µm.(e) Kinetics of SOS localization to endocytic vesicles in EGF stimulated COS-1 cells. EGFP-tagged SOSFL is compared to a full length SOS1 molecule with a functionally impaired allosteric pocket (SOSFL-L687E, R688A). Representative images accompany the bar graph. The results represent an average of two independent experiments (25 cells counted per condition for each experiment).Bar: 10 µm.Source data for plots and graphs are available online.
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Figure 7: PR domain dependent SOS endocytosis mediates signal attenuation(a) Confocal data in 3D rendering showing SOSFL enriched vesicle-like structures appearing away from the cell-bilayer contact zone at late time points (≈10–30 min. after cell landing). Bar: 3 µm.(b) Statistics of cells displaying internal SOS puncta as shown in a. N cells;N SLB samples;N cell cultures: FL 97;4;2, HDPC 75;3;2, HDPC-SH2 78;3;2. Error bars indicate SD over the different SLB samples.(c) Localization of EGFP-tagged full length SOS1 in COS-1 cells stimulated with EGF for the indicated time points. Bar: 10 µm.(d) Colocalzation of internalized SOS1 with the Rab5 endosomal marker in COS-1 cells stimulated for 25 minutes with EGF. Images shown are representative of colocalization pattern observed in >75% of the cells in 3 independent experiments (25 cells analyzed per experiment). The enlarged inset in the merge is also plotted in Supplementary Figure 7d as separate image channels. Bar: 10 µm.(e) Kinetics of SOS localization to endocytic vesicles in EGF stimulated COS-1 cells. EGFP-tagged SOSFL is compared to a full length SOS1 molecule with a functionally impaired allosteric pocket (SOSFL-L687E, R688A). Representative images accompany the bar graph. The results represent an average of two independent experiments (25 cells counted per condition for each experiment).Bar: 10 µm.Source data for plots and graphs are available online.
Mentions: Over time, the initially scattered BCR clusters concatenated and moved toward the center of the synapses formed between the B cells and the SLB. About 15–20 minutes after cell landing, a large central cluster appeared, a phenomenon commonly referred to as ‘BCR capping’56 (Fig. 6c and Supplementary Movie 1). SOSFL initially moved with the activated BCR, but at later time points we found that it was depleted from the central BCR cluster (Fig. 6d,e). Thus, SOSFL leaves the plasma membrane at the site of the central BCR cluster, which also correlates with attenuation of SOSFL-driven Ras-ERK signaling at later time points (Fig. 4g). Confocal fluorescence microscopy revealed the appearance of punctate SOS structures located inside the cells, reminiscent of endocytic vesicles (Fig. 7a). Moreover, these vesicle-like structures appeared only for SOSFL but not for SOSHDPC or the chimeric SOSHDPC–SH2 and only on bilayers displaying the BCR activating antibody (Fig. 7a,b). These observations suggest that removal of SOS1 from the membrane in a BCR signal dependent process requires the C-terminus.

View Article: PubMed Central - PubMed

ABSTRACT

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis.

No MeSH data available.


Related in: MedlinePlus