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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.


The catalytic core of SOS is stably and functionally recruited to Ras-decorated supported lipid bilayers in vitro, independently of Grb2 and lipid binding domains(a) The domain architecture of full-length (FL) human SOS1 (hSOS1). The catalytic unit (Cat) is depicted together with the flanking regulatory domains. Yellow boxes in the C-terminal proline rich (PR) domain indicate PxxP motifs known to interact with Grb2.(b) Classical model of SOS-Ras-ERK signal transduction pathway. In the shown example, SOS is recruited to the plasma membrane downstream of activated B cell receptor via binding of Grb2 to phosphotyrosine motifs on the adaptor protein LAB.(c) Single SOS activity assay based on micro-patterned Ras functionalized fluid supported lipid bilayers.(d) Representative overlay image of fluorescent GDP bound to Ras (red channel) and membrane-recruited SOSCat (green channel) in the single molecule assay depicted in c. Membrane corrals where individual copies of SOSCat were recruited have depleted signal and appear darker in the GDP channel, demonstrating highly processive SOSCat activity (i.e., recruited SOS activates Ras in a sustained manner without dissociating from the membrane surface). The particular experiment was repeated five times.
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Figure 1: The catalytic core of SOS is stably and functionally recruited to Ras-decorated supported lipid bilayers in vitro, independently of Grb2 and lipid binding domains(a) The domain architecture of full-length (FL) human SOS1 (hSOS1). The catalytic unit (Cat) is depicted together with the flanking regulatory domains. Yellow boxes in the C-terminal proline rich (PR) domain indicate PxxP motifs known to interact with Grb2.(b) Classical model of SOS-Ras-ERK signal transduction pathway. In the shown example, SOS is recruited to the plasma membrane downstream of activated B cell receptor via binding of Grb2 to phosphotyrosine motifs on the adaptor protein LAB.(c) Single SOS activity assay based on micro-patterned Ras functionalized fluid supported lipid bilayers.(d) Representative overlay image of fluorescent GDP bound to Ras (red channel) and membrane-recruited SOSCat (green channel) in the single molecule assay depicted in c. Membrane corrals where individual copies of SOSCat were recruited have depleted signal and appear darker in the GDP channel, demonstrating highly processive SOSCat activity (i.e., recruited SOS activates Ras in a sustained manner without dissociating from the membrane surface). The particular experiment was repeated five times.

Mentions: Receptor-triggered activation of SOS is a multilayered process involving membrane recruitment, release of autoinhibition, and allosteric modulation by Ras. The initial membrane recruitment of SOS is thought to occur via association of PxxP motifs in the C-terminal proline-rich (PR) domain with Grb2, which in turn binds phospho-tyrosine motifs on activated receptors or transmembrane adaptor proteins6,7,10,16–21. SOS additionally contains a series of N-terminal domains with homology to Dbl (DH) and Pleckstrin (PH) as well as a Histone Fold (HF) domain (Fig. 1a), which can autoinhibit SOS activity when assayed in solution. On membranes, this autoinhibition is released through interactions with various membrane lipids22–24 (reviewed in ref.9). Full activation of SOS is contingent on binding of Ras to an allosteric pocket situated at the rim of the REM and CDC25 domains25. The REM and CDC25 domains in SOS1 together form the catalytic core, which we term SOSCat throughout the manuscript (Fig. 1a). Mutations in SOS1 that perturb these regulatory functions result in altered signaling behavior and have been implicated in developmental disorders such as Noonan26, Costello and CFC-syndrome27. SOS2 has a very similar domain make-up, but appears somewhat redundant to SOS1 in cells13; in this study we solely focus on SOS1.


One-way membrane trafficking of SOS in receptor-triggered Ras activation
The catalytic core of SOS is stably and functionally recruited to Ras-decorated supported lipid bilayers in vitro, independently of Grb2 and lipid binding domains(a) The domain architecture of full-length (FL) human SOS1 (hSOS1). The catalytic unit (Cat) is depicted together with the flanking regulatory domains. Yellow boxes in the C-terminal proline rich (PR) domain indicate PxxP motifs known to interact with Grb2.(b) Classical model of SOS-Ras-ERK signal transduction pathway. In the shown example, SOS is recruited to the plasma membrane downstream of activated B cell receptor via binding of Grb2 to phosphotyrosine motifs on the adaptor protein LAB.(c) Single SOS activity assay based on micro-patterned Ras functionalized fluid supported lipid bilayers.(d) Representative overlay image of fluorescent GDP bound to Ras (red channel) and membrane-recruited SOSCat (green channel) in the single molecule assay depicted in c. Membrane corrals where individual copies of SOSCat were recruited have depleted signal and appear darker in the GDP channel, demonstrating highly processive SOSCat activity (i.e., recruited SOS activates Ras in a sustained manner without dissociating from the membrane surface). The particular experiment was repeated five times.
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Related In: Results  -  Collection

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Figure 1: The catalytic core of SOS is stably and functionally recruited to Ras-decorated supported lipid bilayers in vitro, independently of Grb2 and lipid binding domains(a) The domain architecture of full-length (FL) human SOS1 (hSOS1). The catalytic unit (Cat) is depicted together with the flanking regulatory domains. Yellow boxes in the C-terminal proline rich (PR) domain indicate PxxP motifs known to interact with Grb2.(b) Classical model of SOS-Ras-ERK signal transduction pathway. In the shown example, SOS is recruited to the plasma membrane downstream of activated B cell receptor via binding of Grb2 to phosphotyrosine motifs on the adaptor protein LAB.(c) Single SOS activity assay based on micro-patterned Ras functionalized fluid supported lipid bilayers.(d) Representative overlay image of fluorescent GDP bound to Ras (red channel) and membrane-recruited SOSCat (green channel) in the single molecule assay depicted in c. Membrane corrals where individual copies of SOSCat were recruited have depleted signal and appear darker in the GDP channel, demonstrating highly processive SOSCat activity (i.e., recruited SOS activates Ras in a sustained manner without dissociating from the membrane surface). The particular experiment was repeated five times.
Mentions: Receptor-triggered activation of SOS is a multilayered process involving membrane recruitment, release of autoinhibition, and allosteric modulation by Ras. The initial membrane recruitment of SOS is thought to occur via association of PxxP motifs in the C-terminal proline-rich (PR) domain with Grb2, which in turn binds phospho-tyrosine motifs on activated receptors or transmembrane adaptor proteins6,7,10,16–21. SOS additionally contains a series of N-terminal domains with homology to Dbl (DH) and Pleckstrin (PH) as well as a Histone Fold (HF) domain (Fig. 1a), which can autoinhibit SOS activity when assayed in solution. On membranes, this autoinhibition is released through interactions with various membrane lipids22–24 (reviewed in ref.9). Full activation of SOS is contingent on binding of Ras to an allosteric pocket situated at the rim of the REM and CDC25 domains25. The REM and CDC25 domains in SOS1 together form the catalytic core, which we term SOSCat throughout the manuscript (Fig. 1a). Mutations in SOS1 that perturb these regulatory functions result in altered signaling behavior and have been implicated in developmental disorders such as Noonan26, Costello and CFC-syndrome27. SOS2 has a very similar domain make-up, but appears somewhat redundant to SOS1 in cells13; in this study we solely focus on SOS1.

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.