A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

Lin, Chun–Wei; Nocka, Laura M.; Stinger, Brittany; DeGrandchamp, Joey; Lew, Nugent; Alvarez, Steven; Phan, Henry T.; Kondo, Yasumasa; Kuriyan, John; Groves, Jay T.

doi:10.1101/2021.12.12.472247

Cited by 4 publications

(7 citation statements)

References 63 publications

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“…More recently, we have shown that the Y160E Grb2 mutant is defective in promoting phase separation of scaffold proteins upon phosphorylation (C.‑W. Lin et al, 2021). In the present study, the use of Grb2-Y160E shows a reduction in the PLCγ2-peptide fusion phosphorylation by Btk, compared to the wild-type Grb2.…”

Section: Resultsmentioning

confidence: 99%

“…The second possibility is that Grb2 alone promotes condensation of LAT (C.‑W. Lin et al, 2021), creating small domains of dense LAT, within which Btk cannot diffuse freely. The addition of SOS-PRR along with Grb2 induces the full phase transition of the phosphorylated LAT.…”

Section: Resultsmentioning

confidence: 99%

“…One possibility is that, Grb2 is able to simultaneously bind Btk and LAT, thus slowing Btk molecules through an additional anchor point to the membrane (via the Grb2-LAT complex). The second possibility is that Grb2 alone promotes condensation of LAT (C.-W. Lin et al, 2021), creating small domains of dense LAT, within which Btk cannot diffuse freely. The addition of SOS-PRR along with Grb2 induces the full phase transition of the phosphorylated LAT.…”

Section: Grb2 Can Recruit Btk To Clusters Of Scaffold Proteinsmentioning

confidence: 99%

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Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Nocka

Groves

Kuriyan

2022

Preprint

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The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH22 TH) module connected by a proline-rich linker to a “Src module”, an SH2-SH3-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by hexakisinositol phosphate (IP6) (Wang et al. 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3- bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues – thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Grb2 Can Recruit Btk To Clusters Of Scaffold Proteinsmentioning

confidence: 99%

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Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Nocka

Groves

Kuriyan

2022

Preprint

Self Cite

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“…One possibility is that Grb2 is able to simultaneously bind Btk and LAT, thus slowing Btk molecules through an additional anchor point to the membrane (via the Grb2-LAT complex). The second possibility is that Grb2 alone has promoted small LAT condensates that are not immediately visible by eye ( Lin et al, 2021 ), creating small domains of dense LAT, within which Btk cannot diffuse freely. The addition of SOS-PRR along with Grb2 induces the full phase transition of the phosphorylated LAT ( Figure 5C and Figure 5—figure supplement 1 ).…”

Section: Resultsmentioning

confidence: 99%

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Nocka

Eisen

Iavarone

et al. 2023

eLife

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The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a ‘Src module’, an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by inositol hexakisphosphate (IP6) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues – thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.

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“…[ 11 ] Recently, liquid–liquid phase separation (LLPS) of proteins induced by multivalent protein‐protein interactions emerged as a key principle driving dynamic receptor clustering. [ 15–21 ] Experimentally pinpointing and characterizing the involvement of LLPS in the formation and activation of signaling complexes in the plasma membrane has remained challenging due to the nanoscale dimensions and the high dynamics of such entities.…”

Section: Introductionmentioning

confidence: 99%

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co‐Condensation of Wnt Signalodroplets

Philippi

Richter

Kappen

et al. 2022

Small

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Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of 300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

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A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

Cited by 4 publications

References 63 publications

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co‐Condensation of Wnt Signalodroplets

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