Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

doi:10.1016/j.molcel.2022.02.005

. 2022 Mar 17;82(6):1089-1106.e12.

doi: 10.1016/j.molcel.2022.02.005. Epub 2022 Feb 28.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

Affiliations

¹ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: c.c.lin@leeds.ac.uk.
² School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
³ School of Mathematics, University of Leeds, Leeds LS2 9JT, UK.
⁴ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
⁵ School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK.
⁶ Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London WC1E 6BT, UK; Francis Crick Institute, London NW1 1AT, UK.
⁷ Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
⁸ Department of Molecular and Cellular Oncology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA.
⁹ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: j.e.ladbury@leeds.ac.uk.

PMID: 35231400
PMCID: PMC8937303
DOI: 10.1016/j.molcel.2022.02.005

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

Chi-Chuan Lin et al. Mol Cell. 2022.

. 2022 Mar 17;82(6):1089-1106.e12.

doi: 10.1016/j.molcel.2022.02.005. Epub 2022 Feb 28.

Authors

Affiliations

¹ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: c.c.lin@leeds.ac.uk.
² School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
³ School of Mathematics, University of Leeds, Leeds LS2 9JT, UK.
⁴ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
⁵ School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK.
⁶ Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London WC1E 6BT, UK; Francis Crick Institute, London NW1 1AT, UK.
⁷ Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
⁸ Department of Molecular and Cellular Oncology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA.
⁹ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: j.e.ladbury@leeds.ac.uk.

PMID: 35231400
PMCID: PMC8937303
DOI: 10.1016/j.molcel.2022.02.005

Abstract

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Keywords: FGFR2; Liquid-liquid phase separation (LLPS); Plcγ1; Receptor tyrosine kinases (RTKs); Shp2; kinase activity; phosphatase activity; phospholipase activity.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1**
Phosphorylated RTK-mediated condensation of protein complexes (A) (Above) Images of recombinant phosphorylated receptors from the EGFR, FGFR, and VEGFR families (Atto-488 labeled) droplet formation upon adding SHP2_C459S (top panel) or SHC (middle panel); phosphorylated RTK proteins alone do not form droplets (third panel). Concentrations of each RTK-SHP2_C459S or SHC pair were shown (x axis: RTK concentration; y axis: SHP2_C459S or SHC concentration) and scale bars, 10 μm. (Below) Schematic diagram with residue numbers shows the defined boundaries of RTK intracellular regions of SHP2, SHC, and PLCγ1 proteins and polypeptides used in this study. (B) Phase diagrams of phosphorylated EGFR, FGFR, and VEGFR family proteins (Atto-488 labeled) with concentrations shown in x axis and SHP2_C459S (y axis) in 20 mM HEPES (pH7.5), 150 mM NaCl, and 1 mM TCEP. The sizes of the circles represent the average sizes of droplets (μm²), and the color scale bars represent the number of droplets in a 0.0256-mm² area.

**Figure 2**
The dynamic LLPS properties of phosphorylated pFGFR2_Cyto-SHP2_C459S-pPLCγ1 condensates (A) The dynamic LLPS properties of pFGFR2_Cyto (10 μM)-SHP2_C459S (60 μM) condensates was assessed by the fusion experiment. Images were taken every 5 min. Scale bars, 100 μm. (B) Quantification of FRAP data (means ± SD, n = 2 experiments) for pFGFR2_Cyto (10 μM)-SHP2_C459S (60 μM) condensates. (C) The dynamic LLPS property of full-length SHP2_C459S-RFP condensates coexpressed with FGFR2_ΔVT and stimulated with 10 ng/ml of FGF9 ligand in HEK293T cells. Images were taken every 30 s. Scale bars, 250 nm. (D) Droplet formation observed between pFGFR2_Cyto Atto-488 (10 μM), SHP2_C459S Atto-550 (60 μM), and pPLCγ1 Atto-647 (12 μM). (i) Individual proteins showed no evidence of droplet formation. Droplet formation was observed after 1 min between different combinations of proteins: (ii) all three proteins; (iii) pFGFR2_Cyto with SHP2_C459S, and (v) SHP2_C459S with pPLCγ1. (iv) No droplet formation was observed for pFGFR2_Cyto with pPLCγ1. Droplet size was diminished with increasing concentrations of NaCl after incubation for 1 min (vi: 250 mM, vii: 500 mM) compared with (ii) and in the presence of 10% 1, 6-hexanediol for 1 min (viii). Addition of 20 μM of lipoamide (ix) or lipoic acid (x) (in 0.1% DMSO) also reduces droplet numbers and sizes while 0.1% DMSO has negligible effect. Scale bars, 10 μm. (E) The dynamic LLPS properties of pFGFR2_Cyto Atto-488 (10 μM)-SHP2_C459S Atto-550 (60 μM)-pPLCγ1 Atto-647 (12 μM) condensates was assessed by the fusion experiment. Images were taken every 2 min. Scale bars, 10 μm.

**Figure 3**
The formation of LLPS pFGFR2-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers and plasma membranes (A) pFGFR2_Cyto-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers. (i) Confocal images of homogeneously distributed pFGFR2_Cyto Atto-488 (20 μM, 6xHis tagged) on membrane bilayers, (ii) pFGFR2_Cyto Atto-488 gradually clustered upon the addition of SHP2_C459S Atto-594 (60 μM, untagged), and (iii) pPLCγ1 Atto-647 (6 μM, untagged), followed by (iv) additional 36 μM of untagged pPLCγ1 Atto-647. Scale bars, 10 μm. (B) FRAP analysis showing the dynamic nature of pFGFR2_Cyto-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers as all pFGFR2_Cyto, SHP2_C459S, and pPLCγ1 exchanged with their counterparts in the dilute phase. Data are presented as mean ± SD, n = 2 experiments. (C) Immunofluorescence staining images showing colocalized SHP2-Alexa 488 and PLCγ1-Alexa 647 droplet formation on plasma membrane in FGF9-stimulated (10 ng/ml, 15 min) Caco-2 cells and Caco-2 FGFR2i cells. Inset image: magnification of regions shown to exemplify endogenous SHP2-PLCγ1 clusters on membranes. Graph (right of image): statistical analysis of droplet formation in parental Caco-2 cells and Caco-2 FGFR2i cells. Only the SHP2-Alexa 488 and PLCγ1-Alexa 647 colocalized droplets were counted. Knocking down FGFR2 reduces SHP2-Alexa 488 and PLCγ1-Alexa 647 colocalized droplets. Degree of colocalization of endogenous PLCγ1 and SHP2 determined by Pearson's R value. Sample numbers = 37 (wild type) or 38 (FGFR2i) from 2 independent experiments. (D) Live cell images showing FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation on plasma membrane upon FGFR2_ΔVT expression and activation in HEK293T SHP2 KO cells. FGFR2_ΔVT, SHP2_C459, and PLCγ1 were tagged with Neptune 2.5, mOrange, and mEGFP, respectively. Alexa 350-conjugated wheat germ agglutinin was used to stain the plasma membrane. (i) Serum-starved (-FGF9) cells show a low level of FGFR2_ΔVT-SHP2_C459S-PLCγ1 droplets colocalized on membrane; this could have been due to protein recruitment by the basally activated FGFR2. Most SHP2 and PLCγ1 proteins are diffused in cytosol. (ii) FGF9-stimulation (+FGF9, 10 ng/ml for 15 min) led to the activation of FGFR2 and enhanced FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation on the plasma membrane (see also Video S1). (iii) Expression of fluorescent tags alone does not initiate the droplet formation. (iv) In the absence of SHP2_C459S-mOrange and PLCγ1-mEGFP, activated FGFR2_ΔVT still forms droplets on the membrane with other endogenous cellular proteins. (v) SHP2_C459S-mOrange does not form droplets in the absence of FGFR2_ΔVT. (vi) PLCγ1-mEGFP does not form droplets in the absence of FGFR2_ΔVT. (vii) FGFR2_ΔVT cannot recruit active PLCγ1-mEGFP to the membrane in the absence of SHP2 expression, resulting in the random diffusion of PLCγ1-mEGFP. (E) Statistical analysis of FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation in the absence (light peach) or presence (dark peach) of FGF9 stimulation. Sample numbers = 40 per condition from 3 independent experiments. Data are presented as mean ± SD.

**Figure 4**
Characterization of the interactions between FGFR2_ΔVT-SHP2_C459S droplets (A) (Left) Pull-down experiments using GST-SHP2_C459S or GST-SHP2_2SH2 (see schematic in Figure 1A) show that the binding of SHP2 requires phosphorylation of FGFR2_ΔVT. FGFR2_ΔVT or FGFR2_ΔVT-KD (double mutant Y656/657F) stably expressing HEK293T cells were unstimulated or FGF9-stimulated (10 ng/ml, 15 min). Arrows highlight GST fusion as part of the SHP2 constructs. The lower level of interaction without FGF9 stimulation due to protein recruitment by the basally activated FGFR2 as shown in the pFGFR2 blot (Input). (Right) Densitometry analysis of GST pull down, n = 3. Data are presented as mean ± SD. Replicate data are shown in Data S1A. (B) (Left) Pull-down experiments using different SHP2 constructs (see schematic in Figure 1A). GST-SHP2, GST-SHP2_C459S, and GST-SHP2_2SH2 pull down pFGFR2_Cyto and pFGFR2_C58 while pFGFR2_kinase was pulled down at a significantly lower level. Red arrows: input of recombinant pFGFR2_Cyto, pFGFR2_kinase, and pFGFR2_C58. Long exposure was required to observe pFGFR2_C58. Black arrows: fusion protein loading control indicating different SHP2 constructs. (Right) Densitometry analysis of GST pull down, n = 2. Data are presented as mean ± SD. Replicate data are shown in Data S1B. (C) (Left) C-terminal 58 residues of FGFR2, GST-FGFR2_C58 with individual Y to F substitutions, were phosphorylated and used to pull down recombinant SHP2_2SH2. The Y769F mutation abrogates binding. (Right) Densitometry analysis of GST pull-down level (salmon) and phosphorylation level (green) of different GST-FGFR2_C58 Y/F mutants, n = 4. Data are presented as mean ± SD. Replicate data are shown in Data S1C. (D) (Left) Pull-down experiment using GST-SHP2_C459S. FGFR2_ΔVT and FGFR2_ΔVT-Y769F were transfected into HEK293T cells: unstimulated or FGF9-stimulated for 5 min, 15 min, or 60 min. GST-SHP2_C459S binds to FGFR2_ΔVT, but not to FGFR2_ΔVT-Y769F, confirming that the interaction is mediated by pY769. The lower level of interaction in the absence of FGF9 stimulation is due to protein recruitment by the basally activated FGFR2 as shown in the pFGFR2 blot (Input). (Right) Densitometry analysis of GST pull down, n = 3. Data are presented as mean ± SD. Replicate data are shown in Data S1D.

**Figure 5**
Interactions between SHP2 and PLCγ1 droplets and the formation of ternary complexes (A) Plot of the chemical shift changes (ppm) of the backbone amide peaks of ¹H, ¹⁵N-labeled PLCγ1_2SH2 (200 μM) upon the addition of 3 mol L⁻¹ equivalent of SHP2_2SH2. The residue numbers are indicated on the x axis. (B) CSP of residues mapped on to the crystal structure of the PLCγ1_2SH2 (PDB code: 4FBN). The gradient indicates the strength of the perturbation. The pY binding pockets for NSH2 and CSH2 are shown in cyan (R562, R586, S588, E589, T590, and T596) and green (R675, R694, R696 and A703), respectively. Left hand image shows putative binding region (highlighted by increasing CSP). Right hand image shows the structure rotated into plane by 180° to show the comparatively negligible CSP on the ‘non-binding’ surface. (C) Plot of the chemical shift changes (ppm) of the backbone amide peaks of ¹H, ¹⁵N-labeled SHP2_2SH2 (100 μM) upon the addition of 6 mol L⁻¹ equivalent of PLCγ1_2SH2. The residue numbers are indicated on the x axis. (D) CSP of residues mapped on to the crystal structure of the SHP2_2SH2 (PDB code: 2SHP). The gradient indicates the strength of the perturbation. The critical pY binding residue for NSH2 and CSH2 are shown in cyan (R32) and green (R138), respectively. Left hand image shows mild CSP on the “non-binding” surface. Right hand image shows the structure rotated into plane by 180° to show the putative binding region (highlighted by increasing CSP). (E) Formation of ternary complex revealed by GST pull-down assay. Upper panel blot: unphosphorylated GST-FGFR2_Cyto does not interact with SHP2_2SH2 (lane 4), PLCγ1_2SH2 (lane 5), or pPLCγ1_2SH2 (lane 6). Phosphorylated GST-pFGFR2_Cyto can precipitate SHP2_2SH2 (lane 7), PLCγ1_2SH2 (Lane 8), and coprecipitated SHP2_2SH2 and PLCγ1_2SH2 (lane 9). The addition of ATP/MgCl₂ (5 mM) does not affect GST-pFGFR2_Cyto precipitating SHP2_2SH2 (lane 10), whereas the phosphorylation of PLCγ1_2SH2 (pPLCγ1_2SH2) by GST-pFGFR2_Cyto abolishes the interaction (lane 11). However, pPLCγ1_2SH2 can be precipitated by GST-pFGFR2_Cyto when SHP2_2SH2 is present (lane 12), suggesting an adaptor function of SHP2_2SH2 in the ternary complex formation. Second panel blot: the phosphorylation state of PLCγ1_2SH2. Third panel blot: the phosphorylation state of FGFR2_Cyto. Lower panel blot: total GST-FGFR2_Cyto protein loading control. The figure represents 3 independent experiments. Replicate data are shown in Data S2A. (F) The ternary complex was constituted using BLI. Upper panel: GST-pFGFR2_Cyto captured on an anti-GST sensor was exposed sequentially to excess SHP2_2SH2 (200 μM, 0 s) and pPLCγ1_2SH2 (200 μM, 240 s) in the presence of 5 mM ATP/Mg²⁺. After each binding equilibrium was reached, the sensor was washed (120 s and 360 s). Lower panel: GST-pPLCγ1_2SH2 was captured on an anti-GST sensor. Then sequential binding of SHP2_2SH2 (200 μM, 0 s) and pFGFR2_Cyto (200 μM, 240 s) was measured in the presence of 5 mM ATP/Mg²⁺. Dotted red lines mark the equilibrium binding of each complex. Figures are representative sensorgrams from 3 independent experiments. (G) BiFC was used to study the formation of ternary complex. Inset: schematic depicting the interaction: pFGFR2_Cyto (blue) and pPLCγ1_2SH2 (yellow) with split CFP tag (green). 100 nM of CN173-pFGFR2_Cyto and CC173-pPLCγ1_2SH2 were used for the assay. The addition of increasing concentration (x axis on graph) of SHP2_2SH2 (purple) produces fluorescent signal (cyan), n = 3. Data are presented as mean ± SD. (H) Time evolution of the ternary complexes simulated from the deterministic mathematical model; out of the four possible ternary complexes (pF·S·P, pF·P·S, pF·S·pP, and pF·S·pP), only pF·S·pP prevails. F, FGFR2_Cyto; S, SHP2_2SH2; P, PLCγ1_2SH2.

**Figure 6**
Characterization of the ternary complex formation (A) (i) *In vitro* phase separation assay using Atto-labeled pFGFR2_Cyto (10 μM) and truncated SHP2_2SH2 (30 μM). The addition of a pY769 peptide (ii) or a general pY peptide (ii) to compete SH2 domain binding reduces droplet formation. Scale bars, 10 μm. (B) R to A mutation of residues 32 or/and 138 in the pY binding sites show that both wild-type SH2 domains of SHP2 are required (30 μM of each mutant) for LLPS with pFGFR2_Cyto (10 μM). (i) Wild-type SHP2_2SH2. (ii) SHP2_{2SH2 R32A}. (iii) SHP2_{2SH2 R138A}. (iv) SHP2_{2SH2 R32/138A}. Scale bars, 10 μm. (C) *In vitro* phase separation assay using Atto-labeled pFGFR2_Cyto (10 μM), SHP2_2SH2 (30 μM), and pPLCγ1_2SH2 (12 μM). (i) Individual proteins showed no evidence of droplet formation. Droplet formation was observed after 1 min: (ii) with all three proteins; (iii) with pFGFR2_Cyto and SHP2_2SH2, not with pFGFR2_Cyto with pPLCγ1_2SH2 and (iv) with SHP2_2SH2 with pPLCγ1_2SH2. (v) Droplet size was diminished with increasing concentration of NaCl after incubation for 1 min and (vi) (250 mM compared with 150 mM in (ii)) in the presence of 10% 1, 6-hexanediol for 1 min (vii). Scale bars, 10 μm. (D) (Top panel) An excess of inactive FGFR2_{Cyto K517I} (FGFR2_{Cyto K517I} [2000 μM]:pFGFR2_Cyto [10 μM] = 200:1) was used as the substrate to monitor kinase activity. 500 μM of ATP/MgCl₂ was added and incubated at room temperature for 15 min. In the context of phase-separated droplets (by adding SHP2_C459S [60 μM] and pPLCγ1 [12 μM]; lane 5), the kinase activity of pFGFR2_Cyto was enhanced (compare lanes 3 and 5). The addition of 10% 1,6-hexanediol (lane 9) results in the reduction of kinase activity by dissolving the phase-separated droplets (compare lane 5 and lane 9). (Bottom panel) Densitometry analysis of kinase assay, n = 5. Data are presented as mean ± SD. Replicate data are shown in Data S2C. (E) (Top panel) A synthesized GST-phospho-substrate (600 μM, lane 1) was used to measure SHP2 (60 μM) activity in the context of phase-separated droplets. Upon the addition of FGFR2_Cyto and pPLCγ1 (10 μM and 12 μM, respectively) and incubation for 15 min, the SHP2 activity is reduced in the droplets compared with the isolated phosphatase (compare lane 2 and lane 4). The addition of 10% 1,6-hexanediol (lane 5) results in the upregulation of activity by dissolving the droplets (compare lane 4 and lane 5). (Bottom panel) Densitometry analysis of phosphatase assay, n = 2. Data are presented as mean ± SD. Replicate data are shown in Data S2C. (F) Confocal images of the effect of external phosphatase CIP (10 μM) on pFGFR2_Cyto-SHP2_C459S-pPLCγ1 LLPS formation. Top panel: droplet formation without CIP. Middle panel: the addition of CIP after droplet formation has limited effect on the dephosphorylation of proteins, hence droplets are still present. Lower panel: the addition of CIP to pFGFR2_Cyto before droplet formation (before the addition of SHP2_C459S and pPLCγ1) efficiently dephosphorylates pFGFR2_Cyto; therefore, no droplet can form at the unphosphorylated state. Western blots (below left) confirmed that pFGFR2_Cyto-SHP2_C459S-pPLCγ1 (10 μM, 60 μM, and 12 μM, respectively) droplet formation prevents the dephosphorylation of pPLCγ1 and pFGFR2_Cyto by 10 μM of CIP (exposure 0.5 h). (Below right) Densitometry analysis of the phosphorylation state of pPLCγ1 (salmon) and pFGFR2_Cyto (dark green), n = 2. Data are presented as mean ± SD. Replicate data are shown in Data S2D. (G) The lipase activity of PLCγ1 (50 μM) was dramatically enhanced in the phase-separated environment (by adding pFGFR2_Cyto [10μM] and SHP2_C459S [60 μM]; green curve) compared with PLCγ1 alone (magenta curve). Sample sizes n = 4. Data are presented as mean ± SD.

**Figure 7**
Phase transition of FGFR2-SHP2-PLCγ1 upregulates downstream signaling (A) (Left) Depletion of SHP2 upregulates PLCγ1 through phosphorylation of Y783 but downregulates its downstream effectors (shown by reduced phosphorylation of PKCβII-S660 and AKT-S473) in FGF9-stimulated (10 ng/ml) MCF7 cells and A431 cells. (Right) Densitometry analysis of SHP2 expression and the activation levels of various signaling proteins (dark green: parental cells; light green: SHP2 depletion cells). MCF7 cells: n = 3; A431 cells: n = 2. Data are presented as mean ± SD. Replicate data are shown in Data S3A. (B) Inhibition of calcium response in MCF7 SHP2 KO cells (sample size = 8) and A431 SHP2i cells (sample size = 16) (light cyan) upon FGF9 stimulation (10 ng/ml) for 1 h compared with the parental cells (dark cyan). (C) (Left) Cell motility is reduced in the SHP2 depletion cells upon FGF9 stimulation (10 ng/ml) (MCF7 SHP2 KO cells and A431 SHP2i cells) compared with control the parental cells. (Right) Graphical representation of percentage recovery; (dark cyan) parental cells, (light cyan) SHP2 depletion cells (n = 3). (D) (Left) Knockin SHP2 constructs restore PKCβII activity upon FGF9 stimulation (10 ng/ml) in A431 SHP2i cells. Knockin wild-type SHP2 has lower effect on restoring PKCβII activity, which could have been due to the rapid phospho-turnover mediated by overexpressed SHP2. Both SHP2_C459S and SHP2_2SH2 greatly increase PKCβII activity, indicating that the phosphatase activity is dispensable and only the tandem SH2 domains are required. (Right) Densitometry analysis of PKCβII activity upon knock in of various SHP2 constructs (dark cyan: parental cells; light cyan: SHP2 depletion cells), n = 3. Data are presented as mean ± SD. Replicate data are shown in Data S3B. (E) (Top) Mutations of the PLCγ1 binding interface residues on SHP2 tandem SH2 domains (SHP2_{C459S NMR}) abolish the ability of SHP2_C459S to restore PKCβII activity in MCF7 SHP2 KO cells. (Bottom) Densitometry analysis of PKCβII activity upon knockin of various SHP2 constructs (dark cyan: parental cells; light cyan: SHP2 depleted cells), n = 3. Data are presented as mean ± SD. Replicate data are shown in Data S4C. (F) Schematic diagrams of the domains of FGFR2_Cyto, SHP2, and PLCγ1. (i) Upon ligand stimulation (orange), membrane-localized FGFR2 (blue) recruits the NSH2 domain of PLCγ1 (green) into pY769 on its C terminus. This results in the phosphorylation (pY783), activation dissociation from FGFR2 of PLCγ1. FGFR2 can also recruit SHP2 (purple) CSH2 domain into pY769 (ii). The FGFR2-SHP2 complex is then available to recruit the active PLCγ1 through the tandem SH2 domains of SHP2 and PLCγ1. The “secondary interaction” mediated by the SHP2 NSH2 domain and pYs on the FGFR2 kinase domain further provide the multivalency for the phase separation of the ternary complexes on cellular membrane (iii).

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Comment in

In unity, there is strength: Phase separation controls receptor tyrosine kinase signal transduction.
Jimenez Salinas A, Lee YK. Jimenez Salinas A, et al. Mol Cell. 2022 Mar 17;82(6):1081-1083. doi: 10.1016/j.molcel.2022.03.003. Mol Cell. 2022. PMID: 35303480

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[1] Agazie Y.M., Hayman M.J. Development of an efficient “substrate-trapping” mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates. J. Biol. Chem. 2003;278:13952–13958. - PubMed

[2] Agazie Y.M., Hayman M.J. Development of an efficient “substrate-trapping” mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates. J. Biol. Chem. 2003;278:13952–13958. - PubMed

[3] Ahmed Z., Lin C.C., Suen K.M., Melo F.A., Levitt J.A., Suhling K., Ladbury J.E. Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and SHP2 phosphatase activity. J. Cell Biol. 2013;200:493–504. - PMC - PubMed

[4] Ahmed Z., Lin C.C., Suen K.M., Melo F.A., Levitt J.A., Suhling K., Ladbury J.E. Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and SHP2 phosphatase activity. J. Cell Biol. 2013;200:493–504. - PMC - PubMed

[5] Alberti S., Gladfelter A., Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell. 2019;176:419–434. - PMC - PubMed

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Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

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