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Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

Ahmed Z, Timsah Z, Suen KM, Cook NP, Lee GR, Lin CC, Gagea M, Marti AA, Ladbury JE - Nat Commun (2015)

Bottom Line: Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Center for Biomolecular Structure and Function, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

No MeSH data available.


Related in: MedlinePlus

Binding of phosphotyrosine by Grb2-SH2 domain leads to dissociation.smFLIM shows Grb2 dimerization is disrupted upon phospho-Shc peptide binding by its SH2 domain. GFP-tagged Grb2 immobilized on GFP-trap beads were imaged in 20 mM Tris-HCl, 50 mM NaCl at pH 8.0. The reference average lifetime in the absence of acceptor is 1.6 ns. RFP–Grb2 was added and allowed to form complex for 1 h and the lifetime of GFP–Grb2 shortens as indicated by left-shifted peak with average lifetime centred on 1.5 ns clearly indicating that FRET has occurred. Phospho-Shc peptide (10 μM) containing the pYxN motif was then added, allowed to equilibrate for an hour and the fluorescence lifetime was measured. Addition of the phospho-Shc peptide restores the lifetime to the control values obtained in the absence of acceptor. This clearly shows that the phospho-Shc peptide disrupts dimerization of Grb2. The zoomed image of the bead (right hand column) shows lifetime values mapped to a false colour image. The change in fluorescence lifetime as a function of colour is highly noticeable. The data presented here was consistently reproduced in three independent experiments. Scale bars, 10 μm.
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f3: Binding of phosphotyrosine by Grb2-SH2 domain leads to dissociation.smFLIM shows Grb2 dimerization is disrupted upon phospho-Shc peptide binding by its SH2 domain. GFP-tagged Grb2 immobilized on GFP-trap beads were imaged in 20 mM Tris-HCl, 50 mM NaCl at pH 8.0. The reference average lifetime in the absence of acceptor is 1.6 ns. RFP–Grb2 was added and allowed to form complex for 1 h and the lifetime of GFP–Grb2 shortens as indicated by left-shifted peak with average lifetime centred on 1.5 ns clearly indicating that FRET has occurred. Phospho-Shc peptide (10 μM) containing the pYxN motif was then added, allowed to equilibrate for an hour and the fluorescence lifetime was measured. Addition of the phospho-Shc peptide restores the lifetime to the control values obtained in the absence of acceptor. This clearly shows that the phospho-Shc peptide disrupts dimerization of Grb2. The zoomed image of the bead (right hand column) shows lifetime values mapped to a false colour image. The change in fluorescence lifetime as a function of colour is highly noticeable. The data presented here was consistently reproduced in three independent experiments. Scale bars, 10 μm.

Mentions: From the reported crystal structure13 Y160 appears to form a hydrogen bond with E87 of the partner protomer (Fig. 1a). Since E87 lies within the conserved FLI/VRE motif of the SH2 domain (which is involved in recognition of phosphorylated tyrosine residues), the insertion of a tyrosylphosphate into the binding pocket on the SH2 domain might be expected to perturb the side chain position of E87 and disrupt the interaction between Y160 and E87. Using the previously described smFLIM method we determined that the addition of a tyrosylphosphopeptide based on the sequence of the known Grb2 ligand Shc (pShc) leads to dissociation of Grb2 dimers into the monomeric form (mGrb2) (Fig. 3). The addition of a proline-rich peptide derived from SOS which binds to the SH3 domains of Grb2 had no effect on dimerization on the surface of the Talon beads (Supplementary Fig. 4a). We confirmed this observation by measuring the monomer–dimer equilibrium of Grb2 using MST. Since Grb2 is monomeric, in the presence of a saturating concentration of pShc no apparent change in the oligomeric state of the protein was observed (Supplementary Fig. 4b). As expected similar experiments in the presence of the SOS1 peptide, Grb2 undergoes a dimer-to-monomer dissociation which has an identical apparent Kd (∼0.7 μM) to wild-type protein in the absence of peptide binding (Supplementary Fig. 4c). These results are consistent with previously reported NMR data where Grb2 was found to be monomeric in the presence of a phosphopeptide22.


Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

Ahmed Z, Timsah Z, Suen KM, Cook NP, Lee GR, Lin CC, Gagea M, Marti AA, Ladbury JE - Nat Commun (2015)

Binding of phosphotyrosine by Grb2-SH2 domain leads to dissociation.smFLIM shows Grb2 dimerization is disrupted upon phospho-Shc peptide binding by its SH2 domain. GFP-tagged Grb2 immobilized on GFP-trap beads were imaged in 20 mM Tris-HCl, 50 mM NaCl at pH 8.0. The reference average lifetime in the absence of acceptor is 1.6 ns. RFP–Grb2 was added and allowed to form complex for 1 h and the lifetime of GFP–Grb2 shortens as indicated by left-shifted peak with average lifetime centred on 1.5 ns clearly indicating that FRET has occurred. Phospho-Shc peptide (10 μM) containing the pYxN motif was then added, allowed to equilibrate for an hour and the fluorescence lifetime was measured. Addition of the phospho-Shc peptide restores the lifetime to the control values obtained in the absence of acceptor. This clearly shows that the phospho-Shc peptide disrupts dimerization of Grb2. The zoomed image of the bead (right hand column) shows lifetime values mapped to a false colour image. The change in fluorescence lifetime as a function of colour is highly noticeable. The data presented here was consistently reproduced in three independent experiments. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491180&req=5

f3: Binding of phosphotyrosine by Grb2-SH2 domain leads to dissociation.smFLIM shows Grb2 dimerization is disrupted upon phospho-Shc peptide binding by its SH2 domain. GFP-tagged Grb2 immobilized on GFP-trap beads were imaged in 20 mM Tris-HCl, 50 mM NaCl at pH 8.0. The reference average lifetime in the absence of acceptor is 1.6 ns. RFP–Grb2 was added and allowed to form complex for 1 h and the lifetime of GFP–Grb2 shortens as indicated by left-shifted peak with average lifetime centred on 1.5 ns clearly indicating that FRET has occurred. Phospho-Shc peptide (10 μM) containing the pYxN motif was then added, allowed to equilibrate for an hour and the fluorescence lifetime was measured. Addition of the phospho-Shc peptide restores the lifetime to the control values obtained in the absence of acceptor. This clearly shows that the phospho-Shc peptide disrupts dimerization of Grb2. The zoomed image of the bead (right hand column) shows lifetime values mapped to a false colour image. The change in fluorescence lifetime as a function of colour is highly noticeable. The data presented here was consistently reproduced in three independent experiments. Scale bars, 10 μm.
Mentions: From the reported crystal structure13 Y160 appears to form a hydrogen bond with E87 of the partner protomer (Fig. 1a). Since E87 lies within the conserved FLI/VRE motif of the SH2 domain (which is involved in recognition of phosphorylated tyrosine residues), the insertion of a tyrosylphosphate into the binding pocket on the SH2 domain might be expected to perturb the side chain position of E87 and disrupt the interaction between Y160 and E87. Using the previously described smFLIM method we determined that the addition of a tyrosylphosphopeptide based on the sequence of the known Grb2 ligand Shc (pShc) leads to dissociation of Grb2 dimers into the monomeric form (mGrb2) (Fig. 3). The addition of a proline-rich peptide derived from SOS which binds to the SH3 domains of Grb2 had no effect on dimerization on the surface of the Talon beads (Supplementary Fig. 4a). We confirmed this observation by measuring the monomer–dimer equilibrium of Grb2 using MST. Since Grb2 is monomeric, in the presence of a saturating concentration of pShc no apparent change in the oligomeric state of the protein was observed (Supplementary Fig. 4b). As expected similar experiments in the presence of the SOS1 peptide, Grb2 undergoes a dimer-to-monomer dissociation which has an identical apparent Kd (∼0.7 μM) to wild-type protein in the absence of peptide binding (Supplementary Fig. 4c). These results are consistent with previously reported NMR data where Grb2 was found to be monomeric in the presence of a phosphopeptide22.

Bottom Line: Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Center for Biomolecular Structure and Function, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

No MeSH data available.


Related in: MedlinePlus