Limits...
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

Grb2 tyrosine phosphorylation disrupts dimerization.FRET between Atto488–Grb2 and Atto550–Grb2 measured with FLIM. The panels on the right show the distribution of the average fluorescence lifetime. The middle image is the representation of the measured lifetime as a false colour map. On the right hand side are zoomed images of a bead with a characteristic imperfection. The mean lifetime of Atto488–Grb2 is centred at ∼1.9 ns. The addition of the acceptor Atto550–Grb2 leads to an apparent left shift in the average lifetime. This is clear from the appearance of a new peak at ∼1.6 ns as a result of FRET between Atto488–Grb2 and Atto550–Grb2. Addition of FGFR2 has no effect on the apparent average lifetime. However, further addition of ATP/MgCl2 reversed the average lifetime to 1.9 ns providing evidence for the disruption of FRET and hence the disruption of dimerization on phosphorylation of Grb2. Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4491180&req=5

f2: Grb2 tyrosine phosphorylation disrupts dimerization.FRET between Atto488–Grb2 and Atto550–Grb2 measured with FLIM. The panels on the right show the distribution of the average fluorescence lifetime. The middle image is the representation of the measured lifetime as a false colour map. On the right hand side are zoomed images of a bead with a characteristic imperfection. The mean lifetime of Atto488–Grb2 is centred at ∼1.9 ns. The addition of the acceptor Atto550–Grb2 leads to an apparent left shift in the average lifetime. This is clear from the appearance of a new peak at ∼1.6 ns as a result of FRET between Atto488–Grb2 and Atto550–Grb2. Addition of FGFR2 has no effect on the apparent average lifetime. However, further addition of ATP/MgCl2 reversed the average lifetime to 1.9 ns providing evidence for the disruption of FRET and hence the disruption of dimerization on phosphorylation of Grb2. Scale bars, 50 μm.

Mentions: To demonstrate that phosphorylation of the wild-type Grb2 disrupts dimerization, we devised a pseudo single molecule FLIM (smFLIM) experiment. Bacterially expressed and purified His-tagged WTGrb2 was N-terminally labelled with Atto488 and immobilized on Talon beads, while non-tagged WTGrb2 was labelled with Atto550. After measurement of the reference lifetime of Atto488–WTGrb2, Atto550–WTGrb2 was added and allowed to form a complex. The fluorescence lifetime population showed a distinct left shift to a shorter value indicating that FRET has occurred (Fig. 2). Incubation for one hour in the presence of purified FGFR2 cytoplasmic domain alone had no effect on the FRET lifetime between the Grb2 protomers. However a further hour of incubation in the presence of ATP and MgCl2 in order to phosphorylate Y160 on Grb2 (ref. 20) resulted in a shift of the average fluorescence lifetime towards the original reference lifetime for Atto488–WTGrb2 alone (Fig. 2), demonstrating that the formation of the Grb2 dimer was abrogated by phosphorylation. These findings clearly demonstrate that the disruption of Atto488–Grb2/Atto550–Grb2 complex is FGFR2 activation dependent.


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)

Grb2 tyrosine phosphorylation disrupts dimerization.FRET between Atto488–Grb2 and Atto550–Grb2 measured with FLIM. The panels on the right show the distribution of the average fluorescence lifetime. The middle image is the representation of the measured lifetime as a false colour map. On the right hand side are zoomed images of a bead with a characteristic imperfection. The mean lifetime of Atto488–Grb2 is centred at ∼1.9 ns. The addition of the acceptor Atto550–Grb2 leads to an apparent left shift in the average lifetime. This is clear from the appearance of a new peak at ∼1.6 ns as a result of FRET between Atto488–Grb2 and Atto550–Grb2. Addition of FGFR2 has no effect on the apparent average lifetime. However, further addition of ATP/MgCl2 reversed the average lifetime to 1.9 ns providing evidence for the disruption of FRET and hence the disruption of dimerization on phosphorylation of Grb2. Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Grb2 tyrosine phosphorylation disrupts dimerization.FRET between Atto488–Grb2 and Atto550–Grb2 measured with FLIM. The panels on the right show the distribution of the average fluorescence lifetime. The middle image is the representation of the measured lifetime as a false colour map. On the right hand side are zoomed images of a bead with a characteristic imperfection. The mean lifetime of Atto488–Grb2 is centred at ∼1.9 ns. The addition of the acceptor Atto550–Grb2 leads to an apparent left shift in the average lifetime. This is clear from the appearance of a new peak at ∼1.6 ns as a result of FRET between Atto488–Grb2 and Atto550–Grb2. Addition of FGFR2 has no effect on the apparent average lifetime. However, further addition of ATP/MgCl2 reversed the average lifetime to 1.9 ns providing evidence for the disruption of FRET and hence the disruption of dimerization on phosphorylation of Grb2. Scale bars, 50 μm.
Mentions: To demonstrate that phosphorylation of the wild-type Grb2 disrupts dimerization, we devised a pseudo single molecule FLIM (smFLIM) experiment. Bacterially expressed and purified His-tagged WTGrb2 was N-terminally labelled with Atto488 and immobilized on Talon beads, while non-tagged WTGrb2 was labelled with Atto550. After measurement of the reference lifetime of Atto488–WTGrb2, Atto550–WTGrb2 was added and allowed to form a complex. The fluorescence lifetime population showed a distinct left shift to a shorter value indicating that FRET has occurred (Fig. 2). Incubation for one hour in the presence of purified FGFR2 cytoplasmic domain alone had no effect on the FRET lifetime between the Grb2 protomers. However a further hour of incubation in the presence of ATP and MgCl2 in order to phosphorylate Y160 on Grb2 (ref. 20) resulted in a shift of the average fluorescence lifetime towards the original reference lifetime for Atto488–WTGrb2 alone (Fig. 2), demonstrating that the formation of the Grb2 dimer was abrogated by phosphorylation. These findings clearly demonstrate that the disruption of Atto488–Grb2/Atto550–Grb2 complex is FGFR2 activation dependent.

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