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

In vitro and in vivoWTGrb2 form dimers.(a) Homodimer with individual Grb2 protomers of the dimer depicted using surface (green) and ribbon (blue/purple) representations (derived from PDB 1GRI). Individual SH2 and SH3 domains of the protomers are differentially coloured and labelled. Zoomed box (top)—Y160 of one protomer (purple) is buried in the dimer interface, packing against the CSH3 domain and hydrogen bonding with E87 of the other protomer chain (green). Zoomed box (bottom)—hydrogen bonded Asn188 (green) and Asn214 (purple) in the dimer interface. (b) Grb2 dimerization measured by MST. Unlabelled WTGrb2 protein (7.3 nM to 30 μM) was titrated into a fixed concentration of labelled Grb2 (100 nM). The data for thermophoresis was recorded at 20 °C using the blue LED at 20% and IR-Laser at 40%. The isotherm derived from the raw data and fitted according to the law of mass action to yield an apparent Kd dimer of 0.76±0.20 μM. (c) The data points representing the MST results of the Y160E mutant, which show a scattered distribution and unsuitable for fitting. (d) The thermophoresis results of the N188/214D mutant, as with Y160E the data is unsuitable for fitting analysis. (e) FLIM of Grb2 dimerization in mammalian cells through FRET measurements. CFP- and RFP-tagged wild-type or the Grb2 mutants together with controls were co-transfected in HEK293T cells as indicated on the figure. The results show a clear shortening of the average lifetime distribution for fluorophore in cells expressing the WTGrb2. This is due to FRET between the CFP–Grb2 and RFP–Grb2. The Y160E and N188/214D mutants on the other hand show an average lifetime distribution comparable to the controls indicating no FRET. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: In vitro and in vivoWTGrb2 form dimers.(a) Homodimer with individual Grb2 protomers of the dimer depicted using surface (green) and ribbon (blue/purple) representations (derived from PDB 1GRI). Individual SH2 and SH3 domains of the protomers are differentially coloured and labelled. Zoomed box (top)—Y160 of one protomer (purple) is buried in the dimer interface, packing against the CSH3 domain and hydrogen bonding with E87 of the other protomer chain (green). Zoomed box (bottom)—hydrogen bonded Asn188 (green) and Asn214 (purple) in the dimer interface. (b) Grb2 dimerization measured by MST. Unlabelled WTGrb2 protein (7.3 nM to 30 μM) was titrated into a fixed concentration of labelled Grb2 (100 nM). The data for thermophoresis was recorded at 20 °C using the blue LED at 20% and IR-Laser at 40%. The isotherm derived from the raw data and fitted according to the law of mass action to yield an apparent Kd dimer of 0.76±0.20 μM. (c) The data points representing the MST results of the Y160E mutant, which show a scattered distribution and unsuitable for fitting. (d) The thermophoresis results of the N188/214D mutant, as with Y160E the data is unsuitable for fitting analysis. (e) FLIM of Grb2 dimerization in mammalian cells through FRET measurements. CFP- and RFP-tagged wild-type or the Grb2 mutants together with controls were co-transfected in HEK293T cells as indicated on the figure. The results show a clear shortening of the average lifetime distribution for fluorophore in cells expressing the WTGrb2. This is due to FRET between the CFP–Grb2 and RFP–Grb2. The Y160E and N188/214D mutants on the other hand show an average lifetime distribution comparable to the controls indicating no FRET. Scale bar, 25 μm.

Mentions: We sought to identify the factors regulating Grb2 monomer/dimer equilibrium which might define a switching mechanism for Grb2-mediated signal transduction. Since Y160 lies within the previously reported dimer interface and forms a hydrogen bond with E87 on the partner protomer13 (Fig. 1a), tyrosine kinase post-translational modification of Y160 could perturb dimerization through a repulsive charge or steric clash effect.


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)

In vitro and in vivoWTGrb2 form dimers.(a) Homodimer with individual Grb2 protomers of the dimer depicted using surface (green) and ribbon (blue/purple) representations (derived from PDB 1GRI). Individual SH2 and SH3 domains of the protomers are differentially coloured and labelled. Zoomed box (top)—Y160 of one protomer (purple) is buried in the dimer interface, packing against the CSH3 domain and hydrogen bonding with E87 of the other protomer chain (green). Zoomed box (bottom)—hydrogen bonded Asn188 (green) and Asn214 (purple) in the dimer interface. (b) Grb2 dimerization measured by MST. Unlabelled WTGrb2 protein (7.3 nM to 30 μM) was titrated into a fixed concentration of labelled Grb2 (100 nM). The data for thermophoresis was recorded at 20 °C using the blue LED at 20% and IR-Laser at 40%. The isotherm derived from the raw data and fitted according to the law of mass action to yield an apparent Kd dimer of 0.76±0.20 μM. (c) The data points representing the MST results of the Y160E mutant, which show a scattered distribution and unsuitable for fitting. (d) The thermophoresis results of the N188/214D mutant, as with Y160E the data is unsuitable for fitting analysis. (e) FLIM of Grb2 dimerization in mammalian cells through FRET measurements. CFP- and RFP-tagged wild-type or the Grb2 mutants together with controls were co-transfected in HEK293T cells as indicated on the figure. The results show a clear shortening of the average lifetime distribution for fluorophore in cells expressing the WTGrb2. This is due to FRET between the CFP–Grb2 and RFP–Grb2. The Y160E and N188/214D mutants on the other hand show an average lifetime distribution comparable to the controls indicating no FRET. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: In vitro and in vivoWTGrb2 form dimers.(a) Homodimer with individual Grb2 protomers of the dimer depicted using surface (green) and ribbon (blue/purple) representations (derived from PDB 1GRI). Individual SH2 and SH3 domains of the protomers are differentially coloured and labelled. Zoomed box (top)—Y160 of one protomer (purple) is buried in the dimer interface, packing against the CSH3 domain and hydrogen bonding with E87 of the other protomer chain (green). Zoomed box (bottom)—hydrogen bonded Asn188 (green) and Asn214 (purple) in the dimer interface. (b) Grb2 dimerization measured by MST. Unlabelled WTGrb2 protein (7.3 nM to 30 μM) was titrated into a fixed concentration of labelled Grb2 (100 nM). The data for thermophoresis was recorded at 20 °C using the blue LED at 20% and IR-Laser at 40%. The isotherm derived from the raw data and fitted according to the law of mass action to yield an apparent Kd dimer of 0.76±0.20 μM. (c) The data points representing the MST results of the Y160E mutant, which show a scattered distribution and unsuitable for fitting. (d) The thermophoresis results of the N188/214D mutant, as with Y160E the data is unsuitable for fitting analysis. (e) FLIM of Grb2 dimerization in mammalian cells through FRET measurements. CFP- and RFP-tagged wild-type or the Grb2 mutants together with controls were co-transfected in HEK293T cells as indicated on the figure. The results show a clear shortening of the average lifetime distribution for fluorophore in cells expressing the WTGrb2. This is due to FRET between the CFP–Grb2 and RFP–Grb2. The Y160E and N188/214D mutants on the other hand show an average lifetime distribution comparable to the controls indicating no FRET. Scale bar, 25 μm.
Mentions: We sought to identify the factors regulating Grb2 monomer/dimer equilibrium which might define a switching mechanism for Grb2-mediated signal transduction. Since Y160 lies within the previously reported dimer interface and forms a hydrogen bond with E87 on the partner protomer13 (Fig. 1a), tyrosine kinase post-translational modification of Y160 could perturb dimerization through a repulsive charge or steric clash effect.

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