Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

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.

Grb2 binding induces phosphorylation-independent activation of Shp2

“…Mutation of Y160 to glutamate (a phosphotyrosine charge mimetic) in the Grb2 dimer interface abrogates self-association of the adaptor protein (Ahmed et al, 2015). To characterise the interaction between full-length Shp2 (Shp2 FL ) and monomeric Grb2 (including Y160E mutation, Grb2 Y160E ; Ahmed et al, 2015) we initially used microscale thermophoresis (MST) to measure the affinity of the interaction of full length proteins (K D = 0.33 ± 0.04 µM; Fig 1A).…”

“…Mutation of Y160 to glutamate (a phosphotyrosine charge mimetic) in the Grb2 dimer interface abrogates self-association of the adaptor protein (Ahmed et al, 2015). To characterise the interaction between full-length Shp2 (Shp2 FL ) and monomeric Grb2 (including Y160E mutation, Grb2 Y160E ; Ahmed et al, 2015) we initially used microscale thermophoresis (MST) to measure the affinity of the interaction of full length proteins (K D = 0.33 ± 0.04 µM; Fig 1A). We demonstrated that Grb2 Y160E can bind at two discrete sites on Shp2 using bio-layer interferometry (BLI) on the following four GST-tagged phosphatase constructs: Shp2 FL , the tandem SH2 domains (residues 1-220: Shp2 2SH2 ), the PTP domain (221-524: Shp2 PTP ) and a peptide corresponding to the C-terminal 69 amino acids (525-593: Shp2 C69 ).…”

“…Grb2 consists of an SH2 domain sandwiched between two SH3 domains and is integral to several RTK-mediated signalling pathways. Non-phosphorylated Grb2 exists in a concentration-dependent dimer-monomer equilibrium (K d = 0.8 µM; Ahmed et al, 2015). In addition to the result of depletion of cellular concentration (Timsah et al, 2014), monomeric Grb2 (mGrb2) will also prevail when it is phosphorylated on tyrosine 160 (Y160) in the dimer interface (Ahmed et al, 2015).…”

“…Non-phosphorylated Grb2 exists in a concentration-dependent dimer-monomer equilibrium (K d = 0.8 µM; Ahmed et al, 2015). In addition to the result of depletion of cellular concentration (Timsah et al, 2014), monomeric Grb2 (mGrb2) will also prevail when it is phosphorylated on tyrosine 160 (Y160) in the dimer interface (Ahmed et al, 2015). In the absence of growth factor stimulation Grb2 cycles between the phosphorylated mGrb2, and the non-phosphorylated, typically dimeric state.…”

“…The former is dependent on constitutive, background RTK activity, e.g. fibroblast growth factor receptor 2 (FGFR2; Lin et al, 2012), whilst the latter results from concomitant Shp2 activity (Ahmed et al, 2015).…”