Abstract
The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.
Highlights
The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway
Using the monomeric phosphorylation charge mimetic Grb[2] mutant Y160E (Grb2Y160E)[30], we show that in non-stimulated cells Grb2Y160E is able to greatly enhance Shp[2] phosphatase activity via a bidentate interaction involving two discrete interfaces; (1) between the NSH2 domain of Shp[2] and the Src homology 2 (SH2) of Grb[2], and (2) between the protein tyrosine phosphatase (PTP) domain of Shp[2] and the CSH3 of Grb[2]
We demonstrated that Grb2Y160E can bind at two discrete sites on Shp[2] using biolayer interferometry (BLI) on the following four GST-tagged phosphatase constructs: Shp2WT, the tandem SH2 domains, the PTP domain (251–524: Shp2PTP) and a peptide corresponding to the C-terminal 69 amino acids (525–593: Shp2C69)
Summary
The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. In non-stimulated cells we find that binding of the adaptor protein Grb[2], in its monomeric state, initiates Shp[2] activity independent of phosphatase phosphorylation. Grb[2] forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp[2], releasing the phosphatase from its auto-inhibited conformation. 1234567890():,; The reciprocal process of phosphorylation by kinases, and dephosphorylation by phosphatases, of selected residues regulates the intensity and longevity of intracellular tyrosine kinase-mediated signal transduction. Shp[2] is ubiquitously expressed in vertebrate cells and consists largely of, in sequential order: two Src homology 2 (SH2) domains (NSH2 and CSH2, respectively); a protein tyrosine phosphatase (PTP). NSH2 forms an intramolecular interaction with the PTP domain, directly blocking access to the catalytic site, resulting in a ‘closed’
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