Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphoryla...

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Bibliographic Details
Published in:Journal of molecular recognition 2019-11, Vol.32 (11), p.e2803-n/a
Main Authors: Bradford, Andrew M., Koirala, Rajan, Park, Chad K., Lyons, Barbara A.
Format: Article
Language:English
Subjects:
Binding
cancer
cell migration
Dimerization
Grb7 regulation
Growth factors
In vitro methods and tests
Kinases
Light scattering
oligomerization
Phosphorylation
Photon correlation spectroscopy
Protein structure
Proteins
Residues
Signaling
Size exclusion chromatography
Tyrosine
tyrosine phosphorylation
Ultracentrifugation
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Summary:It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization Kd of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation. Characterization of the full‐length human Grb7 protein, and a phosphorylation mimic mutant, was completed with a goal of understanding the functionally active Grb7 protein conformation. The data support a model for Grb7 activation, whereby Grb7 becomes primarily monomeric upon tyrosine phosphorylation, and makes the internal RA‐PH domains available for downstream signaling events.
ISSN:0952-3499
1099-1352
DOI:10.1002/jmr.2803