Characterization of the full-length human Grb7 protein and a phosphorylation representative mutant
Affiliation
Univ Arizona, Dept Chem & Biochem, Analyt Biophys CoreIssue Date
2019-07-28
Metadata
Show full item recordPublisher
WILEYCitation
Bradford, AM, Koirala, R, Park, CK, Lyons, BA. Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant. J Mol Recognit. 2019;e2803. https://doi.org/10.1002/jmr.2803Journal
JOURNAL OF MOLECULAR RECOGNITIONRights
© 2019 John Wiley & Sons, Ltd.Collection Information
This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.Abstract
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.Note
12 month embargo; published online: 28 July 2019ISSN
0952-3499PubMed ID
31353673DOI
10.1002/jmr.2803Version
Final accepted manuscriptSponsors
NIH [P20GM103451, SC3 GM116728]ae974a485f413a2113503eed53cd6c53
10.1002/jmr.2803
Scopus Count
Collections
Related articles
- Dimerization in the Grb7 protein.
- Authors: Peterson TA, Benallie RL, Bradford AM, Pias SC, Yazzie J, Lor SN, Haulsee ZM, Park CK, Johnson DL, Rohrschneider LR, Spuches A, Lyons BA
- Issue date: 2012 Aug
- Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain.
- Authors: Spuches AM, Argiros HJ, Lee KH, Haas LL, Pero SC, Krag DN, Roller PP, Wilcox DE, Lyons BA
- Issue date: 2007 Jul-Aug
- Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation.
- Authors: Siamakpour-Reihani S, Peterson TA, Bradford AM, Argiros HJ, Haas LL, Lor SN, Haulsee ZM, Spuches AM, Johnson DL, Rohrschneider LR, Shuster CB, Lyons BA
- Issue date: 2011 Mar-Apr
- Grb7 protein RA domain oligomerization.
- Authors: Godamudunage MP, Foster A, Warren D, Lyons BA
- Issue date: 2017 Aug
- Tyrosine phosphorylation of growth factor receptor-bound protein-7 by focal adhesion kinase in the regulation of cell migration, proliferation, and tumorigenesis.
- Authors: Chu PY, Huang LY, Hsu CH, Liang CC, Guan JL, Hung TH, Shen TL
- Issue date: 2009 Jul 24