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

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 dimerizationdependent 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 tyrosinephosphorylated 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 K d 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. Abbreviations: 6His, six histidine residue protein tag used for purification; AUC, analytical ultracentrifugation; BPS, between pleckstrin and SH2; DLS, dynamic light scattering; DTT, dithiothreitol;ErbB2, erythroblastosis oncogene B2; FL, full length; Grb7, growth factor receptor-bound protein 7; GST, glutathione S-transferase protein tag used for purification; MES, 2-(N-morpholino) ethanesulfonic acid; PH, pleckstrin homology; RA, Ras associating; SEC, size exclusion chromatography; SH2, Src homology 2; Ve/Vo, the ratio of the elution volume to the void volume of the column; WT, wild type

La proteína 7 unida al receptor del factor de crecimiento (GRB7) en cáncer de mama

“…Se ha sugerido que la dimerización del dominio SH2 afecta la estabilidad estructural y la habilidad de unión del péptido tirosina fosforilado de GRB7. 34 El GRB7 es fosforilado en residuos serina y treonina en células quiescentes y en las células estimuladas con factor de crecimiento. La fosforilación en el residuo tirosina de GRB7 induce la unión a receptores tirosina quinasa.…”