Related Experiment Videos
Identification of a second Grb2 binding site in the v-Fms tyrosine kinase
A Mancini1, R Niedenthal, H Joos
1Institut für Physiologische Chemie, Medizinische Hochschule Hannover, Germany.
Abstract:
Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins containing Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, Y696KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. Here, we mapped Y921TNL within the C-terminal domain of Fms as a novel autophosphorylation site. We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cell extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696. A yeast two-hybrid system which allowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2. Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increased for proteins carrying both sites. In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of fibronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.
Insights
Researchers identified a new binding site (Y921) on the Fms protein that interacts with growth factor receptor bound protein 2 (Grb2). This discovery impacts understanding of cell signaling pathways and cancer development.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncogenesis
Background:
- The v-Fms oncogene product undergoes tyrosine autophosphorylation, creating binding sites for Src homology 2 (SH2) domain-containing proteins that mediate mitogenic and morphogenic signals.
- Previously, Y696 in the kinase insert domain of v-Fms was identified as a Grb2 binding site, crucial for Ras/Raf1 pathway stimulation.
Purpose of the Study:
- To identify and characterize novel autophosphorylation sites on the Fms protein.
- To investigate the role of these sites in binding to growth factor receptor bound protein 2 (Grb2).
- To elucidate the functional consequences of Fms-Grb2 interactions on cellular phenotypes.
Main Methods:
- Mapping of novel autophosphorylation sites using biochemical techniques.
- Assessment of Grb2 binding affinity using recombinant fusion proteins and cell extracts.
- Quantification of Grb2 binding via a yeast two-hybrid system with a functional Fms tyrosine kinase.
- Analysis of cellular phenotypes, including fibronectin network content and growth characteristics, in NIH3T3 cells expressing Fms mutants.
Main Results:
- A novel autophosphorylation site, Y921, was identified in the C-terminal domain of Fms.
- Phosphorylated Y921 demonstrated efficient binding to Grb2, comparable to Y696, with enhanced binding when both sites were present.
- Simultaneous mutation of Y696 and Y921 to phenylalanine abolished Grb2 binding.
- NIH3T3 cells expressing Y921F mutant Fms exhibited increased fibronectin network and reduced serum-independent growth.
Conclusions:
- Y921 represents a second functional Grb2 binding site on the Fms protein.
- The Fms-Grb2 interaction, mediated by Y696 and Y921, plays a significant role in regulating cellular fibronectin content and growth phenotypes.
- Targeting these phosphorylation sites could offer therapeutic strategies for Fms-driven cancers.