Related Experiment Videos
A 77-kDa protein associates with pp125FAK in mast cells and becomes tyrosine-phosphorylated by high affinity IgE
M M Hamawy1, K Minoguchi, W D Swaim
1Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-1188, USA.
Abstract:
The focal adhesion kinase, pp125FAK, is a novel non-receptor protein tyrosine kinase expressed in different cells including mast cells. Here we report that a 77-kDa protein associates with pp125FAK in the mast cell analog, rat basophilic leukemia (RBL-2H3) cells. When pp125FAK immunoprecipitates were subjected to an in vitro kinase assay, there was prominent phosphorylation on tyrosine of pp125FAK and of a 77-kDa protein. By V8 protease digestion mapping and by immunoblotting with two different anti-pp125FAK antibodies, the 77-kDa protein was distinct from pp125FAK. This Fak Associated Protein or FAP was detected in RBL-2H3 cells but not in fibroblasts. The aggregation of the high affinity IgE receptor, Fc epsilon RI, induced the in vivo tyrosine phosphorylation of FAP. However, there was a marked decrease in the in vitro phosphorylation of FAP in the immunoprecipitates from Fc epsilon RI aggregated cells. Both of these Fc epsilon RI-mediated effects were enhanced by cell adhesion. There was strong association of FAP with non-tyrosine-phosphorylated pp125FAK. Thus this interaction does not appear to be mediated by the Src homology 2 domain. Together the data indicate that FAP associates with pp125FAK and suggest that FAP may play a role in Fc epsilon RI signaling.
Insights
A novel 77-kDa Fak Associated Protein (FAP) interacts with focal adhesion kinase (pp125FAK) in mast cells. FAP phosphorylation is regulated by Fc epsilon RI aggregation, suggesting a role in mast cell signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Focal adhesion kinase (pp125FAK) is a non-receptor protein tyrosine kinase found in various cells, including mast cells.
- Mast cells play a crucial role in allergic responses, mediated by receptors like Fc epsilon RI.
Purpose of the Study:
- To identify and characterize proteins interacting with pp125FAK in mast cells.
- To investigate the role of a novel associated protein in Fc epsilon RI signaling.
Main Methods:
- Immunoprecipitation of pp125FAK from rat basophilic leukemia (RBL-2H3) cells.
- In vitro kinase assays to assess protein phosphorylation.
- V8 protease digestion mapping and immunoblotting to identify and distinguish proteins.
- Analysis of protein phosphorylation and association following Fc epsilon RI aggregation and cell adhesion.
Main Results:
- A 77-kDa protein, termed Fak Associated Protein (FAP), was identified and found to associate with pp125FAK.
- FAP was detected in RBL-2H3 cells but not fibroblasts.
- Fc epsilon RI aggregation induced in vivo tyrosine phosphorylation of FAP, with decreased in vitro phosphorylation.
- FAP associated with non-tyrosine-phosphorylated pp125FAK, independent of the Src homology 2 domain.
Conclusions:
- FAP is a novel protein that associates with pp125FAK in mast cells.
- FAP phosphorylation and association with pp125FAK are modulated by Fc epsilon RI signaling and cell adhesion.
- FAP may play a significant role in Fc epsilon RI-mediated mast cell signaling pathways.