Reconstitution of High Affinity IgE Receptor-mediated Secretion By Transfecting Protein Tyrosine Kinase pp125 FAK

Majed M. Hamawy, Mark Swieter, Stephan E. Mergenhagen, Reuben P. Siraganian
1997 Journal of Biological Chemistry  
To study the role of the focal adhesion tyrosine kinase (FAK) in receptor-mediated secretion, we transfected FAK cDNA into a variant (3B6) of the RBL-2H3 mast cell line. This 3B6 cell line expressed low levels of FAK and was defective in high affinity IgE receptor (Fc⑀RI) but not Ca 2؉ ionophore-mediated secretion. Fc⑀RI-mediated secretion was reconstituted after transfection of wildtype FAK. Histamine release was also enhanced by the stable expression of two mutants of FAK: a kinase-inactive
more » ... rm in which the ATP binding site Lys-454 was replaced by Arg or a mutant in which the autophosphorylation site Tyr-397 was replaced by Phe. Therefore, the catalytic activity and the autophosphorylation site of FAK are not essential for secretion. Fc⑀RI aggregation increased the tyrosine phosphorylation of both mutants of FAK to the same extent as wild-type FAK. Therefore, tyrosine kinases activated by Fc⑀RI aggregation are phosphorylating FAK and some of these phosphorylation sites are other than Tyr-397. These results strongly suggest that FAK plays a role in Fc⑀RI-induced secretion by functioning as an adapter or linker molecule. The focal adhesion kinase (FAK) 1 is a widely expressed tyrosine kinase that localizes with integrins and several proteins to intracellular sites where cells are in contact with the extracellular matrix (1, 2). FAK also becomes tyrosine-phosphorylated in response to the activation of various receptors including the high affinity IgE receptor (Fc⑀RI) and Ag receptors on T-cells (1-4). FAK is essential for development as the inactivation through homologous recombination of the FAK gene results in early embryonal mesodermal defects, retarded growth, and prenatal death (5). Previously, we reported that the aggregation of Fc⑀RI results in tyrosine phosphorylation of FAK in rat basophilic leukemia RBL-2H3 cells, a mast cell line that has been extensively used to study Fc⑀RI-mediated secretion (3). This receptor-induced phosphorylation of FAK and secretion from these cells are enhanced by adherence of the cells to fibronectin (3, 6). Furthermore, a monoclonal antibody to a ganglioside present on the RBL-2H3 cells induces many of the same intracellular reactions initiated by Fc⑀RI aggregation but fails to result in tyrosine phosphorylation of FAK and does not result in secretion (7). These experiments suggest that FAK may play a role in secretion from mast cells and basophils. To further define the role of FAK in secretion, we transfected FAK cDNA into a variant of the RBL-2H3 mast cell line that expressed low levels of this kinase and had decreased Fc⑀RI, but not Ca 2ϩ ionophore-mediated secretion. Fc⑀RI-induced histamine release was reconstituted in the cell lines that expressed increased levels of FAK. These studies demonstrate a function for FAK in Fc⑀RI-mediated secretion.
doi:10.1074/jbc.272.48.30498 pmid:9374543 fatcat:vzpmerjv2nhzlngwwrftewaafu