Reconstitution of high affinity IgE receptor-mediated secretion by transfecting protein tyrosine kinase pp125FAK.
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 (FcepsilonRI) but not Ca2+ ionophore-mediated secretion. FcepsilonRI-mediated secretion was reconstituted after transfection of wild-type FAK. Histamine release was also enhanced by the stable expression of two mutants of FAK: a kinase-inactive form 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. FcepsilonRI aggregation increased the tyrosine phosphorylation of both mutants of FAK to the same extent as wild-type FAK. Therefore, tyrosine kinases activated by FcepsilonRI 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 FcepsilonRI-induced secretion by functioning as an adapter or linker molecule.
Hamawy
M M
MM
Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Swieter
M
M
Mergenhagen
S E
SE
Siraganian
R P
RP
D019943:Amino Acid Substitution; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006636:Histamine Release; D008407:Mast Cells / Q000201:enzymology / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011940:Receptor Aggregation; D017455:Receptors, IgE / Q000502:physiology*; D011994:Recombinant Proteins; D015398:Signal Transduction; D014162:Transfection
D015815:Cell Adhesion Molecules; D017455:Receptors, IgE; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat