title: Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.
abstract: High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity.
authors.affiliation: Molecular Allergy and Immunology Section/NIAID/NIH, Rockville, MD 20852, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Scharenberg
authors.forename: S
authors.initials: S
authors.lastname: Lin
authors.forename: B
authors.initials: B
authors.lastname: Cuenod
authors.forename: H
authors.initials: H
authors.lastname: Yamamura
authors.forename: J P
authors.initials: JP
authors.lastname: Kinet
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011940:Receptor Aggregation; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013552:Swine; D000072377:Syk Kinase; D014616:Vaccinia virus
chemical_list: D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D011994:Recombinant Proteins; C086249:lynA protein, rat; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat