RECONSTITUTION OF INTERACTIONS BETWEEN TYROSINE KINASES AND THE HIGH-AFFINITY IGE RECEPTOR WHICH ARE CONTROLLED BY RECEPTOR CLUSTERING

High affinity IgE receptor (Fc epsilon RI) signaling after contact wit h antigen occurs in response to receptor clustering, This paper descri bes methodology, based on vaccinia virus driven protein expression, fo r probing signaling pathways and its application to Fc epsilon RI inte ractions with the lyn and syk tyrosine kinases, Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hemat opoietic 'null' cell line is sufficient to reconstruct clustering-cont rolled receptor tyrosine phosphorylation and activation of syk, withou t apparent requirement for hematopoietic specific phosphatases, The sr c family kinase lyn phosphorylates Fc epsilon RI in response to recept or clustering, resulting in syk binding to the phosphorylated Fc epsil on RI, Lyn also participates in the tyrosine phosphorylation and activ ation of syk in a manner which is dependent on phosphorylated Fc epsil on RI. Using overexpression of active and dominant negative syk protei ns in a mast cell line which naturally expresses Fc epsilon RI, we cor roborate syk's role downstream of receptor phosphorylation, and demons trate that syk SH2 domains protect receptor ITAMs from ongoing dephosp horylation, Based on these results, we propose that receptor clusterin g controls lyn-mediated Fc epsilon RI. tyrosine phosphorylation by shi fting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI t yrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allow ing clustering to indirectly control syk activity.