Tyrosine phosphorylation of phospholipase C γ1 couples the FCη receptor mediated signal to mast cells secretion

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Abstract
Mast cells respond to clustering of the type I FC receptor (FcRI) on their membranes by mediator secretion. Recently, a marked enhancement of tyrosine phosphorylation on several proteins has been observed as a result of antigen-induced FcRI aggregation on these cells. We report here that the phosphatidyl inositide specific phospholipase Cγ1, (PLCγ1) is one of the prime proteins that undergoes tyrosine phosphorylation as a result of this stimulus. This was determined by immunoprecipitation of phosphotyrosine containing proteins from detergent tysates of rat mucosal mast cells (rat basophilic leukemia cells, subline 2H3; RBL-2H3) and Western blotting analysis of the separated components. A fast appearance of phosphorylated tyrosine residues on PLCγ1 was observed, reaching its maximal intensity at -1–3 mln after stimulation and declined afterwards to basal levels. Moreover, the phosphorylation depended on maintaining the aggregated FceRI as did other cellular responses (e.g. phosphatidyl inositides hydrolysis and secretion). The time course of both FceRI Induced phospholipase Cγ1 activation, as monitored by the formation of inositol phosphates, and of the secretory response of the cells followed that of the PLCγ, phosphorylation. Furthermore, the tyrphostln AG490, a protein tyrosine kinase inhibitor, caused similar inhibition of the FceRI-induced PLCγ1 phosphorylation, inositol phosphates formation, and mediator secretion. Significantly, no tyrosine phosphorylation of PLCγ1 was induced by the Ca2+ ionophore, ionomycin, even at doses that cause optimal secetory response. Taken together, these results clearly suggest that PLCγ1 phosphorylation on tyrosine residues constitutes a key element in the cascade coupling the FcRI clustering stimulus to the secretory response. Moreover, FCRI clustering is dynamically coupled through tyrosine phosphorylation/dephosphorylation to the PLCγ, activity and to the ensuing secretory response.

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