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dc.description.abstractPhospholipase C (PLC)-γ isozymes play crucial roles in transmembrane signaling of many receptors that couple to protein tyrosine kinases. Regulatory mechanisms for the hematopoietic cell-specific isozyme, PLC-γ2, are unclear compared to those for ubiquitously expressed PLC-γ1. In the present study, Tyr residues in PLC-γ2 phosphorylated upon receptor stimulation of cells and kinases responsible for the phosphorylation were identified in the signaling of B cell antigen receptor (BCR). Stimulation of various cell types all led phosphorylation at two Tyr residues, Tyr753 and Tyr759. Significant phosphorylation of Tyr 1217 in the C-terminal tail was also observed in B cells, but not in other cell types. A phosphoinositide (PI) 3-kinase-dependent protein tyrosine kinase Btk has been implicated in BCR-mediated phosphorylation of PLC-γ2. We found that phosphorylation at Tyr753 and Tyr759 were largely, mediated by Btk, whereas that at Tyr1217 was totally independent of Btk. It was also found that activation of protein kinase C, as a consequence of PLC-meditated PI hydrolysis, led to inhibition of Btk-dependent PLC-γ2 phosphorylation, constituting a negative feedback loop to regulate PLC activity. Furthermore, we revealed that a product of PI 3-kinase directly regulates activity of the PLC other than via its phosphorylation by Btk: while Btk silencing lowered BCR-induced PI hydrolysis approximately by half with concomitant reduction in PLC-γ2 phosphorylation, additional treatment with PI 3-kinase inhibitor further reduced the apparent PLC activity with little effect on the phosphorylation.-
dc.description.tableofcontentsABSTRACT = i CONTENTS = iii LIST OF FIGURES & TABLE = v INTRODUCTION = 1 Phospholipase C = 1 PLC-γ1/2 isozymes = 7 Phosphorylation sites of PLC-γ2 = 11 Phosphorylation mechanism of PLC-γ2 by immunoreceptor = 16 Function of Btk in B cells = 21 Roles of PI3-kinase for PLC-γ2 activation in B-cells = 23 Research Scope = 24 MATERIALS AND METHODS = 26 Materials and cells = 26 Antibodies = 26 Preparation of mouse splenic B cells and human platelets = 28 Expression of PLC-γ2 mutants = 29 Cell stimulation, lysis and immunoblotting = 29 RNA interference = 31 Measurement of [^3 H]inositol phosphate generation = 31 RESULTS = 33 Development and characterization of phospho antibodies against tyrosine sites in PLC-γ2 = 33 Differential phosphorylation patterns of PLC-γ2 phosphorylation in various cell types = 38 Identification of responsible kinase(s) for PLC-γ2 in B cells = 48 Roles of PI3-kinase in PLC-γ2 activation in B cells = 68 DISCUSSION = 78 Identification of phosphorylation sites of PLC-γ2 in various cell types = 78 Role of each phosphorylation site in PLC-γ2 activation = 82 Identification of responsible kinase(s) for PLC-γ2 in B cells = 84 Roles of PI3-kinase in PLC-γ2 activation in B cells = 91 REFERENCES = 94-
dc.format.extent723233 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleMechanism of phosphorylation and activation of PLC-γ2 isozyme in immune cells-
dc.typeDoctoral Thesis-
dc.format.pagevi, 105 p.-
dc.identifier.major대학원 분자생명과학부- 2-
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